Book - Comparative Embryology of the Vertebrates 1

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Nelsen OE. Comparative embryology of the vertebrates (1953) Mcgraw-Hill Book Company, New York.

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Part I The Period of Preparation

The events which precede the initiation of the new individual's development are:

( 1 ) The preparation of the male and female parents and their reproductive structures for the act of reprcxluction (Chaps. 1 and 2).

(2) The preparation of the gametes (Chap. 3).

The anterior lobe of the pituitary gland, because of its secretion of the gonadotrophic (gonad-stimulating) hormones, is the pivotal structure in the reproductive mechanism.

The gonadotrophic hormones are:

1. Follicle-stimulating hormone, FSH;
2. Luteinizing hormone, LH (ICSH), and
3. Luteotrophin, LTH.

The Testis and. Its Relation to Reproduction

A. Introduction

1. General description of the male reproductive system

2. Importance of the testis

B. Anatomical features of the male reproductive system

1. Anatomical location of the testis

2. Possible factors involved in testis descent

3. General structure of the scrotum and the testis in mammals

a. Structure of the scrotum

b. General structure of the testis

4. Specific structures of the mammalian testis which produce the reproductive cells and the male sex hormone

a. Seminiferous tubules

b. Interstitial tissue

5. The testis of vertebrates in general

6. Accessory reproductive structures of the male

a. The reproductive duct in forms utilizing external fertilization

b. The reproductive duct in species practicing internal fertilization

C. Specific activities of the various parts of the male reproductive system

1. Introduction

a. Three general functions of the male reproductive system

b. Some definitions

2. Activities of the testis

a. Seasonal and non-seasonal types of testicular activity

b. Testicular tissue concerned with male sex-hormone production

c. Testicular control of body structure and function by the male sex hormone

1) Sources of the male sex hormone

2) Biological effects of the male sex hormone

a) Effects upon the accessory reproductive structures

b) Effects upon secondary sex characteristics and behavior of the individual

c) Effects upon the seminiferous tubules

d. Seminiferous-tubule activity and formation of sperm

e. The seminiferous tubule as a sperm-storing structure

3. Role of the reproductive duct in sperm formation

a. Vertebrates without a highly tortuous epididymal portion of the reproductive duct

b. The epididymis as a sperm-ripening structure

c. The epididymis and vas deferens as sperm-storage organs

d. Two types of vertebrate testes relative to sperm formation

4. Function of the seminal vesicles (vesicular glands)

5. Function of the prostate gland

6. Bulbourethral (Cowper’s) glands

7. Functions of seminal fluid

a. Amount of seminal fluid discharged and its general functions

b. Coagulation of the semen

c. Hyaluronidasc

d. Accessory sperm

e. Fructose

f. Enzyme-protecting substances

D. Internal and external factors influencing activities of the testis

1. Internal factors

a. Temperature and anatomical position of the testis

b. Body nourishment in relation to testicular function

c. The hypophysis and its relation to testicular function

2. External environmental factors and testis function

a. Light as a factor

b. Temperature influences

E. Internal factors which may control seasonal and continuous types of testicular function

F. Characteristics of the male reproductive cycle and its relation to reproductive conditions in the female

A. Introduction

1. General Description of the Male Reproductive System

The male reproductive system of most vertebrate animals consists of two testis with a sperm-conveying duct and attendant auxiliary glands associated with each testis. In some species, such as the frog and many teleost fishes, the sperm-conveying duct is a simple structure, but in most vertebrate forms there is a tendency for the duct to be complicated. The cyclostomatous fishes do not possess sperm-conveying ducts from the testis to the outside.

In reptiles, some birds and all mammals, in gymnophionan amphibia and in the “tailed” frog, Ascaphus, in sharks and certain teleost fishes, an intromittent organ is added to the sperm-conveying structures for the purpose of internal fertilization. But an intromittcnt organ is not present in all species which practice internal fertilization. In many salamanders, internal fertilization is effected by the spawning of a spermatophore filled with sperm; the latter is picked up by the cloaca of the female. The sperm in these salamanders are stored in special pockets or tubules within the dorsal wall of the cloaca. These storage tubules form the spermatheca (fig. 10). Direct transfer of sperm to the female by cloacal contact may occur in some species.

2. Importance of the Testis

The word testis or testicle was formerly applied to the ovary of the female, as well as to the male sperm-producing organ, and the term "female testicle" was used in reference to the female organ. The use of the word “ovary” was introduced by Steno in 1667, and also by de Graaf (fig. 1 ) in 1672 in his work on the female generative organs. To quote from de Graaf: “Thus, the general function of the female testicles is to generate the ova, to nourish them, and to bring them to maturity, so that they serve the same purpose in women as the ovaries of birds. Hence, they should rather be called ovaries than testes because they show no similarity, either in form or contents, with the male testes properly so called.” (See Corner, -43.) From the time of de Graaf the word “testis” has been restricted to designate the male organ essential to reproduction.

The phrase "essential to reproduction" does not describe fully the importance of testicular function. As we shall see later on, the testis not only assumes the major role in the male's activities during the period of reproduction, but also, in the interim between specific reproductive periods, it governs in many instances male behavior leading to protection and preservation of the species. Thus, the testis is the organ responsible for maleness in its broader, more vigorous sense.

B. Anatomical Features of the Male Reproductive System

Before endeavoring to understand the general functions of the testis in relation to reproduction, it is best to review some of the structural relationships of the testis in the vertebrate group.

Fig. 1. Reinier de Graaf. Born in Holland, 1641; died in Delft, Holland, 1673. Author of important works on the generative organs of the female. Described the Graafian follicle in the ovary of mammals but erroneously believed it to be the mammalian egg. (From Corner, '43.)

1. Anatomical Location of the Testis

In most vertebrates other than mammals, the testes are suspended well forward within the peritoix eal^cayity. In the Mammalia, however, the condition is variable. In the monotrematous mammals. Echidna and Ornithorynchus, the testes are located within the peritoneal cavity near the kidneys. In the elephant the testes also are located in this area. Schulte (’37) describes the position of the testes in an Indian elephant (Elephas indicus), 20 years old, as being “retroperitoneal lying on each side medial to the lower pole of the kidney.” (The kidneys were found to lie retroperitoneally on either side of the lower thoracic and lumbar vertebrae, and each measured about 275 mm. in length.) However, in the majority of mammals the testes descend posteriad from the original embryonic site, the extent varying with the species. In some there is a slight posterior migration, and the testes of the adult are situated well forward in the pelvic region. Examples of this condition are found in conies, whales, sea cows, African jumping shrews, and in armadillos. In sloths and American anteaters, the testes may descend into the pelvic cavity and lie in the area between the urinary bladder and the posterior body wall. However, in most of the eutherian and marsupial mammals, a dual outpushing of the postero-ventral body wall occurs into which the testes come to lie either permanently, or, in some forms, temporarily during the breeding season. This outward extension of the body-wall tissues is known as the scrotum; it involves not only the skin, muscle and connective tissues of the body wall but the peritoneal lining as well (fig. 2). (The interested student may consult Weber (’28) and Wislocki (’33) for data concerning the extent of testis descent in mammals.)

The peritoneal evaginations into the scrotal sac are two in number, one for each testis; each evagination is known as a processus vaginalis (figs. 3E, F; 4A, B). In many mammals this evagination becomes separated entirely froln the peritoneal cavity, and the testis, together with a portion of the sperm-conveying duct, lies suspended permanently in a small antechamber known as the inguinal bursa or serous cavity of the scrotum (fig. 4B). (See Mitchell, ’39.) This condition is found in the horse, man, opossum, bull, ram, dog, cat, etc. In certain other mammals, such as the rat, guinea pig, and ground hog, the inguinal bursa does not become separated from the main peritoneal cavity, and a persistent inguinal canal remains to connect the inguinal bursa with the peritoneal cavity (fig. 4C). In some rodents the testes pass through this persisting inguinal canal into the scrotum as the breeding season approaches, to be withdrawn again after the breeding period is terminated. The ground squirrel, Citellus tridecemlineatus (Wells, ’35) and the ground hog, Marrnota monax (Rasmussen, ’17) are examples of mammals which experience a seasonal descent of the testis.

In the majority of those mammals possessing a scrotum, it is a permanent structure. In a few, however, it is a temporary affair associated with the breeding season, as in the bat, Myotis, where the testes pass into a temporary perineal pouch or outpushing of the posterior abdominal wall during the reproductive season, to be withdrawn again together with the scrotal wall when the breeding period is past (fig. 4D). A similar periodic behavior is true of many insectivores, such as the common shrews, the moles, and the European hedgehog (Marshall, ’ll).

Fig. 2. Sketch of male reproductive system in man.

The permanent scrotum is a pendent structure, in some species more so than others. In the bull and ram, it extends from the body for a considerable distance, whereas in the cat, hippopotamus, tapir, guinea pig, etc., it is closely applied to the integumentary wall. In primates, including man, in most carnivores, and many marsupials, the pendency of the scrotum is intermediate between the extremes mentioned above.

An exceptional anatomical position of the testes in the lower vertebrates is found in the flatfishes, such as the sole and flounder, where they lie in a caudal outpouching of the peritoneal cavity (fig. 5). The testis on either side may even lie within a special compartment in the tail. (The ovaries assume the latter position in the female.)

Fig. 3. Diagrammatic representations of the urogenital structures in the developing male pig, with special emphasis upon testicular descent. (A) Early relationship of the genital fold (genital ridge), mesonephric kidney and its duct, together with the metanephric kidney and the ureter in 20-mm. pig embryo. The relationship of the mesonephric and metanephric ducts to the urogenital sinus is shown. The Miillcrian duct is omitted. (B) Male pig embryo about 45-mm., crown-rump length, showing relationship of gonad and metanephric kidney. I he metanephric kidney is shown below (dorsal to) the mesonephric kidney. The gonad (testis) is now a well-defined unit. The portion of the genital fold tissue anterior to the testis becomes the anterior suspensory ligament of the testis, while the genital fold tissue caudal to the testis continues back to join the inguinal ligament of the mesonephros (the future gubernaculum). (C) About 80-mm., crown-rump, pig embryo. Observe that the metanephros is now the dominant urinary organ and has grown cephalad, displacing the mesonephric kidney which is regressing and moving caudally with the testis. The remains of the mesonephric kidney at this time are gradually being transformed into epididymal structures. (D) About 130~mm.. crown-rump, pig embryo. Observe that the testis is approaching the internal opening of the inguinal canal. The anterior suspensory ligament is now an elongated structure extending over the lateroventral aspect of the metanephric kidney; the gubernacular tissue is shown extending downward into the inguinal canal. (E) Later stage in testicular descent. The anterior suspensory ligament of the testis is a prominent structure, while the gubernaculum is compact and shortened. (F) The condition found in the full-term, fetal pig. The testis is situated in the scrotal swelling; the gubernaculum is much shortened, while the anterior suspensory ligament remains as a prominent structure, extending cephalad to the caudal portions of the metanephric kidney.

2. Possible Factors Involved in Testis Descent The descent of the testis within the peritoneal cavity and into the scrotum poses an interesting problem. In embryonic development extensive migration of cell substance, or of cells, tissues, and organ structures is one of many processes by which the embryonic body is formed. That is to say, the dynamic movement or displacement of developing body structures from their original position is a part of the pattern of development itself. The casual factors involved in such movements are still unknown, and the study of such behavior forms one of the many interesting aspects of embryological investigation awaiting solution.

Fig. 4. Diagrammatic drawings portraying the relationship of the testis to the processus vaginalis (peritoneal evagination) and the scrotum. The testis is at all times retroperitoneal, i.e., outside the peritoneal cavity and membrane. (A) Earlier stage of testicular descent at the time the testis is moving downward into the scrotum. (B) Position of the testis at the end of its scrotal journey in a form possessing permanent descent of the testis, e.g., man, dog, etc. (C) Testis-peritoneal relationship in a form which does not have a permanent descent of the testis — the testis is withdrawn into the peritoneal cavity at the termination of each breeding season. Shortly before the onset of the breeding period or “rut,” the testis once again descends into the scrotum, e.g., ground hog. (D) Position of testis in relation to body wall and peritoneum in the mole, shrev/, and hedgehog in which there is no true scrotum. The testis bulges outward, pushing the body wall before it during the breeding season. As the testis shrinks following the season of rut, the bulge in the body wall recedes. True also of bat, Myotis.

Fig. 5. Opened peritoneal cavity of a common flounder, Limanda ferruginea, showing the position occupied by the testes. Each testis is situated partly in a separate compartment on either side of the hemal processes of the tail vertebrae.

Various theoretical explanations have been proposed, however, to explain the movement of the testis posteriad from its original embryonic site. Classical theory mentions the mechanical pull or tightening stress of the gubernaculum, a structure which develops in relation to the primitive genital fold or genital ridge (figs. 3B, C; 351C-7).

The genital ridge extends along the mesial aspect of the early developing mesonephric kidney from a point just caudal to the heart to the posterior extremity of the mesonephric kidney near the developing cloacal structures (Hill, ’07). Anteriorly, the genital ridge (fold) merges with the diaphragmatic ligament of the mesonephros (fig. 3A). The gonad (testis or ovary) develops in a specialized region of the more cephalic portion of the genital ridge (Allen, ’04). (See fig. 3A.) The caudal end of the mesonephric kidney eventually becomes attached to the posterior ventral body wall by means of a secondary formation of another cord of tissue, the inguinal fold (fig. 3A). The latter is attached to the posterior ventral body wall near the area where the scrotal outpushing (evagination) later occurs. This inguinal fold later becomes continuous with the genital fold (fig. 3B). The inguinal fold thus becomes converted into a ligament, the inguinal ligament of the mesonephros, uniting the caudal portion of the mesonephric kidney and adjacent genital fold tissue with the area of scrotal evagination (fig. 3B). The gubernaculum represents a later musculo-connective tissue development of the inguinal ligament and the adjacent genital fold tissue. It contains smooth muscle fibers as well as connective tissue. As the scrotal evagination forms at the point where the gubernaculum attaches to the body wall, the gubernaculum from the beginning of its formation is connected with the developing scrotal sac.

As the testis migrates posteriad, the anterior suspensory ligament of the testis elongates and the gubernaculum shortens (fig. 3A~F). This decrease in length of the gubernaculum is both real and relative. It is real in that an actual shortening occurs; it is relative because the rapid enlargement of the developing pelvic cavity and its contained organs makes the length of the gubernaculum appear less extensive. This enlargement of the pelvic space and increase in size of its contained structures and a corresponding failure of the gubernaculum to elongate, certainly are factors in bringing about the intra-abdominal descent of the testis; that is, testis descent within the peritoneal cavity itself (Felix, ’12).

Developmental preparations precede the extra-abdominal descent of the testes, for the scrotal chambers must be prepared in advance of the arrival of the testes. These developmental events are:

(1) two outpocketings of the abdominal wall which come to lie side by side below the skin to form the walls of the scrotal chamber, and

(2) an evagination of the peritoneum into each of the abdominal outpocketings which act as peritoneal linings for each pocket.

It is worthy of mention that the above outpushings of the abdominal wall and of the peritoneum precede the movement of the testes into the scrotum. They serve to illustrate the theory that a shortening of the gubernaculum is not sufficient to explain testis descent. Rather, that in this descent a whole series of developmental transformations are involved; the shortening of the gubernaculum and scrotal development merely represent isolated phases of the general pattern of movement and growth, associated with this descent.

More recent research emphasizes the importance of certain physiological factors relative to the descent problem. It has been determined, for example, that administration of the gonadotrophic hormone of pregnancy urine (chorionic gonadotrophin) or of the male sex hormone, testosterone, aid the process of extra-abdominal descent (i.c., descent from the inguinal ring area downward into the scrotum) . Hormone therapy, using chorionic gonadotrophin together with surgery, is used most often in human cryptorchid conditions. The androgen, testosterone, aids testicular descent mainly by stimulating the growth of the scrotal tissues and the vas deferens; however, it is not too successful in effecting the actual descent of the testis (Robson, ’40; Wells, ’43; Pincus and Thimann, ’50).

The phenomenon of testicular migration thus is an unsolved problem. Many activities and factors probably play a part in ushering the testis along the pathway to its scrotal residence.

3. General Structure of the Scrotum and the Testis in Mammals a. Structure of the Scrotum

The scrotal modification of the body wall generally occurs in the posteroventral area between the anus and the penial organ. However, in marsupials it is found some distance anterior to the latter.

Each scrotal evagination consists of three general parts: the skin with certain attendant muscles, the structures of the body wall below the skin, and the peritoneal evagination. The skin, with its underlying tunica daitos muscle tissue and superficial perineal fascia, forms the outer wall of the scrotum (fig. 6). Within this outer cutaneous covering lie the two body-wall and two peritoneal evaginations. The body-wall evaginations involve connective and muscle tissues of the external oblique, internal oblique, and trans versus muscles. The caudal part of each peritoneal outpocketing forms the serous cavity or inguinal bursa in which the testis is suspended after its descent, and its more anterior portion forms the inguinal canal (figs. 2, 4B, 6). The oblique and transversus layers of tissues thus are molded into a musculo-connective tissue compartment around each serous cavity. The median septum of the scrotum represents the area of partial fusion between the two musculo-connective tissue compartments, whereas the median raphe of the scrotum denotes the area of fusion of the two cutaneous coverings of the body-wall outpushings (fig. 6).

Consequently, passing inward from the superficial perineal fascia of the skin or outer wall, one finds the following tissue layers surrounding the testis:

( 1 ) The external spermatic fascia represents the modified fascia of the external oblique muscle layer of the embryo.

(2) The middle spermatic fascia is a modification of the internal oblique muscular layer, whose tissue forms the cremaster muscle loops within the scrotum (fig. 6). (Some of the cremasteric musculature may be derived from the transversus layer.)

(3) The internal spermatic fascia or tunica vaginalis communis is derived from the transverse muscle layer of the embryo.

(4) Along the inner surface of the tunica vaginalis communis is the peritoneal membrane. The latter is reflected back over the surface of the suspended testis, and thus forms the visceral peritoneal covering of the testis. This lining tissue of the common vaginal tunic and the peritoneal membrane which covers the testis are derived from the original peritoneal evagination into the scrotal pocket; as such it forms the tunica vaginalis propria*

b. General Structure of the Testis

The testis is composed of the following structural parts:

(1) The inner layer of the tunica vaginalis propria, the tunica vaginalis internus, envelops the testis. The cavity between the outer and inner layers of the tunica vaginalis propria is the inguinal bursa. Obliteration by injury or infection of this inguinal bursa may cause degenerative changes in the testis. In other words, the testis normally must be free to move within its serous (peritoneal) cavity.

(2) Within the tunica vaginalis internus of the testis is a thick fibrous layer of connective tissue, the tunica albuginea (fig. 7). From this tunic, connective tissue partitions, the septula of the testis, extend inward and converge toward that testicular zone where supplying blood vessels enter and leave, including the lymphatics. The latter zone is known as the mediastinum testis and it represents a regional thickening of the tunica albuginea. Here the connective tissue fibers form a latticework which acts as a framework for the larger blood and lymph vessels and efferent ducts of the testis. The testis is attached to the scrotal wall in the mediastinal area.

(3) The spaces between the various septula partitions form the septula compartments. In the human testis there are about 250 septula compartments, each containing a lobule of the testis. The lobuli testis contain the convoluted portions of the seminiferous tubules. From one to three seminiferous tubules are found in each lobule; they may anastomose at their distal ends. The combined length of all the seminiferous tubules approaches 250 meters in the human. The convoluted portions of the seminiferous tubules empty into the straight tubules (tubuli recti) and these in turn unite with the rete tubules located within the substance of the mediastinum. Connecting with the rete tubules of the testis, there are, in man, from 12 to 14 ductuli efferentes (efferent ductules of the epididymis) of about 4 to 6 cm. in length which emerge from the mediastinum and pass outward to unite with the duct of the epididymis. The epididymal duct represents the proximal portion of the reproductive duct which conveys the male gametes to the exterior.

Fig. 6. Schematic drawing of the testis and its relationship within the scrotum. On the right side of the drawing the muscle and connective-tissue layers surrounding the inguinal bursa and testis are shown; on the left side may be seen the loops of the cremaster muscle surrounding the tunica vaginalis communis.

Fig. 7. Diagrammatic representation of the general structural relationship of the parts of the human testis. (Modified from Corner, 1943, after Spalteholz and Huber.)

4. Specific Structures of the Mammalian Testis Which Produce THE Reproductive Cells and the Male Sex Hormone

Two very essential processes involved in reproduction are the formation of the sex cells or gametes and the elaboration of certain humoral substances, known as sex hormones. Therefore, consideration will be given next to those portions of the testis which produce the sperm cells and the male sex hormone, namely, the seminiferous tubules and the interstitial tissue.

a. Seminiferous Tubules

The seminiferous tubules lie in the septula compartments (fig. 7). The word seminiferous is derived from two Latin words: semen, denoting seed, and ferre, which means to bear or to carry. The seminiferous tubule, therefore, is a male “seed-bearing” structure. Within this tubule the male gametes or sperm are formed, at least morphologically. However, the word semen has a broader implication in that it is used generally to denote the entire reproductive fluid or seminal fluid. The seminal fluid is a composite of substances contributed by the seminiferous tubules and various parts of the accessory reproductive tract.

The exact form and relationship of the various seminiferous tubules (tubuli seminiferi) which occupy each testicular compartment have been the object of much study. It is a generally accepted belief at present that the tubules within each testicular lobule are attached at their distal ends; that is, that they anastomose (fig. 7). Some investigators also believe that there may be other anastomoses along the lengths of these very much contorted and twisted structures. Moreover, it appears that the septula or testicular compartmental partitions are not always complete; the seminiferous tubules of one lobule thus have the opportunity to communicate with those of adjacent lobules. The seminiferous tubules of any one lobule join at their proximal ends and empty into a single straight seminiferous tubule. The straight tubules or tubuli recti pass into the mediastinum and join the anastomosing rete tubules of the rete testis.

The convoluted portions of the seminiferous tubules produce the sperm (spermia; spermatozoa). In the human testis, the length of one of these tubules is about 30 to 70 cm. and approximately 150 /a to 250 ix in diameter. Each tubule is circumscribed by a basement membrane of connective tissue and contains two cell types:

( 1 ) supporting or Sertoli cells, and

(2) spermatogenic cells or spermatogonia (see fig. 8 and Chap. 3).

The cells of Sertoli are relatively long, slender elements placed perpendicularly to the basement membrane to which they firmly adhere. These cells may undergo considerable change in shape, and some observers believe that they may form a syncytium, known as the “Sertolian syncytium.” Others believe them to be distinct elements. It is said that Sertoli cells may round up and form phagocytes which become free from the basement membrane and move, ameba-like, in the lumen of the seminiferous tubule, phagocytizing degenerating sperm cells. However, their main function appears to be associated with the development of sperm during the period when the latter undergo their transformation from the spermatid condition into the adult

Fig. 8. Semidiagram matic representation of section of cat testis, showing seminiferous tubules and interstitial tissue, particularly the cells of Leydig.

Sperm form. The Sertolian cells thus may act as nursing elements during sperm metamorphosis.

The spermatogenic cells or spermatogonia (germinal epithelium of the tubule) lie toward the outer portion of the seminiferous tubule between the various Sertoli elements. As a rule spermatogonia lie apposed against the basement membrane of the tubule (see fig. 8 and Chap. 3).

b. Interstitial Tissue

The interstitial tissue of the testis is situated between the seminiferous tubules (fig. 8). It consists of a layer of connective tissue applied to the basement membrane of the seminiferous tubule and of many other structures, such as small blood and lymph vessels, connective tissue fibers, connective tissue cells, mast cells, fixed macrophages, etc. The conspicuous elements of this tissue are the so-called interstitial cells or cells of Leydig (fig. 8). In man, cat, dog, etc., the cells of Leydig are relatively large, polyhedral elements, possessing a granular cytoplasm and a large nucleus.

5. The Testis of Vertebrates in General

In the vertebrate group, the testis shows marked variations in shape and size. In many fishes, the testes are irregular, lobular structures, but in other fishes, amphibia, reptiles, birds, and mammals, they assume an ovoid shape. The size of the testis is extremely variable, even in the same species. The testis of the human adult approximates 4 to 5 cm. in length by 3 cm. wide and weighs about 14 to 19 Gm. The testis of the horse averages 11 cm. long by 7 cm. wide with a weight of 30 to 35 Gm., while that of the cat is 1.6 cm. long and 1.1 cm. wide with a weight of 1.5 Gm. In the mud puppy, Necturus, the testis is approximately 3.5 cm. long and 0.8 cm. wide with a weight of 0.3 Gm. The testis of the large bullfrog is 1 .2 cm. by 0.5 cm. with a weight of 0.8 Gm. In comparison to the foregoing, Schulte (’37) gives the weight of each testis of an Indian elephant as two kilograms!

Regardless of size or shape, the presence of seminiferous tubules and interstitial tissue may be observed in all vertebrate testes. In some species the seminiferous tubule is long; in others it is a short, blunt affair. The interstitial cells may be similar to those described above, or they may be small, inconspicuous oval elements.

6. Accessory Reproductive Structures of the Male a. The Reproductive Duct in Forms Utilizing External Fertilization

The accessory reproductive organs of the vertebrate male are extremely variable in the group as a whole. A relatively simple reproductive duct (or in some no duct at all) is the rule for those forms where fertilization is effected in the external medium. In cyclostome fishes, for example, the reproductive cells are shed into the peritoneal cavity and pass posteriad to emerge externally by means of two abdominal pores. Each pore empties into the urogenital sinus. In teleost fishes (perch, flounder, etc.) the conveying reproductive duct is a short, simple tube continuous with the testis at its caudal end and passing posteriorly to the urogenital sinus (fig. 9A). In frogs and toads, as well as in certain other fishes, such as Amia and Polypterus, the male reproductive duct is a simple, elongated tube associated with the testis by means of the efferent ductules of the latter, coursing posteriad to open into the cloaca (frogs and toads) or to the urogenital sinus (Amia; Polypterus) (fig. 9B, C). Simplicity of sperm duct development and external union of the gametes are associated reproductive phenomena in the vertebrate group.

b. The Reproductive Duct in Species Practicing Internal Fertilization

An entirely different, more complex male reproductive duct is found (with some exceptions) in those vertebrates where gametic union occurs within the protective structures of the maternal body. Under these circumstances there may be a tendency for one male to serve several females. Enlargement of the duct with the elaboration of glandular appendages, and structures or areas for sperm storage is the rule under these conditions (fig. 9D--F). This form of the male genital tract is found not only in those species where an intromittent organ deposits the sperm within the female tract, but also where the sperm are deposited externally in the form of spermatophores (fig. 10).

In many species, the reproductive duct is greatly lengthened and becomes a tortuous affair, especially at its anterior or testicular end. In fact, the cephalic end of the duct may be twisted and increased to a length many times longer than the male body itself. This coiled, cephalic portion is called the duct of the epididymis (epididymides, plural). (See figs. 7, 9E.) The word epididymis is derived from two Greek words: epi = upon, and didymis = testicle. The epididymis, therefore, is the body composed of the tortuous epididymal duct and the efferent ducts of the testis which lie upon or are closely associated with the testis. The complex type of reproductive duct is composed thus of two main portions, an anterior, contorted or twisted portion, the epididymal duct, and a less contorted posterior part, the vas deferens or sperm duct proper (fig. 9D, E).

In some vertebrates, in addition to the above complications, the caudal end of the reproductive duct has a pronounced swelling or diverticulum, the seminal vesicle (e.g., certain sharks and certain birds). The latter structures are true seminal vesicles in that they store sperm during the reproductive period.

The epididymal duct in man is a complex, coiled canal composed of a head (caput), a body (corpus), and a tail (cauda). (See fig. 7.) It is C-shaped with its concavity fitting around the dorsal border of the testis, the head portion being located at the anterior end of the latter. The total length of the epididymal duct in man is said to be about 4 to 7 m. In other mammals the epididymal duct may be much longer. For example, in the ram, from 40 to 60 m.; in the boar, 62 to 64 m.; in the stallion, 72 to 86 m. (Asdell, ’46). At its caudal end it becomes much less tortuous and gradually passes into the vas deferens (ductus deferens).

Fig. 9. Various vertebrate testes and reproductive ducts, emphasizing the relative simplicity of the duct where external fertilization is the rule while complexity of the duct is present when internal fertilization is utilized. There are exceptions to this rule, however. (A) Flounder (Limanda ferruginea). (B) Frog (Rana catesbiana). (C) Urodele (Cryptobranchus alleganiensis). (D) Dog shark (Squalus acanthias), (E) Urodele (Necturus rnaculosus). (F) Rooster (G alius domestic us).

The ductus deferens has a length of about 30 to 35 cm. in man. Leaving the scrotum it passes anteriad together with accompanying nerves and blood vessels in the subcutaneous tissue over the front of the pelvic bone into the peritoneal cavity through the inguinal ring (fig. 2). Here it separates from the other constituents of the spermatic cord (i.e., it separates from the nerves and blood vessels) and passes close to the dorsal aspect of the bladder and dorsally to the ureter. It then turns posteriad along the dorsal aspect of the neck of the bladder and the medial region of the ureter, and accompanied by its fellow duct from the other side, it travels toward the prostate gland and the urethra. Just before it enters into the substance of the prostate, it receives the duct of the seminal vesicle. The segment of the vas deferens from the ureter to the seminal vesicle is considerably enlarged and is called the ampulla. After receiving the duct of the seminal vesicle, the vas deferens becomes straightened and highly muscularized — as such it is known as the ejaculatory duct. The latter pierces the prostate gland located at the caudal end of the bladder and enters the prostatic portion of the urethra; from this point the urethra conveys the genital products.

The auxiliary glands associated with the genital ducts of the human male consist of the seminal vesicles, the prostate gland, Cowper’s glands, and the glands of Littre.

The seminal vesicles are hollow, somewhat tortuous bodies (fig. 2). Each vesicle arises in the embryo as an outpushing (evagination) of the vas deferens. The prostate gland has numerous excretory ducts which empty into the urethra. It represents a modification of the lining tissue of the urethra near the urinary bladder together with surrounding muscle and connective tissues. Cowper’s (bulbourethral) glands are small pea-shaped structures placed at the base of the penial organ; their ducts empty into the urethra. The glands of Littre are small, glandular outgrowths along the urethra and are closely associated with it.

To summarize the matter relative to the structural conditions of the reproductive duct in the male of those species which practice internal fertilization:

( 1 ) A lengthening and twisting of the duct occurs.

(2) A sperm-storage structure is present, either as a specialized portion of the duct or as a sac-like extension.

(3) Certain auxiliary glands may be present. These glands are sometimes large and vesicular structures, such as the seminal vesicles of the human duct, or they may be small glands distributed along the wall of the duct, such as the glands of Littre.

C. Specific Activities of the Various Parts of the Male Reproductive System

1. Introduction

a. Three General Functions of the Male Reproductive System

The activities of the testes and the accessory parts of the male reproductive system result in the performance of three general functions as follows:

( 1 ) formation of the semen,

(2) delivery of the semen to the proper place where the sperm may be utilized in the process of fertilization, and

(3) elaboration of the male sex hormone.

b. Some Definitions

Semen or seminal fluid is the all-important substance which the male contributes during the reproductive event. It is the product of the entire reproductive system, including special glands of the accessory reproductive structures. The semen is composed of two parts:

(1) The sperm (spermatozoa, spermia) are the formed elements which take part in the actual process of fertilization.

(2) The seminal plasma, a fluid part, is a lymph-like substance containing various substances dissolved or mixed in it. These contained substances are important as a protection for the sperm and as an aid to the process of fertilization.

With regard to the second function of the male genital system, namely, the delivery of sperm to the site of fertilization, it should be observed that

Fig. 10. Spermatophores of common urodeles. (Redrawn from Noble: Biology of the Amphibia, New York, McGraw-Hill.) (A) Triturus viridescens. (After Smith.) (B) Desmognathus fuscus, (After Noble and Weber.) (C) Eiirycea hislineata.

in some vertebrates this is a more simple problem than in others. In those forms which practice external fertilization, the male system simply discharges the seminal fluid into the surrounding external medium. However, in those vertebrates where internal fertilization is the rule, the female system assumes some of the burden in the transport of the semen to the region where fertilization is consummated, thus complicating the procedure. In these instances, the male genital tract is called upon to produce added substances to the seminal fluid which aid in protecting the sperm en route to the fertilization site.

The elaboration of the androgenic or male sex hormone is a most important function. Androgenic or male sex hormone substances are those organic compounds which induce maleness, for they aid the development of the male secondary sex characteristics, enhance the growth and functional development of the male accessory reproductive structures, and stimulate certain aspects of spermatogenesis.' Like the estrogens, androgens are not confined to a particular sex; they have been extracted from the urine of women and other female animals. The androgens derived from urinary concentrates are androsterone and dehydroisoandrosterone. These two androgens are not as powerful as that prepared from testicular tissue. Testicular androgen was first isolated from testicular tissue in 1935 and was given the name testosterone. It also has been synthesized from cholesterol. It is the most powerful of the androgens and probably similar, if not identical, with the substance produced in the testis (Koch, ’42).

2. Activities of the Testis

a. Seasonal and N on-seasonal Types of Testicular Activity

The testis has two main functions: the production of sperm and formation of the male sex hormone. In many vertebrates these two activities represent a continuous procedure during the reproductive life of the male animal. This

Fig. II. Seasonal spermatogenesis and accessory gland development in the ground squirrel, Citellus tridecemlineatus. Stippling below base line shows period of hibernation, whereas crosshatching reveals the reproductive period. (From Turner: General Endocrinology, Philadelphia, Saunders, after L, J. Wells.)

condition is found in certain tropical fish, in the common fowl and various wild tropical birds, and in many mammals, such as man, the dog, bull, stallion, cat, etc. On the other hand, in the majority of vertebrates these activities of the testis are a seasonal affair. This condition is found in most fish, practically all amphibia, all temperate -zone-inhabiting reptiles, most birds, and many mammals. Among the latter, for example, are the ferret, deer, elk, fox, wolf, and many rodents, such as the midwestern ground squirrel. Seasonal periodicity is true also of the common goose and turkey.

Sperm-producing periodicity is not correlated with any particular season, nor is spermatogenesis always synchronized with the mating urge, which in turn is dependent upon the male sex hormone. In some forms, these two testicular functions may actually occur at different seasons of the year, as for example, in the three-spined stickleback, Gasterosteus aculeatus (fig. 15). (See Craig-Bennett, ’31.) In general, it may be stated that sperm are produced during the weeks or months which precede the development of the mating instinct. Many species follow this rule. For example, in the bat of the genus Myotis, sperm are produced during the late spring and summer months, while mating or copulation takes place during the fall or possibly early the next spring (Guthrie, ’33). In the common newt, Triturus viridescens, spermatogenesis comes to pass during the warm months of the summer, and sperm are discharged from the testis into the reproductive ducts during the late fall and early spring, while copulation is accomplished in the early spring. The testes in this species are quiescent during the cool winter months. In the midwestern ground squirrel, Citellus tridecemlineatus, spermatogenesis begins in November and is marked during February and March (fig. 11). The animal hibernates away the winter months and emerges the first part of April in a breeding condition. Mating occurs in the early spring (Wells, ’35). In the garter snake, Tharnnophis radix, sperm are produced in the testes in the summer months, stored in the epididymides during the hibernation period in the fall and winter, and used for copulation purposes in the spring (Cieslak, ’45). Again, in the Virginia deer, Odocoileus virginianus borealis, studied by Wislocki (’43), active spermatogenesis is realized during the summer and early autumn months, while the mating season or “rut” which results from the driving power of the male sex hormone, is at its peak in October and November (fig. 12). In the fox. Bishop (’42) observed spermatogenesis to begin in the late fall months, while mating is an event of the late winter and early spring. In April and May the seminiferous tubules again assume an inactive state (fig. 13). In the common frog, Rana pipiens, spermatogenesis is present in the summer months and morphogenesis of spermatids into sperm happens in large numbers during September, October, and November. Sperm are stored in the testis over the winter, and the mating instinct is awakened in the early spring (Glass and Rugh, ’44). Following the mating season in spring and early summer the testis of the teleost, Fundulus heteroclitus, is depleted of sperm until the next winter and spring (Matthews, ’38).

As the seasonal type of testicular activity is present in a large number of vertebrate species, it seems probable that it represents the more primitive or fundamental type of testicular functioning.

b. Testicular Tissue Concerned with Male Sex-hormone Production

While one cannot rule out the indirect effects which activities of the seminiferous tubules may have upon the functioning of the testis as a whole, including the interstitial tissue, direct experimental evidence and other observations suggest that the interstitial tissue holds the main responsibility for the secretion of the male sex hormone, testosterone, or a substance very closely allied to it. For example, if a testis from an animal possessing a permanent scrotum is removed from the inguinal bursa and placed within the peritoneal cavity, the seminiferous tubules tend to degenerate, but the interstitial tissue remains. The sex hormone, under these circumstances, continues to be produced. Again, males having cryptorchid testis (i.e., testes which have failed in their passage to the scrotum) possess the secondary sex characteristics of normal males but fail to produce sperm cells. Also, it has been demonstrated that the mammalian fetal testis contains the male sex hormone. However, in this fetal condition, the seminiferous tubules are present only in an undeveloped state, whereas interstitial tissue is well differentiated. It is probable in this case that the interstitial tissue of the fetal testis responds to the luteinizing hormone in the maternal blood.

Fig. 12. Sections of the testis of the deer, Odocoileus virginianus borealis. (After Wislocki.) (A) Seminiferous tubules of deer in June. Observe repressed state of tubules and absence of sperm. (B) Epididymal duct of same deer. Observe absence of sperm and smaller diameter of duct compared with (D). (C) Seminiferous tubules of October deer; spermatogenic activity is marked. (D) Epididymal duct, showing well-developed epididymal tube and presence of many sperm.

In hypophysectomized male rats injected with dosages of pure folliclestimulating hormone (FSH) or with small doses of pure luteinizing hormone (LH; ICSH), the seminiferous tubules of the testis respond and spermatogenesis occurs. However, the interstitial tissue remains relatively unstimulated and the accessory structures continue in the atrophic state. If larger doses of the luteinizing factor are given, the interstitial tissue responds and the secondary sexual characters are developed, showing a relationship between interstitial activity and sex-hormone production. (Consult Evans and Simpson in Pincus and Thimann, ’50, pp. 355, 356.)

From certain species whose reproductive activities are confined to a particular season of the year, there also comes evidence that the interstitial tissue is the site of sex-hormone production. In the behavior of testicular tissue in the stickleback, Gasterosteus, as shown by van Oordt (’23) and Craig-Bennett (’31) sperm are produced actively in the seminiferous tubules during one period of the year when the interstitial tissue is in an undeveloped condition. The secondary sex characters also are in abeyance at this season of the year. However, during the months immediately following sperm production, sperm are stored within the seminiferous tubules and active spermatogenesis is absent. When the seminiferous tubules thus have completed their spermatogenic activity, the interstitial tissue begins to increase, followed by a development of secondary sex characteristics (figs. 14, 15). A similar difference in the rhythm of development of these two testicular tissues can be shown for many other vertebrates. All of these suggestive facts thus serve to place the responsibility for male sex-hormone production upon the interstitial tissue, probably the cells of Leydig.

Fig. 13. Sections of seminiferous tubules of silver fox. (After Bishop.) (A) Regressed state of tubules following breeding season. (B) Tubule from fox during the breeding season, characterized by active spermatogenesis.

Fig. 14. Sections of the testis of the stickleback (Gasterosteus pungitius). (Modified from Moore, ’39, after Van Oordt.) Cf. fig. 13, (A) Spermatogenic activity with many

formed sperm in seminiferous tubules before the mating season, interstitial tissue in abeyance. (B) At mating period. Interstitial tissue well developed, spermatozoa stored in the tubules with spermatogenic activity absent.

Fig. 15. Seasonal reproductive cycle in the stickleback {Gasterosteus aculeatus). Cf. fig. 14. Breeding season is indicated by crosshatching below base line. Observe that spermatogenic activity follows rise of temperature, whereas interstitial-tissue and sexcharacter development occur during ascending period of light. (Redrawn from Turner: General Endocrinology, Philadelphia, Saunders, modified from Craig-Bennett, 1931.)

c. Testicular Control of Body Structure and Function by the Male Sex Hormone

1) Sources of the Male Sex Hormone. Testosterone is prepared from testicular extracts. It is the most potent of the androgens and is believed to be the hormone produced by the testis. The chemical formula of testosterone is:

on

ciu

o

CHs

J\J\J

The testis, however, is not the only site of androgen formation. As mentioned above, androgens are found in the urine of female animals, castrates, etc. It seems probable that the suprarenal (adrenal) cortex may secrete a certain androgenic substance, possibly adrenosterone, a weak androgen. Many androgens have been synthesized also in the laboratory (Schwenk, ’44).

2) Biological Effects of the Male Sex Hormone. The presence of the male sex hormone in the male arouses the functional development of the accessory reproductive structures, the secondary sexual characters, and also stimulates the development of the seminiferous tubules.

a) Effects upon the Accessory Reproductive Structures. Castration or removal of the testes from an animal possessing a continuous type of testicular activity produces shrinkage, and a general tendency toward atrophy, of the entire accessory reproductive structures. Injection of testosterone or other androgens under such conditions occasions a resurgence of functional development and enlargement of the accessory structures (fig. 16). Moreover, continued injections of the androgen will maintain the accessories in this functional state (Moore, ’42; Dorfman in Pincus and Thimann, ’50). Similarly, under normal conditions in those vertebrates which possess the seasonal type of testicular function, the accessory reproductive organs shrink in size with a loss of functional activity when the testis undergoes regression during the period immediately following the active season. An enlargement and acquisition of a normal functional condition of the accessories follows testicular development as the breeding season again approaches (Bishop, ’42; Wislocki, ’43; Matthews, ’38; Turner, C. L., ’19). (Compare figs. 12A-D.)

b) Effects upon Secondary Sex Characteristics and Behavior of THE Individual. In addition to the primary effects upon the reproductive system itself, the androgens induce many other secondary structures and alterations of the physiology and behavior of the individual. The influence of the testicular hormone has been demonstrated in all of the vertebrate groups from fishes to mammals (Dorfman in Pincus and Thimann, ’50). Examples of testosterone stimulation are: the singing and plumage of the male bird; hair development of certain mammals; the crowing and fighting, together with spur, comb, and wattle growth in the rooster. The disagreeable belligerency and positive energy drive of the bull, stallion, or human male may be attributed, largely, to the action of testicular hormone. However, lest we disparage this aggressive demeanor unduly, it should be recognized that upon such explosive force rests the preservation of species and races in some instances. As an example, witness that hairy dynamo of the barren northern tundras, the bull muskox, whose fiery pugnaciousness when the need arises undoubtedly has been a strong factor in the preservation of this species.

An excellent example of the effect of testosterone is shown in the development of antlers and change in behavior of the Virginia deer, Odocoileus virginianus borealis (Wislocki, ’43). In the northern climate, the testes and male accessory organs reach a profound condition of regression in April and May. Growth of the new antlers starts at this time, and during the late summer the antlers grow rapidly and begin to calcify. During the summer, also, the testes develop rapidly, and spermatogenesis results. Loss of the “velvet” covering of the antlers is experienced during September, and mating is the rule in October and November. The antlers are shed in midwinter. If the testes are removed after the naked antler condition is reached, the antlers are shed rapidly. Testosterone administered to does or to young males which have been castrated induces the development of antlers. The general scheme of antler development suggests, possibly, that the testicular hormone acts upon an anterior pituitary factor, and this activated factor in turn initiates antler growth. Hardening of the antlers and loss of velvet results from testosterone stimulation. Loss of the antler is synchronized with a decrease in the amount of testosterone in the blood stream, accompanied by the acquisition of a docile, non-belligerent, more timid behavior.

c) Effects upon the Seminiferous Tubules. Testosterone has a stimulating effect upon the seminiferous tubule and sperm formation. This matter is discussed in Chap. 3.

d. Seminiferous-tubule Activity and Formation of Sperm See Chap. 3.

e. The Seminiferous Tubule as a Sperm-storing Structure

See p. 3 1 .

3. Role of the Reproductive Duct in Sperm Formation

a. Vertebrates Without a Highly Tortuous Epididyrnal Portion of the Reproductive Duct

In a large number of vertebrates, morphologically developed sperm pass from the testis through the efferent ductules of the epididymis (vasa efferentia) to the epididyrnal duct where they remain for varying periods. However, in many vertebrates the anterior (proximal) portion of the sperm duct does not form a tortuous epididyrnal structure similar to that found in other vertebrates. This condition is present in the common frog, Rana; in the hellbender, Cryptobranchus; in the bowfin, Amia; etc. Because of this fact, the sperm pass directly into the vas deferens or sperm duct (Wolffian duct) without undergoing a sojourn through a convoluted epididyrnal portion of the duct.

Correlated with the type of testis and sperm-duct relationship in the frog, is the fact that one may obtain viable, fertilizing sperm directly from the testis. For example, if one removes the testis from a living frog and macerates it in pond water or in an appropriate saline solution, active sperm are obtained which are capable of fertilizing eggs in a normal manner. That is, the frog testis matures sperm morphologically and physiologically. This type of testicular maturation is characteristic of many of the lower vertebrates possessing simple reproductive ducts.

Fig. 16. Effects of the male sex hormone upon the functional development of the accessory reproductive structures of the male rat. (After Turner: General Endocrinology , Philadelphia, Saunders, p. 324.) (A) Normal male rat condition produced by injection of crystalline male sex hormone for 20 days into castrate before autopsy. (B) Castrated male litter mate of (A) receiving no replacement therapy.

Fig. 17. Diagrammatic drawings of the two types of testicular-reproductive relationships occurring in the vertebrate group. (A) Simplified type of reproductive duct connected with the testis by means of efferent ductules. The duct-testis relationship of many telepst fishes is similar to this but does not possess the efferent ductules, the sinus-like reproductive duct being attached directly to the testis. Sperm cells (spermatozoa) are matured and stored within the testis. This type of relationship generally is found where fertilization is external or where sperm are discharged all at once during a short reproductive period. (B) More complicated variety of reproductive duct, connected with the testis by means of efferent ducts, but possessing an anterior twisted portion, the epididymal duct in which the sperm are stored and physiologically matured. This type of duct generally is found in those vertebrates which utilize internal fertilization and where sperm are discharged over a short or extended reproductive period.

b. The Epididymis as a Sperm-ripening Structure

On the other hand, in those forms which possess an anterior convoluted epididymal portion of the reproductive duct, the journey of the sperm through this portion of the duct appears to be necessary in order that fertilizable sperm may be produced. In mammals it has been shown that the epididymal journey somehow conditions the physiological ripening of the sperm. Sperm taken from the mammalian testis will not fertilize; those from the caudal portion of the epididymis will, provided they have been in the epididymis long enough. Under normal conditions sperm pass through the epididymis slowly, and retain their viability after many days’ residence in this structure. Sperm prove to be fertile in the rabbit epididymis up to about the thirty-eighth day; if kept somewhat longer than this, they become senile and lose the ability to fertilize, although morphologically they may seem to be normal (Hammond and Asdell, ’26). In the rat, they may live up to 20 to 30 days in the epididymis and still be capable of fertilization (Moore, ’28). It has been estimated that the epididymal journey in the guinea pig consumes about two weeks, although they may live and retain their fertilizing power as long as 30 days in epididymides which have been isolated by constriction (Moore and McGee, ’28; Young, ’31; Young, ’31b). It is said that in the bull, sperm within the epididymis may live and be motile for two months. As a result of these facts, it may be concluded that the epididymal journey normally is a slow process, and that it is beneficial for the development of sperm “ripeness” or ability to fertilize.

c. The Epididymis and Vas Deferens as Sperm-storage Organs

Along with the maturing faculty, the epididymal duct and vas deferens also act as sperm-storage organs. As observed on p. 23, in the bat, Myotis, sperm are formed in great numbers in the seminiferous tubules and pass to the epididymal duct where they are stored during the fall, winter, and early spring months; the epididymal journey thus is greatly prolonged in this species. In the ovoviviparous garter snake, Thamnophis radix, sperm are produced during the summer months; they pass into the epididymides during early autumn and are stored there during the fall and winter. In the mammal, sperm are stored in the epididymal duct.

Aside from its main purpose of transporting sperm to the exterior (see sperm transport, p. 177), the caudal portion of the sperm duct or vas deferens also is capable of storing sperm for considerable periods of time. In the common perch, Perea ftavescens, sperm are developed in the testes in the autumn, pass gradually into the accessory reproductive ducts, and are stored there for five or six months until the breeding season the following spring (Turner, C. L., ’19). Again, in mammals, the ampullary region of the vas deferens appears to be a site for sperm storage. For example, the ampulla of the bull sometimes is massaged through the rectal wall to obtain sperm for artificial insemination. In this form sperm may be stored in the ampulla and still be viable, for as long as three days. Similarly, in lower vertebrates large numbers of sperm may be found in the posterior extremities of the vas deferens during the breeding season. Thus, the reproductive duct (and its epididymal portion when present) is instrumental in many vertebrate species as a temporary storage place for the sperm.

d. Two Types of Vertebrate Testes Relative to Sperm Formation

The importance of the epididymal duct in many vertebrates and its relative absence in others, focuses attention upon the fact that in many vertebrate species sperm are produced, stored, and physiologically matured entirely within the confines of the testis (frog, bowfin, stickleback, etc.). The reproductive duct under these circumstances is used mainly for sperm transport. In many other vertebrate species sperm are morphologically formed in the testis and then are passed on into the accessory structures for storage and physiological maturation. Functionally, therefore, two types of testes and two types of accessory reproductive ducts are found among the vertebrate group of animals (fig. 17). It naturally follows that the testis which produces, stores, and physiologically matures sperm is best adapted for seasonal activity, particularly where one female is served during the reproductive activities. That is, it functions as an “all at one time” spawning mechanism. On the other hand, that testis which produces sperm morphologically and passes them on to a tortuous epididymal duct for storage and physiological maturing is best adapted for the continuous type of sperm production or for the service of several females during a single seasonal period. The sperm, under these conditions, pass slowly through the epididymal duct, and, therefore, may be discharged intermittently.

4. Function of the Seminal Vesicles (Vesicular Glands)

The seminal vesicles show much diversity in their distribution among various mammals. Forms like the cat, dog, opossum, rabbit, sloth, armadillo, whale, do not possess them, while in man, rat, elephant, mouse, they are welldeveloped structures. It was formerly thought that the seminal vesicles in mammals acted as a storehouse for the sperm, hence the name. In reality they are glandular structures which add their contents to the seminal fluid during the sexual act.

5. Function of the Prostate Gland

The prostate gland also is a variable structure and is found entirely in the marsupial and eutherian mammals. In marsupials it is confined to the prostatic portion of the urethral wall; in man it is a rounded, bulbous structure which surrounds the urethra close to the urinary bladder. In many other mammals it is a much smaller and less conspicuous structure. It discharges its contents into the seminal fluid during the orgasm. It is probable that the prostatic and vesicular fluids form the so-called “vaginal plug’’ in the vagina of the rat, mouse, etc.

6. Bulbourethral (Cowper’s) Glands

The bulbourethral glands are absent in the dog but present in most other mammals. In marsupials and monotremes these structures are exceptionally well formed. In the opossum there are three pairs of bulbourethral glands. The mucous contents of these and other small urethral glands are discharged at the beginning of the sexual climax and, as such, become part of the seminal fluid.

7. Functions of Seminal Fluid a. Amount of Seminal Fluid Discharged and Its General Functions

As stated previously, the semen or seminal fluid is composed of two parts, the sperm cells (spermia; spermatozoa) and the seminal plasma. The presence of the sperm cells represents the most constant feature, although they may vary considerably from species to species in size, shape, structure, and number present. The seminal plasma varies greatly as to composition and amount discharged.

The quantity of seminal fluid discharged per ejaculate and the relative numbers of sperm present in man and a few other vertebrate species associated with him are as follows:*

Species

Volume of Single Ejaculate, Most Common Value, in CC.

Sperm Density in Semen, Average Value, per CC.

Boar

250

CC.

100,000,000

per

CC.

Bull

4-5

CC.

1,000,000,000

per

CC.

Cock

0.8

CC.

3,500,000,000

per

CC.

Dog

6

CC.

200,000,000

per

CC.

Man

3.5

CC.

100,000,000

per

CC.

Rabbit

1

CC.

700,000,000

per

CC.

Ram

1

CC.

3,000,000,000

per

CC.

Stallion

70

CC.

120,000,000

per

CC.

Turkey

0.3

CC.

7,000,000,000

per

CC.

• Modified from Mann (’50).

Two important branches of study involving the semen pertain to:

(1) the chemical and physiological nature and numerical presence of the sperm, and

(2) the physiology and biochemistry of the seminal plasma.

(See Mann, ’50, for discussion and bibliography.) As a result of the studies thus far, a considerable body of information has been accumulated.

The main function of the semen, including the plasma and accessory sperm, appears to be to assist the sperm cell whose chance fortune it is to make contact with the egg. Once this association is accomplished, the egg seemingly takes over the problem of fertilization. The seminal plasma and the accessory numbers of sperm appear to act as an important protective bodyguard and also as an aid for this event. Modern research emphasizes, therefore, that the work of the male reproductive system is not complete until this contact is made.

b. Coagulation of the Semen

In many mammalian species, the semen tends to coagulate after its discharge from the male system. In the mouse, rat, guinea pig, opossum, rhesus monkey, etc., the semen coagulates into a solid mass, the vaginal plug, once it reaches the vagina of the female. The probable function of the vaginal plug is to prevent the semen from seeping out of the vagina. The formation of this plug may be due to a protein present in the contents of the seminal vesicle which comes in contact with the enzyme, vesiculase. In the rat and guinea pig this catalyst probably is produced by the “coagulating gland,” a specialized structure associated with the seminal vesicles in these forms. Some of it also may come from the prostate.

Coagulation of the seminal fluid also occurs in man, stallion, and boar but it is entirely absent in the dog, bull, and many other animals. Human semen coagulates immediately after discharge but liquefies a short time afterward. This liquefaction may be due to the presence of two enzymes, fibrinogenase and fibrinolysin, found in human semen and both derived from the prostate. These enzymes are found also in dog semen. In the latter their property of inhibiting blood coagulation may be of use where considerable amounts of blood may be present in the female genital tract at the onset of full estrous conditions. Another important contribution of the prostate gland is citric acid. Its role is not clear but it may enter into the above coagulation-liquefaction process (Mann, ’50, p. 348).

c. Hyaluronidase

Various enzymes have been demonstrated to be present in the semen of certain invertebrates and vertebrates. One such enzyme is hyaluronidase which appears to be produced in the testes of the rat, rabbit, boar, bull, and man. It is not found in the testes of vertebrates below the mammals. Its specific function is associated with the dispersal of the follicle cells surrounding the egg; in so doing it may aid the process of fertilization in mammals.

d. Accessory Sperm

One sperm normally effects a union with the egg in fertilization. Accessory sperm may enter large-yolked eggs, but only one is intimately involved in the union with the egg pronucleus. However, what is meant by accessory sperm here is the large number of sperm which normally clusters around the egg during the fertilization process in many animal species. A suggestion of a function for these accessory sperm follows from the fact that hyaluronidase may be extracted from the semen, presumably from the sperm themselves. Rowlands (’44) and also Leonard and Kurzrok (’46) have shown that a seminal fluid deficient in sperm numbers may fertilize if hyaluronidase extracted from sperm (?) is added to such a weakened sperm suspension. The implication is that the accessory sperm thus may act as “cupbearers” for the one successful sperm in that they carry hyaluronidase which aids in liquefying the follicle cells and other gelatinous coating material around the egg.

e. Fructose

An older concept in embryology maintained that sperm were unable to obtain or utilize nourishment after they departed from the testis. More recent investigation has shown, however, that sperm do utilize certain sugar materials, and that their survival depends upon the presence of a simple sugar in the medium in which they are kept. (See Mann, ’50.)

The sugar that is found normally in semen is fructose. It varies in quantity from species to species, being small in amount in the semen of the boar or stallion but considerably larger in quantity in the seminal fluid of the bull, man, and rabbit. The seat of origin of this sugar appears to be the seminal vesicle, at least in man, although the prostate may also be involved, particularly in the rabbit and also in the dog. The dog, however, has but a small amount of fructose in the seminal discharge. The real function of seminal fructose “might be as a readily utilizable store of energy for the survival of motile spermatozoa” (Mann, ’50, p. 360).

f. Enzyme-protecting Substances

Runnstrom (personal communication) and his co-workers have demonstrated that the fertilizing life of sea-urchin sperm is increased by certain substances found in the jelly coat of the sea-urchin egg. Presumably these substances are protein in nature, and, according to Runnstrom, they may act to preserve the enzyme system of the sperm. Similarly, the seminal fluid may act to preserve the enzyme system of the sperm, while en route to the egg, especially within the female genital tract.

D. Internal and External Factors Influencing Activities of the Testis

Conditions which influence testicular activity are many. Many of the factors are unknown. Nevertheless, a few conditions which govern testis function have been determined, especially in certain mammalian species. The general results of experimental determination of some of the agents which affect testicular function are briefly outlined below.

1. Internal Factors

a. Temperature and Anatomical Position of the Testis

It is well known that in those mammals which have a permanent scrotal residence of the testes failure of the testis or testes to descend properly into the scrotum results in a corresponding failure of the seminiferous tubules to produce sperm. In these instances the testis may appear shriveled and shrunken (fig. 18). However, such cryptorchid (ectopic) conditions in most cases retain the ability to produce the sex hormone at least to some degree. A question therefore arises relative to the factors which inhibit seminiferous tubule activity within the cryptorchid testis.

The failure of cryptorchid testes to produce viable sperm has been of interest for a long time. Observations have demonstrated that the more hidden

Fig. 18. Experimental unilateral cryptorchidism in adult rat. The animal's left testis was confined within the abdominal cavity for six months, whereas the right testis was pernfitted to reside in the normal scrotal position. Observe the shrunken condition of the cryptorchid member. (After Turner: General Endocrinology, Philadelphia, Saunders.)

the testis (i.e., the nearer the peritoneal cavity) the less likely are mature sperm to be formed. A testis, in the lower inguinal canal or upper scrotal area is more normal in sperm production than one located in the upper inguinal canal or inside the inguinal ring. Studies made upon peritoneal and scrotal temperatures of rats, rabbits, guinea pigs, etc., demonstrate a temperature in the scrotum several degrees lower than that which obtains in the abdomen. These observations suggest that the higher temperature of the non-scrotal areas is a definite factor in bringing about seminiferous tubule injury and failure to produce sperm.

With this temperature factor in mind, Dr. Carl R. Moore (in Allen, Danforth, and Doisy, ’39) and others performed experiments designed to test its validity as a controlling influence. They found that confinement alone of an adult guinea pig testicle in the abdomen led to marked disorganization of all seminiferous tubules in seven days. After several months of such confinement the seminiferous tubules experience marked degenerative changes and only Sertoli cells remain (fig. 19A, B). The interstitial tissue, however, is not greatly impaired. If such a testis is kept not too long within the abnormal position and once again is returned to the scrotum, spermatogenesis is rejuvenated (fig. 20A, B). In a second experiment, the scrotum of a ram was encased loosely with insulating material; a rapid degeneration of the seminiferous tubules followed. Young (’27, ’29) in a third type of experiment found that water 6 to 7° warmer than the body temperature applied to the external aspect of the guinea-pig testis for a 15-minute period evoked degenerative

Fig. 19. Sections of experimental, cryptorchid, guinea-pig, seminiferous tubules and interstitial tissue. (Modified from C. R. Moore in Sex & Internal Secretions, Williams & Wilkins, Baltimore, 1939.) (A) Testis confined to abdomen for three months. (B)

Testis confined to abdomen for six months. Observe degenerate state of seminiferous tubule after six months’ confinement. Interstitial tissue not greatly affected by confinement.

changes with temporary sterility (fig. 21). Recovery, however, is the rule in the latter instance. Summarizing the effects of such experiments involving temperature, Moore (in Allen, Danforth, and Doisy, ’39, p. 371) concludes: “The injury developing from applied heat, although more rapidly effective, is entirely similar to that induced by the normal body temperature when the testicle is removed from the scrotum to the abdomen.”

The position of the scrotum and its anatomical structure is such as to enhance its purpose as a regulator of testicular temperature (figs. 2, 6). When the surrounding temperature is cold, the contraction of the dartos muscle tissue of the scrotal skin contracts the scrotum as a whole, while the contraction of the cremaster muscle loops pulls the testes and the scrotum closer to the body, thus conserving the contained heat. When the surrounding temperature is warm, these muscles relax, producing a more pendulous condition to permit heat loss from the scrotal wall.

In accordance with the foregoing description of the scrotum as a necessary thermoregulator for the testis, it has been further shown for those mammals which possess a scrotum that testis grafts fare much better when transplanted to the scrotal wall or into the anterior chamber of the eye (Turner, C. D., '48). The anterior chamber of the eyeball possesses a temperature much cooler than the internal parts of the body.

Fig. 20. Sections of testis during and after abdominal confinement. (Modified from C. R. Moore in Sex & Internal Secretions, Williams & Wilkins, Baltimore, 1939.) (A)

Section of left testis to show degenerate state of seminiferous tubules after 24 days of abdominal confinement. (B) Section of right testis 74 days after replacement in scrotum. Observe spermatogenic activity in tubules.

Fig. 21. Effect of higher temperature applied to external surface of guinea-pig testis. Water, 47®, was applied to surface of scrotum for period of 10 minutes. Testis was removed from animal 12 days after treatment. Seminiferous tubules are degenerate. (Modified from Moore, ’39; see also Young, ’27, J. Exp. Zool., 49.)

Two types of seminiferous tubules are thus found in mammals. In a few mammalian species (see p. 6) the temperature of the peritoneal cavity is favorable to the well-being of the seminiferous tubule; in most mammalian species, however, a lower temperature is required. On the other hand, the activities of the interstitial tissue of the testis appear to be much less sensitive to the surrounding temperature conditions, and the male sex hormone may be produced when the testes are removed from the scrotum and placed within the peritoneal cavity.

With regard to the functioning of the testis within the peritoneal cavity of birds it has been suggested that the air sacs may function to lower the temperature around the testis (Cowles and Nordstrom, ’46). In the sparrow, Riley (’37) found that mitotic activity in the testis is greatest during the early morning hours when the bird is resting and the body temperature is lower, by 3 or 4° C.

b. Body Nourishment in Relation to Testicular Function

The testis is a part of, and therefore dependent upon, the well-being of the body as a whole. However, as observed in the preceding pages the interstitial cells and their activities in the production of the male sex hormone are less sensitive to the internal environment of the body than are the seminiferous tubules.

The separation of these two phases of testicular function is well demonstrated during starvation and general inanition of the body as a whole. A falling off of sperm production is a definite result of starvation diets, although the germinative cells do not readily lose their ability to proliferate even after prolonged periods of starvation. But the interstitial cells and the cells of Sertoli are not as readily affected by inadequate diets or moderate starvation periods. Sex drive may be maintained in a starving animal, while his ability to produce mature, healthy sperm is lost. On the other hand, long periods of inanition also affect sex hormone production and the sexual interests of the animal.

Aside from the abundance of food in a well-rounded dietary regime, adequate supplies of various vitamins have been shown to be essential. Vitamin Bi is essential to the maintenance of the seminiferous tubules in pigeons. Pronounced degenerative changes in the seminiferous tubules of rats and other mammals occur in the absence of vitamins A and E (Mason, ’39). Prolonged absence of vitamin E produces an irreparable injury to the testis of rats; injury produced by vitamin A deficiency is reparable. The B-complex of vitamins seems to be especially important for the maintenance of the accessory reproductive structures, such as the prostate, seminal vesicles, etc. The absence of vitamin C has a general body effect, but does not influence the testis directly. Spme of these effects may be mediated through the pituitary gland. As vitamin D is intimately associated with the mineral metabolism of the body, it is not easy to demonstrate its direct importance.

c. The Hypophysis and Its Relation to Testicular Function

The word “hypophysis” literally means a process extending out below. The early anatomists regarded the hypophysis cerebri as a process of the brain more or less vestigial in character. It was long regarded as a structure through which waste materials from the brain filtered out through supposed openings into the nasal cavity. These wastes were in the form of mucus or phlegm, hence the name “pituitary,” derived from a Latin word meaning “mucus.” The word pituitary is often used synonymously with the word hypophysis.

The hypophysis is made up of the pars anterior or anterior lobe, pars intermedia or intermediate lobe, and a processus infundibuli or posterior lobe. The anterior lobe is a structure of great importance to the reproductive system; its removal (ablation) results in profound atrophic changes throughout the entire reproductive tract.

The importance of the pituitary gland in controlling reproductive phenomena was aroused by the work of Crowe, Cushing, and Homans (TO) and by Aschner (’12) who successfully removed the hypophysis of young dogs. One of the first fruits of this work was a demonstration of the lack of genital development when this organ was removed. Since that time many

the other cohabitants of man — rats, mice, cats, rabbits, etc. — have been hypophysectomized, and in all cases a rapid involution and atrophy of the genital structures results from pituitary removal. The testis undergoes profound shrinkage and regression following hypophysectomy, the degree of change* varying with the species. In the rooster and monkey, for example, regressive changes are more marked than in the rat. (Consult Smith, ’39, for data and references.)

A striking demonstration of the influence of the hypophysis upon the genital tract is the result of its removal from a seasonal-breeding species, such as the ferret. Ablation of the pituitary in this species during the nonbreeding season causes slight if any change in the testis and accessory reproductive organs. However, when it is removed during the breeding season, a marked regression to a condition similar to that present during the nonbreeding season occurs (Hill and Parkes, ’33).

The experimental result of hypophysectomy on many animal species thus points directly to this structure as the site of hormonal secretion, particularly to the anterior lobe (Smith, ’39). The initial work on the relation of pituitary hormones and the gonad was done upon the female animal. The results of these studies aroused the question whether one or two hormones were responsible. The latter alternative was suggested by the work of Aschheim and Zondek (’27) and Zondek (’30) who concluded that two separate substances appeared to be concerned with the control of ovarian changes.

Nevertheless, for a time the concept of only one gonad -con trolling (gonadotrophic) hormone was produced by the pituitary, continued to gain attention, and some workers suggested that the two ovarian elfects of follicular growth and luteinization of the follicle were due to the length of time of administration of one hormone and not to two separate substances. However, this position soon was made untenable by research upon the gonadotrophic substances derived from the pituitary gland. Studies along this line by Fevold, Hisaw, and Leonard (’31) and Fevold and Hisaw (’34) reported the fractionation, from pituitary gland sources, of two gonadotrophic substances, a follicle-stimulating factor or FSH and a luteinization factor or LH. This work has been extensively confirmed. It should be observed in passing that the male pituitary gland contains large amounts of FSH, although, as mentioned below, the function of the testis and the male reproductive system relies to a great extent upon the luteinizing factor. Some investigators refer to the LH factor as the interstitial-cell-stimulating hormone, ICSH. (See Evans, ’47; and also Evans and Simpson in Pincus and Thimann, ’50.)

The action of these two hormones upon testicular tissue, according to present information, is somewhat as follows: If pure follicle-stimulating hormone, FSH, which produces only FSH effects in the female, is injected in low doses into hypophysectomized male rats, the seminiferous tubules are stimulated and spermatogenesis occurs. Under these conditions, the interstitial tissue remains unstimulated and the accessories continue in an atrophic state. It has further been demonstrated that slight amounts of the luteinizing gonadotrophic hormone, LH (ICSH), added to the above injections of FSH, effects a much better stimulation of the spermatogonial tissue, and the interstitial tissue also develops well.

On the other hand, when pure LH (ICSH) is given alone in small doses, spermatogenesis is stimulated with slight or no effect upon the male accessory structures. However, when larger doses of the LH (ICSH) factor alone are injected, the interstitial tissue is greatly stimulated, and the testicular weight increases much more than when FSH alone is given. Furthermore, the accessory reproductive structures are stimulated and become well developed, suggesting the elaboration of the male sex hormone. In agreement with these results, the administration alone of testosterone, the male sex hormone, increases the weight and development of the accessory structures in hypophysectomized animals and it also maintains spermatogenesis. It appears, therefore, that the effects of the LH substance upon the seminiferous tubules and the accessory organs occur by means of its ability to arouse the formation of the male sex hormone.

A summary of the actions of the pituitary gonadotrophic hormones upon testicular tissue may be stated as follows:

( 1 ) Pure FSH in small doses stimulates the seminiferous tubules and spermatogenesis with little or no effect upon the interstitial tissue or the accessory reproductive structures, such as the seminal vesicles or prostate gland;

(2) Small doses of pure LH also stimulate spermatogenesis with little or no stimulation of the accessory structures;

(3) Pure LH (ICSH) in larger doses stimulates the development of the interstitial tissue with the subsequent secretion of the male sex hormone and hypertrophy of the accessory reproductive organs;

(4) The male sex hormone in some way aids or stimulates the process of spermatogenesis, suggesting that the action of LH occurs through the medium of the sex hormone (fig. 22).

(Consult Evans and Simpson in Pincus and Thimann, ’50, for data and references; also Turner, C. D., ’48.)

The foregoing results of the action of the FSH and LH upon testicular function might suggest that the LH substance alone is essential in the male animal. However, it should be observed that without the presence of FSH, LH is not able to maintain the tubules in a strictly normal manner, the tubules showing a diminution of size. Also, in extreme atrophic conditions of the tubules, pure FSH stimulates spermatogenesis better than similar quantities of LH. It is probable that FSH and LH (ICSH) work together to effect complete normality in the male. This combined effect is known as a synergistic effect. It also is of interest that the injection of small doses of testosterone propionate into the normal male, with the pituitary gland intact, results in inhibition of the seminiferous tubules, probably due to the suppression of pituitary secretion by the increased atnount of the male sex hormone in the blood. However, high doses, while they likewise inhibit the pituitary, result in a level of androgen which stimulates the seminiferous tubules directly (Ludwig, ’50).

Aside from the above actions upon testicular tissue by the luteinizing hormone (LH;ICSH) certain other functions of this substance should be mentioned (see fig. 22). One of these is the apparent dependence of the Sertoli cells upon the presence of the interstitial cells (Williams, ’50). Interstitial tissue behavior and development in turn relies mainly upon LH (ICSH) (Fevold, ’39; Evans and Simpson in Pincus and Thimann, ’50). As the sperm are intimately associated with the Sertoli elements during the latter phases of spermatogenesis in which they transform from the spermatid into the form of the adult sperm, a very close association and reliance upon the presence of the luteinizing hormone thus appears to be established in sperm development.

A further study of the LH factor is associated with the maintenance of the seminiferous tubules themselves. In aged males, the interstitial tissue and the seminiferous tubules normally involute and regress with accumulation of large amounts of connective tissue material. In testicular grafts made into the rabbit’s ear, Williams (’50) found, when interstitial tissue was present in the grafts, the seminiferous tubules were more nearly normal; when absent, the tubules underwent fibrosis.

Another function of the LH substance apparently is concerned with release of the sperm from the Sertoli cells. De Robertis, et al. (’46), showed that anterior pituitary hormones possibly cause release of sperm from the Sertoli cells in the toad by the production of vacuoles and apical destruction of the cytoplasm of the Sertoli elements. In testicular grafts Williams (’50) accumulated evidence which suggests that vacuoles and secretion droplets in the Sertoli cells occurred as a result of LH administration. The combined results of these investigators suggest that sperm release from the Sertoli cell is dependent, in some way, upon LH (ICSH) activity.

A final function is concerned with the physiological maturing of sperm in the reproductive duct, at least in many vertebrate species. The well-being of the epididymis and vas deferens is dependent upon the presence of the male sex hormone (Creep, Fevold, and Hisaw, ’36). As the male sex hormone results from stimulation of the interstitial cells by the interstitial-cellstimulating substance, LH (ICSH), the connection between this substance and the physiological maturation of the sperm cell is obvious.

2. External Environmental Factors and Testis Function

As we have seen above, the anterior lobe of the hypophysis acts as the main internal environmental factor controlling the testes and, through them, the reproductive ducts. It has been observed also that food, vitamins, and anatomical position of the testis are important influences in regulating testicular function. Furthermore, general physiological conditions such as health or disease have an important bearing upon the gonads (Mills, ’19). All of the above conditions are contained within the body of the organism, and as such represent organismal conditions.

Fio. 22. Chart showing the effects of the hypophyseal anterior lobe upon the developing gametes. It also suggests the various factors influencing pituitary secretion of the gonadotrophic hormones, FSH and LH. Observe that the primitive gamete in the cortex of the ovary is subjected to the cortical environment and develops into an oocyte, whereas in the medullary or testicular environment it develops into a spermatocyte. Experiments upon sex reversal have demonstrated that the medullary and cortical portions of the gonad determine the fate of the germ cell. In the male area or medulla, the germ cell differentiates in the male direction, while in the cortex, the differentiation is in the direction of the female gamete or oocyte, regardless of the innate sex-chromosome constitution of the primitive germ cell. The fate of the germ cell thus is influenced by four main sets of factors: (1) Internal and external environmental factors, controlling the secretions of the pituitary body, (2) Fnvironment of the testicular tissue (medulla) and possible humoral substances produced in this tissue, (3) Environment of the ovarian tissue (cortex) and possible humoral substances elaborated there, and (4) Secretions of the anterior lobe of the pituitary body.

The following question naturally arises: Do factors or conditions external to the body impinge themselves in such a way as to control pituitary and gonadal function?

a. Light as a Factor

Aside from the supply of nutritive substances or the collision of the many nervous stimuli with the individual which may arouse or depress the sexual activities, two of the most important obvious external factors are temperature and light. Research on the reproductive behavior of many animal species, during the past twenty years, has shown that both of these factors have great significance on the reproductive activities of many vertebrate species. Bissonnette (’30, ’32, ’35, a and b) has accumulated evidence which demonstrates that light is a potent factor in controlling the reproductive behavior of the European starling (Sturnus vulgaris) and also of the ferret (Putorius vulgaris). In the starling, for example, the evidence shows that green wave lengths of the spectrum inhibit testicular activity, while red rays and white light arouse the reproductive function (fig. 23). The addition of electric lighting to each day’s duration produced a total testis size in midwinter which surpassed the normal condition in the spring. In the ferret artificially increased day length beginning at the first part of October brings the testis to maximum size and activity coupled with a normal mating impulse as early as November and December (fig. 24). Under normal conditions the male ferret is able to breed only during February and early March,

These findings relative to the influence of light on the reproductive periodicity of animals confirm a fact which has been known for a long time, namely, that seasonal breeders brought from the northern hemisphere to the southern hemisphere reverse their breeding season. For example, ferrets which normally breed from spring to summer in the northern hemisphere shift their breeding habits to the September-February period when moved to the southern hemisphere. Inasmuch as the hypophysis is instrumental in bringing about secretion of the gonadotrophic hormones responsible for the testicular activity, it is highly probable that light coming through the eyes (see Hill and Parkes, ’33) influences the nervous system in some way arousing the hypophysis and stimulating it to secrete these substances in greater quantity. However, one must keep in mind the caution given by Bissonnette, that light is not the only factor conditioning the sexual cycles of ferrets and starlings.

While numerous animals, such as the migratory birds, ferret, mare, many fish, frogs, etc., normally are brought into a breeding condition during the period of light ascendency, a large number of animals experience a sexual resurgence only during the time of year when the light of day is regressing in span. This condition is found in some sheep, goats, buffalo in nature.

Fig. 23. Sections of testis of the starling (Sturnus vulgaris), showing the effect of electric lighting added to the bird’s normal daily duration of light during the autumn. (After Bissonnette, Physiol. Zool., 4.) (A) Inside young control bird — no light added

— kept inside as control for (B) from November 9 to December 13. (B) Inside young

experimental bird, receiving additional light from “25 watt” bulb from November 9 to December 13. Total treatment, 34 davs.

Fig. 24. Sections of testis and epididymis, showing modification of sexual cycle in the ferret, Putorius vulgaris, by exposure to increasing periods of light. (After Bissonnette, ’35b.) (A) Seminiferous tubules from normal male over 1 year old, made on October

3, no lighting. (B) Epididymis of normal male on October 3, no lighting. (C) Seminiferous tubules of experimental male on November 7, 36 days of added lighting. (D) Epididymis of experimental males on Nov Tiber 7, 36 days of added lighting.

deer, some fish, etc. Bissonnette (’41) working with goats found that: “Increasing daily light periods from January 25 to April 5 — followed by diminishing periods until July 5, while temperatures remained normal for the seasons, with four Toggenburg female goats and one male Toggenburg and one Nubian female — led to cessation of breeding cycles in February instead of March, followed by initiation of breeding cycles in May and June instead of September.” In the ewe, Yeates (’47) also found that a change from increasing daylight to decreasing length of day induced reproductive activity. In a similar manner. Hoover and Hubbard (’37) were able to modify the sexual cycle in a variety of brook trout which normally breeds in December to a breeding season in August.

b. Temperature Influences

In the case of the animals mentioned above, temperature does not appear to be a major factor in inducing reproductive activity. However, in many animals temperature is vitally influential in this respect. For example, in the thirteen-lined spermophile (ground squirrel) Wells (’35) observed that breeding males kept at 40° F. continued in a breeding condition throughout the year. Under normal conditions this rodent hibernates during the winter months and comes forth in the spring ready to breed; sperm proliferation and general reproductive development take place during the period of hibernation. As the temperature rises during the spring and summer, testicular atrophy ensues, followed by a period of spermatogenesis and reproductive activity when the lowered temperatures of autumn and winter come again. Light, seemingly, is not a factor in this sexual cycle. Another instance of temperature control occurs in the sexual phase of the common red newt, Triturus viriciescens. Here it is the rising temperature of the summer which acts as the inducing agent, and sperm thus produced are discharged into the accessory ducts during the fall and winter to be used when copulation occurs in early spring. However, if this species is kept at a relatively low temperature of 8 to 12° C. during the summer months, spermatogenesis is inhibited and the testis regresses. In the stickleback, Gasterosteus aculeatus, as reported by Craig-Bennett (’31), spermatogenesis occurs during July to early September and appears to be conditioned by a rising temperature, whereas the interstitial tissue and the appearance of secondary sexual features reach their greatest development under increased light conditions and slowly rising temperatures (fig. 15). Bissonnette, in his work on ferrets, also observed a difference in the behavior of these two testicular components; the interstitial tissue responds to large increases of daily light periods, whereas the seminiferous tubules are stimulated by small, gradually increasing periods of light.

The above examples emphasize the importance of a single environmental factor on the pituitary-gonadal relationship. However, in the hedgehog, Allanson and Deansley (’34) emphasize temperature, lighting, and hormone injections as factors modifying the sexual cycles, while Baker and Ransom (’32, ’33, a and b) show that light, food, temperature, and locality affect the sexual cycles and breeding habits of the field mouse. In some vertebrates, therefore, a single factor may be the dominant one, whereas in others, numerous factors control the action of the pituitary and reproductive system.

E. Internal Factors Which May Control Seasonal and Continuous Types of Testicular Function

In endeavoring to explain the differences in response to external environmental factors on the part of seasonal and continuous breeders, one must keep in mind the following possibilities:

(1) The anterior lobe of the hypophysis in some forms (e.g., ferret) cannot be maintained in a secretory condition after it has reached its climax; that is, it apparently becomes insensitive to the light factor. As a result, regression of the pituitary and testis occurs (Bissonnette, ’35b).

(2) In the starling, the anterior hypophysis may be maintained by the lighting, but the testis itself does not respond to the presence of the hypophyseal hormones in the blood (Bissonnette, ’35b). The possibility in this instance may be that testicular function wanes because the body rapidly eliminates the hormone in some way (see Bachman, Collip, and Selye, ’34).

(3) Consideration also must be given to the suggestion that the activities of the sex gland by the secretion of the sex hormone may suppress anterior lobe activity (Moore and Price, ’32).

We may consider two further possibilities relative to continuous testicular function :

(4) If the “brake actions” mentioned above are not present or present only in a slight degree, a degree not sufficient to interrupt the activities of the anterior lobe or of the sex gland, a more or less continuous function of the testis may be maintained.

(5) When several or many environmental factors are concerned in producing testicular activity, a slight altering of one factor, such as light, may prove insufficient to interrupt the pituitary-germ-gland relationship, and a continuous breeding state is effected in spite of seasonal changes.

Underlying the above possibilities which may control testicular function is the inherent tendency or hereditary constitution of the animal. In the final analysis, it is this constitution which responds to environmental stimuli, and moreover, controls the entire metabolism of the body. In other words, the above-mentioned possibilities tend to oversimplify the problem. The organism as a whole must be considered; reproduction is not merely an environmentalpituitary-sex gland relationship.

F. Characteristics of the Male Reproductive Cycle and Its Relation to Reproductive Conditions in the Female

As indicated above, reproduction in the male vertebrate is either a continuous process throughout the reproductive life of the individual or it is a discontinuous, periodic affair. In the continuous form of reproduction the activities of the seminiferous tubules and the interstitial or hormone-producing tissues of the testis function side by side in a continuous fashion. In the discontinuous, periodic type of testicular function, the activities of the seminiferous tubules and of the interstitial tissue do not always coincide. The activities of the seminiferous tubules, resulting in the production of sperm for a particular reproductive cycle, tend to precede, in some species by many months, the activities of the sex-hormone-producing tissue. Evidently, the output of the FSH and LH substances from the pituitary gland are spread out over different periods of the year to harmonize with this activity of the testicular components.

It will be seen in the next chapter that a continuous breeding faculty is not present in the female comparable to that of the male. All females are discontinuous breeders. In some species, the cycles follow each other with little rest between each cycle unless the female becomes pregnant or “broody.” Some have a series of cycles over one part of the year but experience sexual quiescence over the remaining portion of the year. However, in most female vertebrates there is but one reproductive cycle per year.

In harmony with the above conditions, the continuous variety of testicular function is always associated with the condition in the female where more than one reproductive cycle occurs per year. Continuous reproductive conditions in the male, therefore, are adapted to serve one female two or more times per year or several different females at intervals through the year. Furthermore, the complicated, highly glandular, greatly extended type of male-reproductive-duct system is adapted to conditions of (1) continuous breeding, or (2) service to more than one female during one breeding season of the year, whereas the simple type of reproductive duct is adapted to the type of service where all or most of the genital products are discharged during one brief period. In other words, the entire male reproductive system and reproductive habits are adapted to the behavior of female reproductive activities.

Bibliography

Allanson, M. and Deanesly, R. 1934. The reaction of anoestrous hedgehogs to experimental conditions. Proc. Roy. Soc., London, s. B. 116:170.

Allen, B. M. 1904. The embryonic development of the ovary and testis of the mammal. Am. J. Anat. 3:89.

Allen, E., Danforth, C. H., and Doisy, E. A. 1939. Sex and Internal Secretions. Consult Chaps. 16, 17, 18, 19. The Williams & Wilkins Co., Baltimore.

Aschheim, S. and Zondek, B. 1927. Hypophysenvorderlappenhormon und Ovarialhormon im Harn von Schwangeren. Klin. Wchnschr. 6:1322.

Aschner, B. 1912. Uber die Fimktion der Hypophyse. PflUger’s Arch. f. d. ges. Physiol. 146:1.

Asdell, S. A. 1946. Patterns of Mammalian Reproduction. Comstock Publishing Co., Inc., Ithaca, New York.

Bachman, C., Collip, J. B., and Selye, H. 1934. Anti-gonadotropic substances. Proc. Soc. Exper. Biol. & Med. 32:544.

Baker, J. R. and Ransom, R. M. 1932. Factors affecting the breeding of the field mouse (Microtus agrestis). I. Light. Proc. Roy. Soc. London, s. B. 110:313.

and . 1933a. Factors affecting the breeding of the field mouse. (Microtus 'agrestis). II. Temperature and food. Proc. Roy. Soc., London, s. B. 112:39.

and . 193,3b. Factors affecting the breeding of the field mouse (Microtus agrestis). 111. Locality. Proc. Roy. Soc., London, s. B, 113:486.

Bishop, D. W. 1942. Germ cell studies in the male fox (Vulpes fulva). Anat. Rec. 84:99.

Bissonnette, T. FI. 1930. Studies on the sexual cycle in birds. I. Sexual maturity, its modification and possible control in the European starling (Sturnus vulgaris). Am. J. Anat. 45:289.

. 1932. Studies on the sexual cycle

in birds. VI. Effects of white, green and red lights of equal luminous intensity on the testis activity of the European starling (Sturnus vulgaris). Physiol. Zodl. 5:92.

. 1935a. Modifications of mammalian sexual cycles. II. Effects upon young male ferrets (Putorius vulgaris) of constant eight and one-half hour days and of six hours of illumination after dark between November and June. Biol. Bull. 68:300.

. 1935b. Modifications of mammalian sexual cycles. III. Reversal of the cycle in male ferrets (Putorius vulgaris) by increasing periods of exposure to light between October second and March thirtieth. J. Exper. Zool. 71:341.

. 1941. Experimental modification

of breeding cycles in goats. Physiol. Zool. 14:379.

Cieslak, E. S. 1945. Relations between the reproductive cycle and the pituitary gland in the snake, Thamnophis radix. Physiol. Zool. 18:299.

Corner, G. W. 1943. On the female testes or ovaries, by Regnier de Graaf, Chap. XII of De Mulierum Organis Generationi Inservientibus (Leyden: 1672). Translated by G. W. Corner in Essays in Biology. The University of California Press, Berkeley and Los Angeles.

Cowles, R. B. and Nordstrom, A. December 1946. A possible avian analogue of the scrotum. Science. 104:586.

Ciaig-Bennett, A. 1931. The reproductive cycle of the three-spined stickleback, Gasterosteus aculeatus. Linn. Philos. Tr. Roy. Soc., London, s. B. 219:197.

Cramer, A. J. 1937. Evaluation of hormone therapy for undescended testes in man. Endocrinology. 21:230.

Crouch, J. E. 1939. Seasonal changes in the testes of the passerine bird, Phainopepla nitens lepida. Proc. Soc. Exper. Biol. & Med. 40:218.

Crowe, S. J., Cushing, H., and Homans, J. 1910. Experimental hypophysectomy. Bull. Johns Hopkins Hosp. 21:127.

De Robertis, E., Burgos, M. H., and Breyter, E. 1946. Action of anterior pituitary on Sertoli cells and on release of toad spermatozoa. Proc. Soc. Exper. Biol. & Med. 61:20.

Dorfman, R. J. 1950. Chap. II. Physiology of androgens in The Hormones. II, by Pincus and Thimann. Academic Press, Inc., New York.

Evans, H. M. 1947. Recent advances in our knowledge of the anterior pituitary hormones. Am. Scientist. 35:466.

and Simpson, M. E. 1950. Chap.

VI. Physiology of the gonadotrophins in The Hormones, II, by Pincus and Thimann. Academic Press, Inc., New York.

Felix, W. 1912. The development of the urinogental organs in Manual of Human Embryology, by Keibal and Mall. J. B. Lippincott Co., Philadelphia and London.

Fevold, H. L. 1939. Chap. XVII in Allen, et al., Sex and Internal Secretions. 2d ed.. The Williams & Wilkins Co., Baltimore.

and Hisaw, F. L. 1934. Interactions of gonad-stimulating hormones in ovarian development. Am. J. Physiol. 109:655.

, , and Leonard, S. L. 1931.

The gonad-stimulating and the luteinizing hormones of the anterior lobe of the hypophysis. Am. J. Physiol. 97:291.

Glass, F. M. and Rugh, R. 1944. Seasonal study of the normal and pituitary stimulated frog (Rana pipiens). 1. Testis and thumb pad. J. Morphol. 74:409.

Creep, R. O., Fevold, H. L., and Hisaw, F. L. 1936. Effects of two hypophyseal gonadotrophic hormones on the reproductive system of the male rat. Anat. Rec. 65:261.

Guthrie, M. J. 1933. The reproductive cycles of some cave bats. J. Mammalogy. 14:199.

Hammond, J. and Asdell, S. A. 1926. The vitality of the spermatozoa in the male and female reproductive tracts. Brit. J. Exper. Biol. 4:155.

Henle, G. and Zittle, C. A. 1942. Studies of the metabolism of bovine epididymal spermatozoa. Am. J. Physiol. 136:70.

Hill, E. C. 1907. On the gross development and vascularization of the testis. (Excellent figures showing migration of the testes in the pig.) Am. J. Anat. 6:439.

Hill, M. and Parkes, A. S. 1933. Studies on the hypophysectomized ferret. Proc. Roy. Soc., London, s. B. 116:221.

Hoover, E. E. and Hubbard, H. F. 1937. Modification of the sexual cycle of trout by control of light. Copeia. 4:206.

Koch, F. C. 1942. Biol. Symp., The excretion and metabolism of the male sex hormone in health and disease. Jaques Cattell Press. 9:41.

Leonard, S. L. and Kurzrok, R. 1946. Inhibitors of hyaluronidase in blood sera and their effect on follicle cell dispersal. Endocrinology. 39:85.

Ludwig, D. J. 1950. The effect of androgens on spermatogenesis. Endocrinology. 46:453.

Mann, T. 1949. Metabolism of semen. Adv. in Enzymology. 9:329.

Marshall, F. H. A. 1911. The male generative cycle in the hedgehogs, etc. J. Physiol. 43:247.

Mason, K. E. 1939. Chap. XXII in Allen, et al.. Sex and Internal Secretions. 2d ed.. The Williams & Wilkins Co., Baltimore.

Matthews, S. A. 1938. The seasonal cycle in the gonads of Fiinditlus. Biol. Bull. 75:66.

Mills, R. G. 1919. The pathological changes in the testes in epidemic pneumonia. J. Exper. Med. 30:505.

Mitchell, G. A. G. 1939. The condition of the peritoneal vaginal processes at birth. J. Anat. 73:658.

Moore, C. R. 1926. The biology of the mammalian testis and scrotum. Quart. Rev. Biol. 1:4.

. 1928. On the properties of the

gonads as controllers of somatic and psychical characteristics. J. Exper. Zool. 50:455.

. 1939. Chap. VII, Part V, in Allen,

et al.. Sex and Internal Secretions. 2d ed.. The Williams & Wilkins Co.. Baltimore.

. 1942. Physiology of the Testis in

Glandular Physiology and Therapy. 2d ed.. Am. M. A. Council on Pharmacy and Chemistry. Chicago.

and McGee, L. C. 1928. On the

effects of injecting lipoid extracts of bull testes into castrated guinea pigs. Am. J. Physiol. 87:436.

and Price, D. 1932. Gonad hormone functions and the reciprocal influence between gonads and hypophysis with its bearing on the problem of sex hormone antagonism. Am. J. Anat. 50:13.

Pincus, G. and Thimann, K. V. 1950. The Hormones, Vol. II. Academic Press, Inc., New York.

Rasmussen, A. T. 1917. Seasonal changes in the interstitial cells of the testis in the woodchuck (Marmota monax). Am. J. Anat. 22:475.

Riley, G. M. 1937. Experimental studies on spermatogenesis in the house sparrow, Passer dornesticus (Tinnaeus). Anat. Rec. 67:327.

Robson, J. M. 1940. Recent Advances in Sex and Reproductive Physiology. J. & A. Churchill, Ltd., London.

Rowlands, J. W. 1944. Capacity of hyaluronidase to increase the fertilizing power of sperm. Nature, London. 154:332.

Sehulte, T. L. 1937. The genito urinary system of the Elephas indie us male. Am. J. Anat. 61:131.

Schwenk, E. 1944. Synthesis of the steroid hormones. Page 129 in The chemistry and physiology of hormones. Publication of Am. A. Adv. Sc.

Smith, P. E. 1939. Chap. XVI in Allen, et al.. Sex and Internal Secretions. 2d ed.. The Williams & Wilkins Co., Baltimore.

Turner, C. D. 1948. Chap. 12 in General Endocrinology. W. B. Saunders Co., Philadelphia.

Turner, C. L. 1919. The seasonal cycle in the spermary of the perch. J, Morphol. 32:681.

van Oordt, G. J. 1923. Secondary sex characters and testis of the ten spined stickleback (Gasterosteus pungitius). Proc. Kon. Akad. Wetensch., Amsterdam. 26:309.

Weber, M. 1928. Die Saiigetiere. Gustav Fischer, Jena.

Wells, L. J. 1935. Seasonal sexual rhythm and its modification in the experimental male of the thirteen-lined ground squirrel (Citellus tridecemlineatus). Anat. Rec. 62:409.

. 1943. Descent of the testis: anatomical and hormonal considerations. Surgery. 14:436.

Williams, R. G. 1950. Studies of living interstitial cells and pieces of seminiferous tubules in autogenous grafts of testis. Am. J. Anat. 86:343.

Wislocki, G. B. 1933. Location of the testes and body temperature in mammals. Quart. Rev. Biol. 8:385.

. 1943a. Studies on the growth of

deer antlers: 1. On the structure and histogenesis of the antlers of the Virginia deer (Odocoileus virginianus borealis). Am. J. Anat. 71:371.

. 1943b. Studies on growth of deer

antlers: 11. Seasonal changes in the male reproductive tract of the Virginia deer (Odocoileus virginianus borealis); with a discussion of the factors controlling antler-gonad periodicity. Essays in Biology In Honor of Herbert H. Evans. The University of California Press, Berkeley and Los Angeles.

. et al, 1947. The effects of gona dectomy and the administration of testosterone propionate on the growth of antlers in male and female deer. Endocrinology. 40:202.

Yeates, N. T. M. 1947. Influence of variation in length of day upon the breeding season in sheep. Nature, London. 160:429.

Young, W. C. 1929. The influence of high temperature on the reproductive capacity of guinea pig spermatozoa as determined by artificial insemination. Physiol. Zodl. 2 : 1 .

1931. A study of the functions of the epididymis. 111. Functional changes undergone by spermatozoa during their passage through the epididymis and vas deferens of the guinea pig. Brit. J. Exper. Biol. 8:151.

Zondek, B. 1930. Uber die Hormone des Hypophysenvorderlappens. 1. Wachstumshormon, Follikelreifungshormon (Prolan A). Luteinisierungshormon (Prolan B) Stoffwechselhormon? Klin. Wchnschr. 8:245.

The Vertetrate Ovary and Its Relationship to Reproduction

A. The ovary and its importance

B. Preformationism, past and present

C. General structure of the reproductive system of the vertebrate female

1. General structure of the ovary

2. General structure of the accessory reproductive organs

D. Dependency of the female reproductive system on general body conditions

1. Inanition

2. Vitamins

a. Vitamin A

b. Vitamin B

c. Vitamin C

d. Vitamin E

3. The hypophysis (pituitary gland)

E. Activities of the ovary in producing the reproductive state

1. The ovary as a “storehouse” of oogonia

2. Position occupied by the primitive female germ cells in the ovarian cortex

3. Primary, secondary, and tertiary follicles of de Graaf

4. Hormonal factors concerned with the development of egg follicles

a. Effects produced by the gonadotrophic hormones of the development of the mammalian egg follicle

b. Stimulating effects of the gonadotrophins on the ovaries of other vertebrates

5. Structure of the vertebrate, mature egg follicle

a. Structure of the mature follicle in metatherian and eutherian mammals

b. Structure of the prototherian egg follicle

c. Egg follicles of other vertebrates

6. Ovulatory process; possible factors controlling ovulation

a. Process of ovulation in higher mammals

1) Changing tissue conditions culminating in egg discharge from the ovary

2) Hormonal control of the ovulatory process

b. Ovulation in vertebrate groups other than the higher mammals

1) Hen

2) Frog

3) Hormonal control of ovulation in lower vertebrates

c. Comparison of the immediate factors affecting egg discharge in the vertebrate group

7. Internal conditions of the ovary as an ovulatory factor

8. Number of eggs produced by different vertebrate ovaries

9. Spontaneous and dependent ovulation in the mammals and in other vertebrates

10. Egg viability after discharge from the ovary

11. History of the egg follicle after ovulation

a. Follicles which do not develop a post-ovulatory body

b. Follicles which develop a post-ovulatory body; formation of the corpus luteum

12. Hormones of the ovary and their activities in effecting the reproductive condition

a. Estrogenic hormone

1) Definition and source of production

2) The ovary as the normal source of estrogen in the non-pregnant female

3) Pituitary control of estrogen formation

4) Effect of estrogen upon the female mammal

5) Effects of estrogen in other vertebrates

b. Progesterone — the hormone of the corpus luteum

1) Production of progesterone

2) Effects of progesterone

F. Reproductive state and its relation to the reproductive cycles in female vertebrates

1. Sexual cycle in the female mammal

a. Characteristics and phases of the reproductive cycle

b. Relation of cstrus and ovulation in some common mammals

1) Spontaneously ovulating forms (Sexual receptivity of male occurs at or near time of ovulation)

2) Dependent ovulatory forms (Sexual receptivity [heat] occurs previous to time of ovulation)

c. Non-ovulatory (anovulatory) sexual cycles

d. Control of the estrous cycle in the female mammal

e. Reproductive cycle in lower vertebrate females

G. Role of the ovary in gestation (pregnancy)

1. Control of implantation and the maintenance of pregnancy in mammals

2. Gestation periods, in days, of some common mammals

3. Maintenance of pregnancy in reptiles and other vertebrates

H. Role of the ovary in parturition or birth of the young

I. Importance of the ovary in mammary-gland development and lactation

J. Other possible developmental functions produced by the ovary

K. Determinative tests for pregnancy

A. The Ovary and Its Importance

One of the editions of the treatise on development, “Exercitationes de Generatione Animalium,” by William Harvey (1578-1657) contains a picture of Jupiter on a throne opening an egg from which various animals, including man, are emerging (fig. 25). Upon the egg (ovum) are engraved the words ovo omnia. At the heading of chapter 62 of this work Harvey placed a caption which explains the phrase ex ovo omnia more explicitly. This heading reads: “Ovum esse primordium commune omnibus animalibus” — the egg is the primordium common to all animals. Published in 1651, this statement still maintains its descriptive force.

Many individual animals arise by asexual reproduction, that is, through a process of division or separation from a parent organism. In the phylum Chordata asexual reproduction is found among the Urochordata, where new individuals may arise by budding from a stolon -like base of the parent (fig. 27). This process often is called gemmation, the formation of a new individual by a protrusion of a mass of cells from the parental body followed by its partial or complete separation. It is a prominent method of reproduction among the lower Metazoa, particularly the coelenterates and sponges. Nevertheless, all animal species among the Metazoa ultimately utilize an egg as the primordium from which the new individual arises. Sexual reproduction, generally associated with the fertilization of an egg by a sperm element, appears to be a needful biological process.

Fig. 25. Copy of the engraved title appearing in one edition of Harvey’s dissertation on generation as shown on p. 139 of Early Theories of Sexual Generation by E. J. Cole. Observe the words “ex ovo omnia” upon the egg which Jupiter is opening. Various animals are emerging from the egg.

Fig. 26. Copy of Hartsoeker’s figure of human spermatozoan, containing the homonculus or “little man,” published in 1694. This figure represents a marked preformationist conception of development. However, it is to be noted that Hartsoeker later abandoned the preformationist concept as a result of his studies on regeneration.

True as the general statement made by Harvey may be, it is not clear what is meant by the word ovum or egg. We know certain of its characteristics, but, for the most part, it must be accepted as an accomplished fact enshrouded in mystery. To Harvey the egg was an indefinite, unorganized association of substance plus a “primordial generative principle” (see Cole, F. J., ’30, p. 140), Other minds have conceived of other meanings. Nevertheless, descriptive and experimental embryology has forced the conclusion that the egg, during its development within the ovary, experiences a profound process of differentiation, resulting in the formation of an invisible organization. Although this organization is invisible, it is imbued with an invincibility which, when set in motion at the time of fertilization, drives the developmental processes onward until final fulfillment is achieved in the fully formed body of the adult organism.

Fig, 27. Forms of asexual reproduction in the subphylum Urochordata 9 #

Chordata. (From MacBride: Textbook of Embryology, Vol. 1, Londo/rt', ^

(A) Budding from "stolon of Perophora listeri, from MacBride after (jR) , (C)

Two stages of budding in an ascidian, from MacBride after Pizon

Beyond the fundamental changes effected in the developing egg while in the ovary, the latter structure has still other roles to maintain. Through the mediation of the hormones produced within the confines of the ovarian substance, the female parent is prepared to assume the responsibilities of reproduction. In addition, in many vertebrates the further responsibility of taking care of the young during the embryonic period stems from the hormones produced in the ovary. In some vertebrates, the instinct of parental care of the young after hatching or after birth indirectly is linked to ovarian-pituitary relationships. Because of these profound and far-reaching influences which the ovary possesses in producing the new individual, it must be regarded as the dynamic center of reproduction for most animal species.

B. Preformationism, Past and Present

The above statement relative to the importance of ovarian influences and of the female parent is a position far removed from that held by some in the past. An ancient belief elevated the male parent and his “seed” or semen. As Cole, F. J., ’30, p. 38, so aptly places the thinking of certain learned sources during the 16th century: “The uterus is regarded as the ‘till’d ground for to sow the seeds on’ — a popular idea, based obviously on the analogy with plants, which prevailed long before and after this period. The seed of the male is therefore the chief agent in generation, but cannot produce an embryo without the cooperation of the female, and whether the result is male or female depends on which side of the uterus the seed falls, the time of the year, temperature, and the incidence of menstruation.” Or, in reference to the Leeuwenhoek’s belief in an intangible preformationism, Cole, F. J., ’30, p. 57, states: “He asserts that every spermatic animalcule of the ram contains a lamb, but it does not assume the external appearance of a lamb until it has been nourished and grown in the uterus of the female.” This statement of A. van Leeuwenhoek (1632-1723) was made as a criticism of N. Hartsoeker (1656-1725) whose extreme adherence to a seminal preformationism led him to picture the preformed body of the human individual, the homonculus, encased within the head of the spermatozoon (fig. 26). Hartsoeker, however, later abandoned this idea.

In fairness it should be observed that the egg during these years did not lack champions who extolled its importance. While the Animalculists considered the sperm cell as the vital element in reproduction, the Ovists, such as Swammerdam (1637-80), Haller (1708-77), Bonnet (1720-93) and Spallanzani (1729-99) believed that the pre-existing parts of the new individual were contained or preformed within the egg.

An extreme form of preformationism was advocated by certain thinkers during this period. For example, Bonnet championed the idea of encasement or “emboitement.” To quote from Bonnet:

The term “emboitement” suggests an idea which is not altogether correct. The germs are not enclosed like boxes or cases one within the other, but a germ forms part of another germ as a seed is a part of the plant on which it develops. This seed encloses a small plant which also has its seeds, in each of which is found a plantule of corresponding smallness. This plantule itself has its seeds and the latter bears plantules incomparably smaller, and so on, and the whole of this ever diminishing series of organized beings formed a part of the first plant, and thus arose its first growths. (Cole, ’30, p. 99.)

On the other hand, there were those who maintained that for some animals, neither the sperm nor the egg were important as “many animals are bred without seed and arise from filth and corruption, such as mice, rats, snails, shell fish, caterpillars, moths, weevils, frogs, and eels” (Cole, ’30, p. 38). This concept was a part of the theory of spontaneous generation of living organisms -a theory ably disproved by the experimental contributions of three men: Redi (1626-97); Spallanzani; and Louis Pasteur (1822-95).

Modern embryology embraces a kind of preformationism, a preformationism which does not see the formed parts of the new individual within the egg or sperm but wi.ich does see within the egg a vital, profound, and highly complex physiochemical organization capable of producing a new individual by a gradual process of development. This organization, this selfdetermining mechanism, is resident in the nucleus with its genes and the organized cytoplasm of the fully developed oocyte or egg. However, as shown later, this organization is dependent upon a series of activating agencies or substances for its ultimate realization. Some of these activating substances come from without, but many of them are produced within the developing organism itself.

C. General Structure of the Reproductive System of the Vertebrate Female

1. General Structure of the Ovary

Morphologically, the ovary presents a series of contrasts in the different vertebrate classes. In teleost fishes the size of the ovary is enormous compared to the body of the female (fig. 28), while in the human (fig. 29), cow, sow, etc., it is a small structure in comparison to the adult body. Again, it may contain millions of mature eggs in the ling, cod and conger, during each breeding season, whereas only a single egg commonly is matured at a time in the cow, elephant, or human. During the reproductive season the ovary may assume a condition of striking colored effects as in the bird, reptile, shark, and frog, only to recede into an appearance drab, shrunken, and disheveled in the non-breeding season.

Fig. 28. Dissection of female specimen of the common flounder, Limanda ferruginea. It particularly shows the ovary with its laterally placed ovarian sinus. Observe that the ovary, during the breeding season, is an elongated structure which extends backward into the tail. There are two ovaries, one on either side of the hemal processes of the caudal vertebrae.

Its shape, also, is most variable in different species. In mammals it is a flattened ovoid structure in the resting condition, but during the reproductive phase it may assume a rounded appearance, containing mound-like protrusions. In birds and reptiles it has the general form of a bunch of grapes. In the amphibia it may be composed of a series of lobes, each of which is a mass of eggs during the breeding season, and in teleost and ganoid fishes it is an elongated structure extending over a considerable area of the body.

Regardless of their many shapes and sizes, the ovaries of vertebrates may be divided morphologically into two main types, namely, compact and saccular forms. The compact type of ovary is found in teleost, elasmobranch, cyclostome, ganoid, and dipnoan fishes, as well as in reptiles, birds and mammals. It has the following regions (figs. 30, 31):

( 1 ) the medulla, an inner zone containing relatively large blood and lymph vessels;

(2) the cortex, an area outside of and surrounding the medulla (except at the hilus), containing many ova in various stages of development;

(3) a tunica albuginea or connective-tissue layer surrounding the cortex; and

(4) the germinal epithelium or the covering epithelium of the ovary.

The germinal epithelium is continuous with the mesovarium, the peritoneal support of the ovary, and the particular area where the mesovarium attaches to the ovary is known as the hilus. Within the mesovarium and passing through the hilus are to be found the blood and lymph vessels which supply the ovary (fig. 30).

The ovary of the teleost fish is a specialized, compact type of ovary adapted to the ovulation of many thousands, and in pelagic species, millions of eggs at one time. It has an elongate hilar aspect which permits blood vessels to enter the ovarian tissue along one surface of the ovary, whereas the opposite side is the ovulating area. In many teleosts the ovulating surface possesses a special sinus-like space or lumen (fig. 28) which continues posteriad to join the very short oviduct. At the time of ovulation the eggs are discharged into this space and move caudally as the ovarian tissue contracts. In other teleosts this ovulatory space is not a permanent structure but is formed only at the time of ovulation. In Tilapia macrocephala, for example, the ovulatory lumen is formed on the side of the ovary opposite the area where the blood vessels enter. The formation of this space at the time of ovulation is described by Aronson and Holz-Tucker (’49) as a rupture of the elastic follicles during ovulation whereupon the follicle walls shrink toward the ovarian midline.

Fig. 29. Diagrammatic representation of a midsagittal section of the reproductive organs of the human female. (Slightly modified from Morris: Human Anatomy, Philadelphia, Blakiston.)

Fig. 30. Schematic three-dimensional representation of the cyclic changes which occur in the mammalian ovary.

carrying the interstitial tissue and immature ova. This shrinking away of the tissues of the ovary leaves a space between these tissues and the outside ovarian wall. A lumen thus is formed along the lateral aspect of the ovary which is continuous with the oviduct. Many teleosts have two ovaries (e.g., flounder); in others there is but one (e.g., perch).

The amphibia possess a true saccular ovary (fig. 32). It has a cortex and germinal epithelium somewhat similar to the compact ovarian variety, but the area which forms the medulla in the compact ovary is here represented by a large lymph space. During early development, the amphibian ovary is a compact structure, but later there is a hollowing out and disappearance of the compact medullary portion, and the cortical area remains as a relatively thin, peripheral region (Burns, ’31; Humphrey, ’29).

Histologically the vertebrate ovary is composed of two general cellular groups, namely:

(1) germ cells, and

(2) general tissue cells of various kinds, such as epithelium, connective tissue, smooth muscle fibers, and the complex of elements comprising the vascular system of the ovary (figs. 30, 32). Some of the general cells form the so-called interstitial tissue of the ovary.

The germ cells differ from the general cells in that each of them has a latent potency for developing a new individual. This latent condition is converted into active potentiality during the differentiation of the primitive germ cell into the mature egg or ovum.

2. General Structure of the Accessory Reproductive Organs

The accessory reproductive structures of the female vertebrate may be separated into three general types, viz.:

( 1 ) the total absence of or the presence of a pair of short funnel-like structures which convey the eggs from the peritoneal cavity through

Fig. 31 . Three-dimensional representation of the bird ovary together with the funnel portion (infundibulum) of the oviduct. Recently ovulated egg is shown in the process of engulfment by the infundibulum. Various stages of developing eggs are shown.

Fig. 32. Anterior half of the saccular ovary of Necturus maculosus.

an opening into the urogenital sinus and thence to the outside as in cyclostome fishes,

(2) a short sinus-like tube attached to each ovary and to the urogenital sinus or to a separate body opening as in many teleost fishes (fig. 28), and

(3) two elongated oviducal tubes variously modified (figs. 29, 33, 34, 35, 36, 37).

Except in the teleost fishes the cephalic end of each oviduct generally is open and is placed near the ovary but not united directly with it (figs. 29, 33) although in some species, such as the rat, it is united with an ovarian capsule (fig. 37). In some vertebrates the anterior orifice of the oviduct may be located a considerable distance from the ovary, as in frogs, toads, and salamanders. In many vertebrates, as in birds and snakes, there is but one oviduct in the adult.

In some vertebrates the oviduct is an elongated glandular tube, as in certain urodele amphibia (fig. 33) and in ganoid fishes; in others, such as frogs, birds or mammals, it is composed of two main parts: ( 1 ) an anterior glandular structure and (2) a more caudally placed uterine portion. The latter may unite directly with the cloaca, as in the frog (fig. 38) or by means of a third portion, the vaginal canal or vagina located between the uterus and the cloaca, as in elasmobranch fishes, reptiles, and birds, or between the uterus and the external urogenital sinus, as in mammals (figs. 35, 36, 37). The vaginal canal may be single, as in eutherian mammals, or double, as in metatherian mammals (figs. 35, 36). In metatherian (marsupial) mammals it appears that a third connection with the oviducts is made by the addition of a birth passageway. This birth canal represents a secondary modification of a portion of the vaginal canals and associated structures (figs. 34, 35, 114). (See Nelsen and Maxwell, ’42.) One of the main functions of the vagina or vaginal canal is to receive the intromittent organ of the male during copulation.

The anterior opening of the oviduct is the ostium tubae abdominale, a funnel-shaped aperture generally referred to as the infundibulum. In the transport of the egg from the ovary to the oviduct the infundibulum, in many species, actually engulfs and swallows the egg.

The portion of the oviduct anterior to the uterus often is called the convoluted glandular part; it is highly twisted and convoluted in many species. In amphibians, reptiles, birds, and in some mammals the glandular portion

Fig. 33. Diagrammatic representation of the reproductive structures of female urodele,

Necturus maculosus.

Fig. 34. Diagrammatic lateral view of female reproductive system of the opossum, showing pseudo-vaginal birth canal.

Fig. 35. Reproductive structures of female opossum shown from the ventral view. Observe that the ovary and infundibular portion of the Fallopian tube lie dorsal to the horn of the uterus.

functions to secrete an albuminous coating which is applied to the egg during its passage through this region. In amphibians, reptiles, and birds it forms the major portion of the oviduct, but in mammals it is much reduced in size and extent. In the latter group it is referred to as the uterine or Fallopian tube.

The uterus is a muscular, posterior segment of the oviduct. Like the anterior glandular portion of the oviduct, it also has glandular functions, but these are subservient to its more particular property of expanding into an enlarged compartment where the egg or developing embryo may be retained. The protection and care of the egg or of the embryo during a part or all of its development, is the main function of the uterus in most vertebrates. In the frogs and toads, however, this structure seems to be concerned with a “ripening” process of the egg. Large numbers of eggs are stored in the uterine sac of the frog for a period of time before spawning.

Various degrees of union between the uterine segments of the two oviducts are found in mammals. In the primates they fuse to form a single uterine compartment with two anterior uterine tubes (fig. 29). In carnivores, there is a caudal body of the uterus with two horns extending forward to unite with the uterine tubes (fig. 36). In the rat and mouse, the uterine segments may be entirely separate, coming together and joining the single vaginal chamber (fig. 37). In the opossum the uterine segments are entirely separated, joining a dual vaginal canal system posteriorly (figs. 34, 35, 114).

D. Dependency of the Female Reproductive System on General Body

Conditions

1. Inanition

In the immature female mammal continued underfeeding results in general retardation of sexual development. The younger follicles may develop, but the later stages of follicular development are repressed. In the adult female, inanition produces marked follicular degeneration and atresia as shown by many records of retarded sexual development, reduced fertility, even cessation of the cyclic activities of menstruation and estrus occurring in man and domestic animals during war-produced or natural famine (Mason in Allen, Danforth, and Doisy, ’39, p. 1153). The ovary thus seems to be especially susceptible to starvation conditions, even more so than the testis. As the condition and well-being of the secondary reproductive structures are dependent upon proper ovarian function, this part of the reproductive system suffers marked changes as a result of ovarian dysfunction during prolonged starvation.

Fig. 36. Schematic representation of reproductive organs of the female cat. On the left side of the illustration, the body of the uterus and uterine horn have been cut open, and the Fallopian tube and ovary are highly schematized. Observe the partial ovarian capsule around the ovary shown on the right and the relatively fixed condition of the infundibular opening of the oviduct lateral to the ovary.

Fig. 37. Diagrammatic representation of the reproductive organs of the female rat, showing the bursa ovarica around each ovary. Observe that uteri open directly into the vagina. (Modified from Turner, ’48.)

Fig. 38. Diagrammatic representation of reproductive structures of the female frog. Observe that the ostium of the oviduct is not an open, mouth-like structure. It remains constricted until the egg starts to pass through.

2. Vitamins

a. Vitamin A

The ovary is not immediately sensitive to a lack in vitamin A in the diet but general epithelial changes in the reproductive tract occur which may aid in producing sterility (Mason, ’39).

b. Vitamin B

Ovarian and uterine atrophy occur as a result of deficiency of this vitamin in monkey, rabbit, mouse and rat (Mason, ’39). This effect may be mediated, at least partly, through the effect of B-deficiency upon the pituitary gland.

c. Vitamin C

During the earlier stages reproductive activity is maintained, but advanced stages of C-deficiency produce regressive effects (Mason, ’39).

d. Vitamin E

E-deficiency in the female rat does not upset the ovarian and general reproductive behavior. However, established pregnancies are disturbed and are terminated by resorption of the embryo (Mason, ’39). In the domestic fowl, unless sufficient amount of vitamin E is present in the egg, embryonic death occurs during early incubation periods of the egg.

3. The Hypophysis (Pituitary Gland)

The ovaries experience pronounced atrophy as a result of hypophysectomy in mammals and non-mammalian species. The earlier stages of follicle formation in the higher mammalian ovary up to the stage of beginning antrum formation are not so much affected, but later follicular development and interstitial tissue growth are inhibited (Smith, P. E., ’39). (See fig. 40.)

E. Activities of the Ovary in Producing the Reproductive State

1. The Ovary as a “Storehouse” of Oogonia

The cortex of the ovary contains many young ova in various stages of development. In the human ovary shortly after birth, the number of oogonia in the cortex of each ovary has been estimated to reach a number as high as 300,000. This figure should not be taken too literally, as the amount of variability in the ovary from time to time is great and degeneration of ova is a common episode. Haggstrdm (’21 ) estimated that each ovary of a 22-yearold woman contained 200,000 young ova. In the ovaries of young rats, Arai (’20, a and b) estimated that there were on the average around 5,000 ova under 20 /x in diameter.

Without entering into the controversy (Chap. 3) relative to the rhythmic origin of germ cells in the ovary, one must accept the conclusion that the normal ovary has within it at all times during its reproductive life large numbers of oogonia in various stages of development. Thus the ovary, aside from its other activities, functions as a storehouse and nursery for young oogonia. Relatively few of these oogonia develop into mature eggs in the mammals. For example, the reproductive life of the human female occurs from about the age of 10 or 14 years to about 48 years. If one egg per monthly cycle is discharged from the ovary which is functional during that cycle, only about 400 eggs would be matured in this way. The number would be less if pregnancies intervened. If one accepts the figures given by Haggstrom, an enormous number of eggs of the human ovary never reach their potential goal. Similarly, according to Corner (’43): “The most prolific egg producer among mammals, the sow, might possibly shed a total of 3,000 to 3,500 eggs, allowing ten years of ovarian activity not interrupted by pregnancy, and assuming the very high average of 20 eggs at each three weekly cycle, but she has vastly more than this in the ovaries at birth.”

2 . Position Occupied by the Primitive Female Germ Cells IN THE Ovarian Cortex

Within the cortex the definitive germ cells or oogonia are found in or near the germinal epithelium (figs. 39, 64). Some authors regard the oogonium as originating from the cells of the germinal epithelium. (See Chap. 3, section on “germ cell origin.”) The definitive germ cell soon becomes associated with small epithelial cells (fig. 41). This complex of a germ cell with its associated epithelial cells is found somewhat deeper in the cortex, within or below the tunica albuginea. As the oogonium begins to experience the changes propelling it toward a state of maturity, it is regarded as an oocyte (Chap. 3). Characteristics of the primitive oocyte arc:

( 1 ) an enlargement of the nucleus,

(2) changes within the chromatin material of the nucleus pertaining to meiosis (Chap. 3), and

(3) a growth and increase in the cytoplasmic substances (fig. 41).

Fig. 40. Effects produced by hypophysectomy on the rat ovary and of replacement therapy utilizing injections of pituitary gonadotrophins. (After Evans, Simpson, and Penchaez: Symposia of Quantitative Biology, Vol. 5, 1937. The Biological Laboratory, Cold Spring Harbor, L. 1., N. Y.) (A) Ovary of hypophysectomized animal. Observe

that Graafian follicles are small. They do not proceed further in their development than the beginning of antral vacuole formation unless replacement therapy is applied. (B) Ovarian condition of hypophysectomized animal receiving replacement therapy in the form of injections of the LH (ICSH) gonadotrophic factor of the anterior lobe of the hypophysis. Interstitial tissue is well developed. (C) Ovarian condition of hypophysectomized animal receiving the FSH gonadotrophic factor. Note follicular growth and antral vacuole formation; interstitial tissue between the follicles remains somewhat deficient. (D) Ovarian condition of hypophysectomized animal receiving injections of FSH plus LH. Corpora lutea are evident (as well as enlarged follicles not shown in the figure). Interstitial tissue remains deficient.

Fig. 41. Development of primary condition of the Graafian follicle in the opossum ovary. (A) Young oocyte with associated epithelial (granulosa) cells which in (B) have encapsulated the oocyte. (C) Encapsulating granulosa cells have increased in number and are assuming a cuboidal shape. (D) Fully developed condition of the primary Graafian follicle. Cf. secondary condition shown in fig. 42.

Fig. 42. Secondary conditions of the Graafian follicle in the opossum ovary. Cf. that of

the rat ovary in fig. 40.

As these changes are initiated, the associated epithelial cells increase in number and eventually encapsulate the oocyte (fig. 41B). This complex of the oocyte with its surrounding layer of follicle cells is known as an egg follicle.

3. Primary, Secondary, and Tertiary Follicles of de Graaf

In the mammalian ovary the developing egg with its associated cells is called the Graafian follicle, so named after the Dutch scientist, Reinier de Graaf (fig. 1), who first described this structure in mammals in 1672-1673. De Graaf was in error, partly, for he believed that the whole follicular complex was the egg. The mammalian egg as such was first described in 1827 by Karl Ernst von Baer (1792-1876). The following statement is taken from de Graaf relative to egg follicles.

We may assert confidently that eggs are found in all kinds of animals, since they may be observed not only in birds, in fishes, both oviparous and viviparous, but very clearly also in quadrupeds and even in man himself. Since it is known to everyone that eggs are found in birds and fishes, this needs no investigation; but also in rabbits, hares, dogs, swine, sheep, cows, and other animals which we have dissected, those structures similar to vesicles exhibit themselves to the eyes of the dissectors like the germs of eggs in birds. Occurring in the superficial parts of the testicles, they push up the common tunic, and sometimes shine through it, as if their exit from the testis is impending. (See fig. 48; also Corner, ’43, page 128.)

The mammalian egg with a single layer of epithelial cells surrounding it is known as a primary Graafian follicle (fig. 41B-D). As the egg and follicle grow, the number of epithelial cells increase and eventually there are several

Fig. 43. Tertiary conditions of the Graafian follicle in the opossum ovary. Similar conditions are found in other mammalian ovaries. (A) Follicle in which the antral vacuoles are beginning to form. (B) This is a follicle in which the antral vacuoles are more numerous and are beginning to coalesce. (C) Condition of the Graafian follicle in the opossum ovary approaching maturity. Observe that the antral space is large and is filled with fluid, the liquor folliculi, while the egg and its surrounding cumulus cells are located at one end of the follicle. The thecal tissue around the follicle is well developed.

Fig. 44. Cellular wall of the mature Graafian follicle in the opossum.

layers of epithelial or granulosa cells surrounding the egg. It may now be regarded as a secondary Graafian follicle (fig. 42 A, B). When a stage is reached where the granulosa cells form a layer five to seven or more cells in thickness extending outward from the egg to the forming thecal layers, small antral vacuoles begin to appear among the granulosa cells. The latter follicle, which is capable of forming antral vacuoles, may be regarded as a tertiary Graafian follicle (fig. 43A).

4. Hormonal Factors Concerned with the Development OF Egg Follicles

The ovary with its contained egg follicles is greatly affected by the gonadotrophic hormones produced in the pituitary body. The removal of the pituitary body (hypophysectomy) causes profound regression of the ovary and accessory reproductive structures. Accordingly, the response of the ovarian tissues to these hormonal substances produced by the hypophysis is responsible for development of the Graafian follicle beyond the early tertiary stage. (See fig. 40 A.) The relationships between the pituitary hormones and the ovary have been studied most intimately in the mammals; the pituitary and eggfollicle relationship in lower vertebrates is more obscure, and probably varies with the particular group.

a. Effects Produced by the Gonadotrophic Hormones on the Development of the Mammalian Egg Follicle

The follicle-stimulating hormone, FSH, appears to increase the number of oogonia and to aid the growth and differentiation of the older follicles. It is possible that some of the effects of FSH upon follicular growth are mediated through its ability, together with small amounts of the luteinizing hormone, LH (ICSH), to cause the formation of estrogen or the female sex hormone, although some investigators believe that estrogen production depends mainly upon the action of LH (ICSH). (See Evans and Simpson in Pincus and Thimann, ’50, p. 355.) In harmony with the idea that estrogen is involved in follicular growth there is some evidence which suggests that introduction of estrogens into the peritoneal cavities of fishes and mammals results in a stimulation of mitotic activity in the germinal epithelium of the ovary. It also has been shown that estrogenic substances retard ovarian atrophy in hypophysectomized immature rats.

When the Graafian follicles of the mammalian ovary reach the proper morphological and physiological conditions (i.e., when they reach the tertiary follicular stage) an increased sensitivity of the follicle cells to FSH occurs. As a result, antral vacuoles filled with fluid appear among the granulosa cells; these eventually coalesce and form the large antral cavity typical of the mature Graafian follicle of the mctatherian and eutherian mammal (fig. 43). The presence of LH (ICSH) is necessary to augment the action of FSH during the latter part of follicle development. The beneficial action of FSH and LH together in later follicular development is shown by the fact that the injection of pure FSH alone is incapable of stimulating growth of the follicle to its full size or to initiate an increased secretion of estrogen. LH aids the maturing process of the follicle only when present in very minimal amounts during the early stages of follicle development and in larger amounts during the later stages of follicular growth. Large amounts of LH in the earlier phases of the follicle’s development bring about a premature luteinization of the follicle with ultimate atresia. A proper quantitative balance of these hormones, therefore, is necessary, with FSH being in the ascendency during the earlier phases of follicle development, followed by increased amounts of LH with decreasing amounts of FSH as the follicle reaches maturity (figs. 22, 53, 59). (For references, consult Evans and Simpson, ’50; Turner, ’48.)

h. Stimulating Effects of the Pituitary Gonadotrophins on the Ovaries of Other Vertebrates

The hormonal control of the developing follicle of other vertebrate ovaries follows similar principles to those outlined above for the mammalian ovary, although data obtained from studies upon other vertebrates in no way compares with the large quantity of information obtained in mammalian studies. In the hen, FSH and LH injected together cause a rapid development of the follicles and premature discharge of the egg from the ovary (Fraps, Olsen, and Neher, ’42). However, in the pigeon. Riddle (’38) reports that another pituitary hormone, prolactin, appears to decrease the production of these hormones and stops egg production with a subsequent atrophy of the ovary. This may be a special means which reduces the number of eggs laid at each nesting period. In regard to accessory reproductive structures, an estrogenic hormone is produced in the ovary of the hen which has profound stimulating effects upon the growth of the oviduct (Romanoff and Romanoff, ’49, pp. 242-244). In the frog, Rana pipiens, mammalian pituitary gonadotrophins are able to effect ovulation (Wright and Hisaw, ’46). Pituitary gonadotrophins have been shown also to have profound stimulative effects on the ovaries of fishes, salamanders, and reptiles.

5. Structure of the Vertebrate, Mature Egg Follicle

As a result of the differentiation and growth induced by the gonadotrophic hormones of the anterior lobe of the hypophysis described in the preceding paragraphs, the egg follicle reaches a state of maturity (fig. 43C). This state is achieved when the follicle is about to rupture with the resultant discharge of the egg. The size of the mature egg follicle varies greatly in different metatherian and eutherian mammals, although the size of the follicle is not related to the size of the egg. On the other hand the size of the mature egg follicle in prototherian mammals and in other vertebrate species shows great divergences, being dependent in this group upon the size of the egg at the time of ovulation (fig. 46).

a. Structure of the Mature Follicle in Metatherian and Eutherian Mammals"'^

The structural pattern of the mature Graafian follicle in the human is strikingly similar to the follicles in other members of this group. It is a vesicular structure with a diameter approximating five millimeters. Externally, the follicle is composed of two connective-tissue layers, an inner cellular layer containing blood capillaries, the theca interna, and an external, fibrous layer, the theca externa (figs. 43C, 44). These two layers are not clearly separable. Passing inward from the theca interna is the basement membrane. Resting upon this membrane are several layers of epithelial cells comprising the membrana granulosa. The latter membrane borders the cavity or antrum of the follicle, which is filled with the liquor folliculi. This liquid is under considerable pressure in the follicle at the time of egg discharge or ovulation.

Projecting inward into the antrum on one side is a small, mound-like mass of granulosa cells, the cumulus oophorus (fig. 43C). Within this hillock of epithelium, is the egg, which measures in the human about 130 /x to 140 fx in diameter. In the opossum, the fully developed Graafian follicle is about 1.25 by 2 mm. in diameter, while the slightly oval egg approximates 120 by 135 ii. The egg of the rat and mouse is small, having a diameter of 75 ju, while that of the dog is about 140 /x; sow, 120 to 140 /x; rabbit, 120 to 130 /x; monkey, 110 to 120 /x; deer, 115 /x; cat, 120 to 130 (x\ mare, 135 /x; armadillo, 80 /X (Hartman, ’29).

• According to Strauss, ’39, the mature Graafian follicle of Erkulus is not a vesicular

structure, as in other higher mammals, but is filled with a loose meshwork of granulosa cells.

While one Graafian follicle in only one ovary is generally developed in the human, monkey, cow, ewe, elephant, etc., at each reproductive period, a multiple condition is found in many other mammals. Each ovary in the opossum may ripen seven or more follicles, in the bitch (female dog) from 2 to 7 follicles, and in the sow from 4 to 10 follicles at each reproductive period.

b. Structure of the Prototherian Egg Follicle

The follicle of the prototherian mammals contains a relatively large egg, while the surrounding fluid and follicular tissue in comparison is small in quantity (fig. 46). In these mammals the egg fills most of the follicular cavity, with the exception of a small fluid-filled space intervening between it and the zona pellucida which lies contiguous to the granulosa cells. Internal and external thecal tissues surround the granulosa cells as in the Graafian follicle of the higher mammals.

c. Egg Follicles of Other Vertebrates

The fully-developed egg follicle in most vertebrates is similar to that found in the prototherian mammals in that the egg tends to fill the entire follicle. The general structural relationships also are similar (figs. 45, 47).

6. Ovulatory Process; Possible Factors Controlling Ovulation

The following description of the ovulatory process in the mammal and in other vertebrates should not be construed as a description of the mechanism, as the exact mechanism is unknown. However, a certain amount of general information has been obtained concerning ovulation and the factors involved. Much of this information has been obtained from studies of the ovulatory

Fig. 45. (A) Young egg follicle of Cryptobranchus alleganiensis, a urodele. (From Noble: “Biology of the Amphibia,” New York, McGraw-Hill, after Smith.) (B) Diagrammatic representation of ovarian events in the frog resulting in egg discharge. (From Turner: “General Endocrinology,” Philadelphia, W. B. Saunders, slightly modified.)

Fig. 46. Diagrammatic representation of the egg of the prototherian mammal. Echidna.

Fig. 47. Diagrammatic drawings of the pendent egg follicle in the ovary of the hen. (A) Low magnification of the entire egg follicle. (B) More detailed view of the blastodisc portion of the egg, nearing maturity, in relation to the pedicle. The latter supports the follicle and permits the blood vessels to pass into and out of the follicle. Compiled from sections of the developing ovary of the hen.

process in higher mammals, especially the rabbit. Among other vertebrates ovulation in the hen and frog have been the objects of considerable study.

a. Process of Ovulation in Higher Mammals

1) Changing Tissue Conditions Cuhninating in Egg Discharge from the Ovary. As the Graafian follicle enlarges and matures under the influence of the follicle-stimulating and luteinizing hormones, it moves closer to the ovarian surface (fig. 30). The surface of the ovary over the ripening follicle bulges outward, forming a mound-like protuberance (fig. 30). In the rabbit as shown by Walton and Hammond (’28) and Hill, Allen, and Cramer (’35) the central part of the original protuberance pushes out still further and forms a papilla-like swelling (fig. 48A-D). As the papilla develops, it becomes avascuiar, and the underlying tissues become thin and greatly distended. The tunica albuginea of the ovary and the two thecal layers of the follicle also are involved in this thinning-out process. As the distended papillary area continues to grow thinner, a small amount of blood followed by some of the follicular fluid containing the egg emerges from the follicle and passes into the surrounding area in close proximity to the infundibulum of the Fallopian tube (fig. 48 E, F). The entire process is a gradual one and may be described as gently but not violently explosive (Hill, Allen, and Cramer, ’35). It is of interest and significance to observe that Burr, Hill, and Allen (’35) were able to detect a change in electromotive force preceding and during the known period of ovulation.

Fig. 48. Process of ovulation in the rabbit. (A-C) Early external changes of the surface of the ovary overlying the bulging Graafian follicle. (D) Formation of a secondary papilla. (E) Rupture of the secondary papilla with discharge of egg and follicular fluid, the latter oozing down over ovarian surface of the follicle. (F) Area of rupture with oozing follicular fluid and egg greatly magnified. (G) Follicle after egg discharge. (A-E and G, slightly modified from Walton and Hammond, Brit. J. Exp. Biol., 6; F, modifier from Hill, Allen, and Kramer, Anat. Rec., 63.)

The process of papillary rupture in the rabbit occupies about five seconds; egg discharge with the surrounding liquor folliculi occurs in approximately 30 to 60 seconds. After the egg has emerged, the follicle as a whole may collapse. The slit-like opening through which the egg and follicular fluid passed during ovulation soon is filled with a clot composed of coagulated blood and follicular fluid (fig. 48G).

While the foregoing processes, visible on the ovarian surface, are consummated, certain internal changes occur which form a part of the ovulatory procedure. These changes arc as follows: At about the time the egg is to be extruded, the follicular fluid reaches its maximum in quantity. This increase produces considerable follicular turgidity which may be associated with an endosmotic effect due to an increase in the salt content of the contained fluid. Shortly before the surface of the follicle ruptures, the cumulus begins to disintegrate, and the egg lies free in the antral fluid. At about this time the first maturation division of the oocyte occurs in the majority of mammals, and the first polar body is extruded.

Concerning the internal changes accompanying rupture of the mammalian follicle, passing mention should be made of the theory that bursting blood vessels discharge their contents into the follicular fluid and thus cause sufficient pressure to rupture the follicle (Heape, ’05). Considerable blood discharge into the follicle seems to be present in some forms, e.g., the mare, quite absent in others such as the human, and present slightly in the opossum.

2) Hormonal Control of the Ovulatory Process. The hormonal mechanism involved in ovulation in the spontaneously-ovulating mammals probably is as follows: The follicle-stimulating hormone causes the growth and development of the follicle or follicles. Estrogen is released by the growing follicles and possibly by other ovarian tissues due to the presence of small amounts of LH, and, in consequence, the estrogenic hormone reaches a higher level in the blood stream (figs. 53; 59).

In the meantime, it is probable that the corpus luteum hormone, progesterone, is produced in small amounts. The exact source of this hormone is not clear. It may be produced by old corpora lutea or by the interstitial tissue of the ovary under the influence of luteotrophin, LTH. The presence of progesterone, in small quantities together with increasing amounts of estrogen, stimulates the anterior lobe to discharge increased amounts of the luteinizing hormone, LH (ICSH). (See figs. 22, 53, 59.) The elevated level of estrogen, according to this theory also causes a decreased output of FSH until it reaches a minimal level at the period shortly before egg discharge (figs. 53, 59). As a result, the increased quantity of LH together with FSH has an added effect upon the follicle which brings about the chain of events leading to egg discharge. Evans and Simpson in Pincus and Thimann (’50) give the proportion of 10 parts of FSH to 1 of LH (ICSH) as the proper hormonal balance in effecting ovulation in the hypophysectomized rat.

In those mammalian species where ovulation is dependent upon the act of copulation, a nervous stimulus is involved which increases the output from the pituitary gland of the gonadotrophic factors, particularly LH.

b. Ovulation in Vertebrate Groups Other Than the Higher Mammals

The physical mechanism involved in the ovulatory procedure in the lower vertebrate classes is different from that found in higher mammals. Two forms, the hen and the frog, have been studied in detail. These two animals represent somewhat different types of ovulatory behavior.

1) Hen. As the hen’s egg develops in the ovary, it gradually pushes the ovarian surface outward; it ultimately becomes suspended from the general surface of the ovary by means of a narrowing stalk, the pedicle (figs. 31, 47). When the ovulatory changes are initiated, the musculature of the ovarian wall overlying the outer surface of the egg appears to contract, and an elongated narrow area along this outer surface becomes avascular. This avascular area represents the place where the ovarian surface eventually ruptures to permit the egg to leave the ovary; it is called variously, the rupture area, stigma, or cicatrix. Gradually, the cicatrix widens and finally a slit-like opening is formed by a tearing apart of tissues in the central region of the cicatrix. Contractions of the smooth muscle fibers appear to be responsible for this tearing procedure (Phillips and Warren, ’37). The egg eventually is expelled through the opening and in many instances it rolls into the infundibular funnel of the oviduct which at this time is actively engaged in an endeavor to engulf or “swallow” the egg (fig. 31).

2) Frog. The egg of the frog projects into the ovarian cavity within the ovary and is attached to the ovarian wall by means of a broad area or stalk (fig. 45B). As the egg enlarges, it tends to push the ovarian surface outward, and the egg and its follicle thus forms a mound-like protuberance from the ovarian surface (figs. 45A, B; 72F). The egg and the surrounding ovarian tissue thus lies exposed on one aspect to the outer surface of the ovary. The outer surface of exposure is the stigma or area of rupture, and in the older follicles this area does not contain blood vessels (fig. 72F). As ovulation

approaches, an opening suddenly appears in the area of rupture. The musculature within the theca interna around the follicle then contracts, and the 

egg rolls out through the opening in the rupture area like a big ameba (fig. 45B). As the egg passes through the aperture, it may assume an hourglass shape (Smith, B. G., T6). After the egg is discharged, the follicle contracts to a much smaller size (fig. 45B). It has been suggested that the rupture of the external surface of the follicle might be produced by a digestive enzyme (Rugh, ’35, a and b).

3) Hormonal Control of Ovulation in Lower Vertebrates. The hormonal mechanism regulating ovarian rupture and egg discharge in the lower vertebrate groups has not been as thoroughly explored in all of the vertebrate groups as it has in the mammals. However, sufficient work has been done to demonstrate that pituitary hormones are responsible in all of the major vertebrate groups, including the fishes. Amphibian pituitary implants under the skin or macerated anterior-lobe pituitary tissue injected into the peritoneal cavity of various amphibia have been effective in producing ovulatory phenomena (Rugh, ’35a). More recently, purified mammalian follicle-stimulating hormone, FSH, and luteinizing hormone, LH, have been used to stimulate egg discharge in frog ovarian fragments, as well as in normal and hypophysectomized females. However, the follicle-stimulating hormone alone will not elicit ovulation (Wright, ’45; Wright and Hisaw, ’46). Accordingly, both factors are necessary in the frog, as in mammals. In the hen, these two pituitary hormones have been shown to bring about ovulation when injected intravenously (Fraps, Olsen, and Ncher, ’42; Romanoff and Romanoff, ’49, pp. 208-215). Also, Neher and Fraps (’50) present evidence which suggests that progesterone plays a part in the physiological chain which elicits ovulation in the hen. A close relationship between the physiological procedures effecting ovulation in the hen and the mammal thus appears to exist.

c. Comparison of the Immediate Factors Effecting Egg Discharge in the

Vertebrate Group

In the vertebrates thus far studied contraction of muscle tissue of the follicle following the rupture of surface tissues presumably is the main factor which brings about egg expulsion. In higher mammals, associated with muscle contracture, there also may be an increase in follicular turgidity due to endosmotic phenomena associated with the contained follicular fluid (Walton and Hammond, ’28). In the frog, hen, and mammal the changes involved in the surface tissues leading to their rupture are associated with the following sequence of events:

( 1 ) avascularity of the surface tissues,

(2) a thinning of the surface tissues, and finally

(3) a rupture of these tissues.

7. Internal Conditions of the Ovary as an Ovulatory Factor

Internal conditions of the ovary undoubtedly are important in controlling follicular growth and ovulation. For example, in the Northern fur seal, Callorhinus ur sinus, the female begins to breed at the age of two years. These seals travel north once a year to the Pribilof Islands in the Bering Sea where they go on land to give birth to the single young and also to breed. Most of the cows arrive between the middle of June and the middle of July. Heavy with young, the females give birth to their offspring within a few hours or days after their arrival. Breeding again takes place about six days after parturition. However, lactation continues, and the young are taken care of during the summer months.

Accordingly, these seals mate each year and it appears that for any particular year the mating behavior and ovulation of the egg are controlled by the ovary, which does not have a corpus luteum. As the corpus luteum, which forms after ovulation in the site of the Graafian follicle, from which the egg is discharged, remains intact for a considerable portion of the year, the ovary which does not have the corpus luteum develops the Graafian follicle for the next summer period. The following year the other ovary will function, and so on, alternating each year (Enders, et al., ’46). Thus, the corpus luteum appears to function as a suppressor of follicular growth within the ovary in which it lies. In the human female, one ovary functions to produce an egg one month, while the following month the other ovary ovulates its single egg. It is possible that here also the large corpus luteum suppresses follicular growth within the particular ovary concerned.

During gestation, the presence of the corpus luteum and its hormone, progesterone, suppresses follicle growth and ovulation in most of the mammalian group. (The placenta may be the source of progesterone during the later phases of pregnancy in forms such as the human.) On the other hand, in the mare, according to Cole, Howell, and Hart (’31 ), ovulation may occur during pregnancy. Species differences, therefore, exist relative to the control of ovulation by the corpus luteum and its hormone, progesterone.

8. Number of Eggs Produced by Different Vertebrate Ovaries

The number of eggs produced during the lifetime of the female varies with the species and is correlated generally with the amount of care given to the young. In many fishes which experience little or no parental care, enormous numbers of eggs may be produced, as for example, in the cod where several millions of eggs are spawned in one season. However, in many of the elasmobranch fishes (i.e., the shark group) the eggs develop within the oviduct, and the young are born alive. Therefore, only six to a dozen eggs produced each reproductive period is sufficient to keep the shark species plentiful. In the hen, where careful breeding and selection have been carried out with a view to egg production, a good layer will lay from 250 to 300 eggs a year. The deer, moose, fur seal, etc., ovulate one egg per year over a life span of a few years. As stated previously, the human female might ovulate as many as 400 eggs in a lifetime. In some species the reproductive life is brief. For example, in the Pacific salmon (Oncorhynchus) females and males die after their single spawning season, and a similar demise occurs in the eel (Anguilla).

9. Spontaneous and Dependent Ovulation in the Mammals AND IN Other Vertebrates

Spontaneous ovulation without apparent stimulation from external sources occurs commonly throughout the vertebrate series. However, dependent ovulation conditioned by psychic or other nervous stimuli also is found extensively. In certain mammals ovulation has been shown to be dependent upon the stimulus induced by copulation, as, for example, the ferret, mink, rabbit, cat, shrew, etc. The stimulus, carried through the nervous system, affects in some way the anterior lobe of the pituitary gland which then produces increased amounts of LH in addition to FSH. These females experience estrus spontaneously, but later follicle growth and egg discharge are dependent upon the added stimulation afforded by copulation.

The element of nervous stimulation has a fundamental relationship to the ovulatory phenomena in the vertebrates. Dependent ovulation occurs in certain birds, such as the pigeon, where mating provides a psychic or nervous stimulation which effects ovulation. The presence of two eggs in the nest tends to suppress ovulation. The removal of these eggs will arouse the ovulatory procedures. However, the pigeon may sometimes lay eggs without the presence of a male. In wild birds in general, the mating reaction is linked to the stimulus for egg laying. The hen, on the other hand, is not dependent upon copulation, but in many of the domestic varieties the presence of a number of eggs in the nest appears to suppress egg laying. In the lower vertebrates nervous stimuli also appear to have an influence upon ovulation. The mating antics of many fish and amphibia may be connected with ovulatory phenomena.

10. Egg Viability after Discharge from the Ovary

The length of time that the egg may survive and retain its capacity for fertilization after leaving the ovary depends upon the nature of the egg and its membrane and the surrounding environment. In the urochordate, Styela, the egg may remain for 3 to 4 hours after it is discharged into the sea water and still be capable of fertilization. In the elasmobranch fishes, reptiles, and birds the conditions of the oviduct are such that fertilization must take place in the upper part of the oviduct within a few seconds or minutes after the egg reaches the infundibular portion. In Fundulus hetewclitus and possibly many other teleost fishes, the egg must be fertilized within 15 to 20 minutes after spawning. In the frog, the egg passes to the uterus at the lower end of the oviduct shortly after it leaves the ovary. Under ordinary reproductive temperatures which obtain in the spring, the egg may remain there for 3 to 5 days without producing abnormalities. If kept at very cool temperatures, the period may be extended. Among the mammals the viability after ovulation varies considerably. In the mare, fertilization must occur within about 2 to 4 hours; rabbit, 2 to 4 hours (Hammond and Marshall, ’25); rat, about 10 hours; mouse, 12 to 24 hours (Long, ’12; Charlton, ’17); opossum, probably within the first hour or so because of the deposition of the albuminous coating in the oviduct; fox, probably only a few hours; sow, about 24 hours or less; man, probably 24 hours or less. In the guinea pig, functional degeneration may begin within 4 to 8 hours after ovulation (Blandau and Young, ’39) .

11. History of the Egg Follicle after Ovulation a. Follicles Which Do Not Develop a Post-ovulatory Body

The changes which occur within the egg follicle after the egg has departed are most variable in different vertebrate species. In most of the fish group the ovary as a whole shrinks to a fraction of its previous size, and many very small, immature eggs, interstitial tissue, and collapsed, contracted, empty follicles make up its composition. Similarly, in frogs, toads, and salamanders the collapsed follicle which follows ovulation does not develop an organized structure. The thecal tissue contracts into a small rounded form within which are a few follicle cells (fig. 45B). These bodies soon disappear.

In many snakes and in turtles, the follicle collapses after ovulation, and it is questionable whether organized bodies develop in the site of the ovulated follicle. A similar condition appears to be the case in birds. However, Pearl and Boring (’18) described an abbreviated form of a corpus luteum in the hen in both discharged and atretic follicles. Also, Rothschild and Traps (’44) found that the removal of the recently ruptured follicle or of this follicle together with the oldest maturing follicle, at a time when the egg which originated from the ruptured follicle is in the oviduct, retarded the laying of the egg from 1 to 7 days. Removal of other portions of the ovary in control hens “practically never” resulted in egg-laying retardation. The ruptured follicle, therefore, is believed, by these investigators, to have some influence on the time of lay of the egg. Whether the hormone progesterone or something similar to it may be produced by the ruptured follicle of the hen is questionable, although present evidence appears to suggest that it does (Neher and Traps, ’50).

b. Follicles Which Develop a Post-ovulatory Body; Formation of the

Corpus Luteum

Post-ovulatory bodies or corpora lutea (yellow bodies) develop in the ovaries of elasmobranch fishes which give birth to their young alive. Also in viviparous snakes of the genera Natrix, Storeria, and Thamnophis, it has been shown that the removal of the ovaries with their corpora lutea invariably results in resorption of the young during the first part of gestation and abortion of the young during the midgestational period, while their removal during the close of gestation permits normal birth to occur (Clausen, ’40). The differentiation of the corpus luteum in the snake involves the granulosa cells of the follicle and possibly the theca interna. The differentiated organ appears similar to that of the mammal (Rahn, ’39).

The function of the corpus luteum which develops in the site of the ruptured follicle in all mammals, including the Prototheria (fig. 49), has been the subject of a long series of studies. (See Brambell, ’30, Chap. 9; Corner, ’43, Chap. V.) Its function during the reproductive period of the female mammal is described below under the section of the ovarian hormones. The events leading to the formation of the corpus luteum in the mammalian ovary may be described as follows: After the discharge of the egg, the follicle collapses. The opening of the follicle at the ovarian surface through which the egg emerged begins to heal. A slight amount of blood may be deposited within the antrum of the follicle during the ovulation process in some mammals. If so, the follicle in this condition is known as the corpus hemorrhagicum.

Fig. 49. (A) Luteal cells of the corpus luteum of the opossum. The cellular conditions in other higher mammals are similar. The centsal core has not yet been invaded and resorbed by the phagocytes accompanying the ingrowing luteal cells and blood vessels. This central core is composed of coagulated blood, blood cells, and connective tissue fibrils. (B) Corpus luteum of the platypus (Ornithorhynchiis).

Then, under the influence of the luteinizing hormone, LH, the granulosa cells of the follicle and also cells from the theca interna, together with blood capillaries, proliferate and grow inward into the antral space (figs. 22, 30, 49). Phagocytes remove the blood clot within the antral space if present, during the inward growth of these structures. As the ingression of cells and capillaries into the follicle continues, the granulosa cells begin to form large, polyhedral lutein cells, while the epithelioid cells of the theca interna form a mass of smaller cells which resemble the true lutein cells; the latter are formed in the peripheral area of the corpus luteum and are called paralutein cells. The small spindle-shaped cells of the theca interna, together with blood capillaries, become dispersed between the lutein cells, forming a framework for the latter.

If the egg is fertilized, the corpus luteum persists and is known as the corpus luteum of pregnancy; if fertilization does not take place, it is called the corpus luteum of ovulation. The latter body soon degenerates. Histologically, both types of corpora are identical when first formed. Eventually the corpus luteum undergoes involution, and its site becomes infiltrated with connective tissue. The latter structure is sometimes referred to as the corpus albicans.

12. Hormones of the Ovary and Their Activities in Effecting the Reproductive Condition

The ovary produces two important hormones which have a profound effect upon the reproductive process. These two hormones are the female sex hormone, estrogen, and the gestational hormone, progesterone.

a. Estrogenic Hormone

1) Definition and Source of Production. The induction of estrus (see p. 93 ) or conditions simulating this state is a property of a relatively large number of organic compounds. Because of this estrus-inducing power, they are spoken of as estrogenic substances or estrogens. Estrogens are widely distributed in nature. Two of the most potent natural estrogens are estradiol and estrone (theelin). Both have been extracted from the mammalian ovary and are regarded as primary estrogenic hormones. The most powerful estrogen is estradiol, and it is regarded at present as the compound secreted by the ovary. During pregnancy it also is found in the placenta. These structures are not the only sources of estrogens, however, for it is possible to extract them from urine after ovariectomy, and they occur in the urine of males as well as that of females. The urine of the stallion is one of the richest sources of estrogens, and the testis contains a high estrogenic content (Pincus and Thimann, ’48, p. 381 ). Estrogens are found also in various plants, such as the potato, pussy willow, etc.

The structural formulae of estradiol and of estrone are as follows:

OH ()

Estradiol Estrone

2) The Ovary as the Normal Source of Estrogen in the Non-pregnant Female. Aside from the fact that estradiol and estrone are readily extracted from the ovary, certain experiments tend to focus attention on the ovary as an important site of estrogen production. For example, the removal of the ovaries of a normal, adult female mammal causes the accessory reproductive organs to undergo profound atrophy. The administration of appropriate amounts of estrogen will restore the accessories of such a female to the condition normal for the resting state. (Consult Pincus, ’50, in Pincus and Thimann, Chap. I.) The injection of follicle-stimulating hormone with small amounts of the luteinizing hormone into the diestrous (i.e., sexually-resting) female with intact ovaries results in follicular development within the ovaries, accompanied by hypertrophy of the accessory reproductive organs to the full estrous condition (Nelsen and White, ’41 ; Pincus, ’50, in Pincus and Thimann) . These and similar experiments point to the ovary as the main site of estrogen formation in the body of the non-pregnant female.

The exact structures of the ovary responsible for estrogen elaboration are not easily determined. Estrogen is found in all parts of the ovary, but certain observations and experimental results suggest that it is formed in relation to the follicular tissues and also by the so-called interstitial tissue of the ovary. For example, when tumors occur within the thecal tissue of the egg follicle in women who have experienced the menopause, there is often an accompanying hypertrophy of the accessory organs. This relationship suggests that thecal gland tissue of the follicle may have the ability to elaborate estrogen (Geist and Spielman, ’43). On the other hand, the normal hypertrophy of the granulosa cells of the egg follicle during the normal reproductive cycle, with the presence of follicular fluid containing estrogen in the antral space of the follicle, points to the granulosa cells as a possible source of estrogen. Also, it has been observed that tumorous growths of the granulosa cells of the follicle produce an excess of estrogenic substance (Geist and Spielman, ’43). Thus, these observations point to the granulosa cells of the egg follicle of the ovary as being capable of estrogen formation. Another possible source of estrogen secretion in the ovary is the interstitial cells, derived in part from theca interna tissue and atretic follicles. These cells are large polyhedral epithelioid cells scattered between the follicles. Their growth appears to be directly stimulated by the injection of pure luteinizing hormone (LH; ICSH) in hypophysectomized rats (fig. 40). A rapid production of estrogen results from such injections and this may mean that these cells are involved in estrogen production within the ovary (Evans and Simpson in Pincus and Thimann, ’50).

In the pregnant female mammal the placenta appears to be a source of estrogen production (Pincus and Thimann, ’48, p. 380; Turner, ’48, p. 422). This is suggested by the successful extraction of estrogen from the placenta of the human and the mare and also by the fact that in these females removal of the ovaries during the middle or latter phase of gestation does not result in estrogen diminution in urinary excretion.

3) Pituitary Control of Estrogen Formation. The removal of the anterior lobe of the pituitary gland of the female results in marked atrophy of ovarian structures (figs. 40, 50) and of the accessory reproductive organs. Replacement therapy (i.e., the injections of the pituitary gonadotrophins, FSH and LH) produces a normal reconstitution of the ovarian and reproductive duct tissues, effecting a normal appearance and functioning of these structures

Fig. 50. Follicular atresia in guinea pig ovary. (Redrawn from Asdell, ’46.) This atresia is a sporadic but not uncommon event in the normal ovary of the mammal. However, after removal of the pituitary gland, marked atresia and degeneration of the more mature follicles occur. (A) Fragmentation of granulosa cells is shown. (B) Beginning invasion of the antral space by theca interna tissue is depicted. (Cf. fig. 40A.) (C) Late stage of atresia with invasion of the antral space by internal thecal cells.

Fig. 51. Effects of estradiol (estrogen) upon the female genital tract of the opossum. (After Risman, J. Morphol., 81.) (A) Reproductive tract of an ovariectomized female.

(B) Hypertrophied condition of a female experiencing the normal estrous changes. (C) Reproductive tract of an ovariectomized female injected with estradiol (0.9 mm.) 36 days after the ovaries were removed.

(fig. 40). This evidence suggests that the pituitary gonadotrophins, FSH and LH, control the development of the ovary and, through their influence upon the ovarian tissues, promote the secretion of estrogen with the subsequent hypertrophy of the female accessory reproductive structures. It is to be observed that it is not at all clear that FSH in pure form is able to elicit estrogen production without the presence of LH (ICSH). (See Evans and Simpson in Pincus and Thimann, ’50, p. 355.)

4) Effect of Estrogen upon the Female Mammal. The changes in the mammalian accessory reproductive organs produced by estrogen are marked. An increase in vascularity and great hypertrophy of the accessory structures result from its injection into ovariectomized females. (See figs. 51, 52, 53.) Increased irritability and activity of the accessory structures also occur. This increased activity appears to be an important factor in the transportation of sperm upward within the female accessory organs to the region where the egg awaits the sperm’s arrival.

The alterations in behavior of the female as a result of estrogen stimulation may be considerable. Females actually seek the presence of a male during the period of strong estrogenic influence. The long journey of the female fur seal to the mating grounds in the Bering Sea, the bellowing and tireless search of the cow moose, the almost uncontrollable demeanor of seeking the male on the part of the female dog or of the cow in “heat” — these are a few illustrations of the regnant power of this stimulant upon the female mammal.

The culmination of these changes in behavior, resulting in a receptive attitude toward the male, is reached at about the time when the egg is discharged from the ovary in many mammalian species. In certain other mammals the period of heat may precede the ovulatory phenomena.

5) Effects of Estrogen in Other Vertebrates. In the hen, estrogenic hormone causes enlargement and functional activity of the oviduct. Estrogenic substance, when injected into female chicks from the eighteenth to the fortieth day, causes an enlargement of the oviduct to about 48 times the natural size. Estrogen also has a profound effect upon the activities of the full-grown hen and aids in egg production (Romanoff and Romanoff, ’49; Herrick, ’44). Estrogen has a pronounced effect upon the oviducts of other vertebrate forms.

b. Progesterone — The Hormone of the Corpus Luteum

1) Production of Progesterone. The luteinizing hormone, LH, of the anterior lobe of the pituitary gland is concerned not only with the development of the egg follicle, but also, after ovulation or the discharge of the egg from the egg follicle, the remaining granulosa cells, and also, some of the theca interna cells of the follicle are induced by the LH factor to form the corpus luteum (figs. 30, 49). Corpora lutea also may be induced by estrogens. This, however, appears to be an indirect stimulus aroused through estrogenic stimulation of the pituitary gland to secrete added amounts of the LH factor (Evans and Simpson in Pincus and Thimann, ’50, p. 359).

Fig. 52. Characteristic histological changes in the female reproductive tract under the influence of estrogen and progesterone. (A-C) Vaginal cyclic changes in the rat. In (A) is shown the condition of the vaginal wall in the diestrus (resting) condition; (B) shows changes in vaginal wall structure during estrus. Observe cornification of outer layer of cells; (C) shows vaginal wall tissue immediately following estrus, i.e., during metestrus. The presence of progesterone tends to suppress the action of estrogen. (After Turner: General Endocrinology, Philadelphia, Saunders.) (D, E) Cyclic changes of the Fallopian tube of the human female during the reproductive cycle. In (D) is shown the midinterval of the cycle, i.e,, at a time paralleling estrus in mammals in general; (E) shows the cellular condition of the lining tissue of the Fallopian tube just before menstruation. In (D) the tissue has responded to the presence of estrogen; (E) effect of progesterone is shown. (After Maximow and Bloom: A Textbook of Histology, Philadelphia, Saunders.) (F, G) Cyclic changes in the uterine-wall tissue during the reproductive cycle in the human female. In (F) is shown general character of the uterine wall during the follicular phase, i.e., responses to estrogen; (G) shows the general condition of the uterine wall following ovulation. The uterus is now responding to the presence of progesterone added to the follicular or estrogenic stimulation. (After Maximow and Bloom: A Textbook of Histology, Philadelphia, Saunders.)

A further pituitary principle, however, seems to be involved in the functional behavior of the corpus luteum. This principle, referred to as luteotrophin (LTH), is associated with the lactogenic-hormone complex produced by the anterior lobe of the pituitary body; it induces the morphologically developed corpus luteum to secrete progesterone. (Consult Evans and Simpson in Pincus and Thimann, ’50, pp. 359, 360; Turner, ’48, p. 379, for references.)

The structural formula of progesterone is as follows:

2) Effects of Progesterone. Progesterone reduces the irritability of the accessory structures and stimulates the mucosa of the uterus to undergo further development. This increased developmental and functional condition of the

Fig. 53. Relationship of the pituitary gonadotrophins and ovarian hormones to the developing Graafian follicle and reproductive-duct change in a polyestrous female mammal.

The Graafian follicle responds to the pituitary gonadotrophins, FSH and LH, with the subsequent growth and ultimate rupture of the follicle and ovulation. Ovulation terminates the follicular phase of the cycle. Under the influence of the LH factor the corpus luteum is established. The latter becomes functional as a result of stimulation by the luteotrophic (lactogenic) hormone. The progestational hormone (progesterone) then is elaborated by the luteal cells. The activity of the latter together with estrogen controls the luteal phase of the cycle.

The rising level of estrogen in the blood suppresses FSH secretion, and together possibly with small amounts of progesterone stimulates LH secretion. Estrogen and small amounts of progesterone also probably stimulate the secretion of large quantities of LTH, and the latter stimulates the secretion of progesterone from the recently formed corpus luteum. When the estrogen level falls, FSH again is secreted.

When the estrogen level rises, the endometrium of the uterus and vaginal mucosa are stimulated. The presence of progesterone suppresses vaginal development, but the uterine mucosa is stimulated to greater activity. Observe that the involution of the endometrial lining in most mammals is gradual but in primates it is precipitous and violent, resulting in menstruation (Cf. fig. 59). (The diestrous period on this chart is shown as a relatively brief period compared to the other aspects of the reproductive cycle. However, it may be very long in females which do not experience a polyestrous condition and in some species it may last a good portion of a year.) (Compiled from various sources in the literature. The portion of the chart showing pituitary and gonadal hormonal relationships is based on data obtained from The Schering Corporation, Bloomfield, N. J.)

accessory reproductive structures added normally to the estrogenic effects during the reproductive cycle constitutes the luteal phase of the cycle. In this phase of the cycle the uterine glands elongate and begin secretion, and the uterus as a whole is prepared for gestation as a result of the action of the progestational hormone, progesterone, associated with estrogen. (See figs. 53, 59.)

F. Reproductive State and Its Relation to the Reproductive Cycle in Female Vertebrates

The changes in the female reproductive organs resulting in structural growth and development referred to above (70-74, 85-88) are consummated in the ability of the female to fulfill the reproductive functions. The phase of the reproductive events characterized by the ability to reproduce is known as the reproductive climax. This period of culmination remains for a brief period, to be followed by recession and involution once again to a resting condition. This developmental progression to a state of reproductive climax followed by regression to a resting condition constitutes a cycle of changing events. When conditions again are right, the cycle is repeated. Each of these cyclic periods is known as a reproductive or sexual cycle (figs. 53-59). The reproductive life of all female vertebrates is characterized by this series of cyclic changes.

In most vertebrate species, the female experiences one sexual cycle per year, which corresponds to the seasonal cycle in the male. However, in various mammals and in certain birds, such as the domestic hen, several or many reproductive cycles may occur during the year. The male, under these conditions, is a continuous breeder; that is, he produces sperm continuously throughout the year.

1. Sexual Cycle in the Female Mammal a. Characteristics and Phases of the Reproductive Cycle

The estrous cycle in mammals is a complex affair composed of a number of integrated subcycles. The changes occurring in the ovary are called the ovarian cycle; the cellular changes in the uterine (Fallopian tube) form a cycle; the responses in the mammary glands constitute the mammary cycle; the cyclic events in the uterus make up the uterine cycle, while those in the vagina form the vaginal cycle (figs. 53, 54, 57).

The entire estrous cycle may be divided by ovarian changes into two main phases: the follicular phase and the luteal phase (fig. 53). The former is under the immediate influence of the enlarging Graafian follicle, which in turn is stimulated by the follicle-stimulating and luteinizing hormones of the pituitary gland, with the subsequent production of estrogen. It is probable that the luteinizing hormone, LH, is mainly responsible for estrogen secretion. (See Evans and Simpson in Pincus and Thimann, ’50, p. 355.) The luteal phase on the other hand is controlled by the activities of the corpus luteum, which has replaced the Graafian follicle under the influence of the luteinizing hormone. The production of progesterone by the corpus luteum is effected as stated previously by the pituitary hormone, luteotrophin (LTH). Ovulation is the pivotal point interposed between these two phases. The follicular phase may occur without ovulation, but the true luteal phase of a normal or fertile reproductive cycle is dependent upon the ovulatory phenomena. Certain luteal conditions may be elaborated in an anovulatory cycle, but we are here concerned with the normal events of the fertile reproductive cycle.

The follicular phase includes that portion of the reproductive cycle known as proestrus and a considerable part of estrus. Proestrus is the period of rapid follicular growth and elaboration of the estrogenic substance which precedes the period of estrus. Estrogen stimulates developmental changes in the cellular structure of the accessory reproductive organs, particularly the vagina and the uterus (figs. 52, 53). Estrus represents the climax of the follicular phase. As such, it is a period of sexual receptivity of the male, and, in spontaneously ovulating forms, of ovulation. During other periods of the cycle the female is indifferent or even antagonistic to the male. The period of estrus is often called period of heat, or period of rut. Estrus is followed by pregnancy if mating is allowed and is successful, or, in many species, by a period of pscudopregnancy if mating is not permitted or if the mating is sterile (figs. 53-57). In some animals, such as the dog, pseudopregnancy is a prolonged normal event even if mating does not occur, continuing over a period almost as long as that of normal pregnancy (fig. 54). In other animals, such as the opossum, pseudopregnancy forms but a brief episode.

Pseudopregnancy is, generally speaking, intermediate in duration between that of a normal luteal phase of the cycle and that of gestation. In those female mammals where it does not occur normally, it is aroused by such procedures as sucking of the nipples, stimulation of the vagina and cervix by the natural mating process, or by artificially stimulating these structures. In some forms, such as the rabbit, pseudopregnancy is aroused by mere handling or even by sight of a male. (For discussion, see Selye, ’48, p. 813.)

The general changes of growth and development of the accessory organs which occur during pregnancy and pseudopregnancy are controlled largely by the secretions of the corpus luteum. The conditions thus imposed by the corpus luteum comprise the luteal or progestational phai^e of the cycle (fig. 57).

In most mammals, if pregnancy does not occur, the ovary and accessory organs again gradually return to the sexually-resting condition known as diestrus (fig. 53). In man and other primates the changes within the uterus are not gradual but are precipitous, and most of the endometrial lining, together with considerable amounts of blood, is discharged to the outside (figs. 53, 59). This phenomenon is called menstruation. The causes of menstruation are largely problematical; it is related to the fall of the level of either or both of the ovarian hormones, progesterone and estrogen. Why certain mammals should experience violent endometrial changes evident in menstruation and others a gradual involution and resorption is a question for the future. The general period of change following estrus in a non-fertile cycle is known as metestrus (fig. 53). In the rat and mouse, metestrus is short, about one or two days; in the human and opossum it occupies approximately ten days to two weeks of the cycle; in the dog, about 40 to 50 days, depending upon the pseudopregnant conditions experienced in different females. The word anestrus is applied to a prolonged diestrus or sexual quiescence between two sexual cycles. However, the involution experienced by the sexual organs in anestrus is somewhat more profound than that prevailing during a brief diestrus. The term lactational diestrus is used to refer to the prolonged diestrous condition in forms such as the rat, wherein estrus is suppressed in the mother while suckling the young.

The length of the sexual cycle varies with the species. When females of the rat or mouse are kept away from a male, the estrous or sexual cycle will repeat itself every 4 to 5 days. In the sow it occurs every 17 to 20 days. In the opossum there is a prolonged anestrous period during the summer and autumn months followed by a polyestrous period during the winter and spring when the estrous cycle reoccurs about every 28 days. In the human female, the sexual cycle occupies about 28 days, and there arc probably about ten normal ovulatory cycles in a year. Some human females may have more, while others experience a slightly smaller number of true ovulatory cycles per year.

Many mammals have one estrous cycle per year. This condition, known as monestrus, is true of most wild mammals, such as the deer, wolf, fox, moose, and coyote. In the shrew, mink, and ferret the moncstrous period may be prolonged if the female is kept away from the male.

Various types of polyestrous conditions exist. In the female dog, for example, there are two or three estrous periods per year about 4 to 6 months apart. In the cat there are several cycles about two weeks apart during the autumn, winter, and spring. In the domestic sheep there is a polyestrous period from September to February in which the cycles occur about every 17 days, followed by an anestrous period from early March to September. In the mare in North America, estrous cycles of about 19 to 23 days occur from March to August. In South America the breeding season is reversed, corresponding to the reversed seasonal conditions south of the equator. In England many mares breed in autumn and winter (Asdell, ’46).

In some mammals estrus may follow immediately after parturition or birth of the young. This may occur occasionally in the rat. Under normal conditions in the fur seal, the female lactates and gestates simultaneously. It is not a common procedure.

It should be observed that there are two aspects of the female reproductive cycle of the mammal relative to fertilization or the bringing together of the male and female reproductive cell. One aspect is the sexual receptivity of the female; the other is the time of ovulation of the egg. In most female mammals sexual receptivity and ovulation are intimately associated and occur spontaneously in the cycle; in others the two events may be separated. In the former group, the development of “heat” and the maturing of the egg follicle are closely associated, while in the latter the conditions favoring sexual receptivity or heat are developed considerably in advance of the maturation of the follicle, as noted in the table below.

b. Relation of Estrus and Ovulation in Some Common Mammals

1) Spontaneously Ovulating Forms (Sexual Receptivity of Male Occurs at or near Time of Ovulation):

Length of Estrus or Period of Heat

Time of Ovulation

Dog

True period of heat about 5-10 days in the middle of a 21 -day estrous period

Variable: 1st day; 2nd day; 5th day; etc., of true period of heat

Guinea pig

6-1 1 hrs.

Views vary: 1-2 hrs. after heat or estrus begins; 10 hrs. after; at end of estrus

Man

Receptivity not always related to cyclic events

12-17 days after onset of preceding menstruation; average around 14th day

Mare

2-11 days; average length 5-6 days

About 1-2 days before end of estrus; best breeding about 3 days after heat begins

Sheep

About 36 hrs.

Late in estrus or just after estrus

ends; presumably about 20-36 hrs. after estrus begins

Sow

Silver fox

Rat

15 days

1-5 days; occurs once a year in February

One determination estimates estrus to be 9-20 hrs.; most receptive to male about first

3 hrs. of heat. Another determination estimates estrus to be 12-18 hrs.

About 1-3 days after onset of estrus 1st or 2nd day of estrus

8 -11 hrs. after beginning of heat

2) Dependent Ovulatory Forms (Sexual Receptivity (Heatl Occurs Previous to Time of Ovulation);

Length of Estrus or Period of Heat

Time of Ovulation

Cat

2-3 days

Time of ovulation uncertain but is

dependent upon copulation

Length of Estrus or Period of Heat

Time of Ovulation

Rabbit (tame)

Estrus prolonged indefinitely during the breeding season from spring to summer; a series of different sets of egg follicles matured; each series lasts about a week, then becomes atretic

Ovulation 10-14 hrs. after mating

Shrew

Estrus prolonged

About 55 -70 hrs. after mating

Ferret

Estrus prolonged

About 30 hrs. after mating

If ovulation and subsequent pregnancy are not permitted by mating, ovarian involution occurs, and an anestrous interlude is established. Anestrus in the common rabbit, Oryctolagus cuniculus, occurs from October to March, but is not absolute.

c. Non-ovulatory (Anovulatory) Sexual Cycles

Not all of the cyclic changes referred to above in those species which normally experience spontaneous ovulation are related to definite egg discharge. Some cycles occur, more or less abortively, without ovulation of the egg. This may happen in the human or in other mammals, such as the dog and monkey. Cycles without ovulations are called non-ovulatory cycles. Menstruation may follow non-ovulatory cycles in the human female.

d. Control of the Estrous Cycle in the Female Mammal

In the control of a reproductive cycle in the vertebrate animal, three main categories of factors appear to influence its appearance and course. These are:

(1) external environmental factors, such as light and temperature,

(2) external factors governing food supply, and

(3) internal factors resulting from an interplay of the activities of the pituitary gland, the ovary, general body health, and of the particular hereditary constitution of the animal.

These factors should be considered not alone in terms of the immediate production of fertile conditions in the parent, but rather, in view of the total end to be achieved, namely, the production of a new individual of the species. For example, the reproductive cycle in the deer reaches its climax or estrus in the autumn after a long period of lush feeding for the mother. The young are born the next spring amid favorable temperatures, followed by another period of bountiful food supply for the mother during lactation and for the fawn as it is weaned. A receding light factor in the late summer and early fall thus may be correlated with the period of heat, which in turn proves to be an optimum time of the year for conception with the resulting birth the following spring. Similarly, light ascendency is a factor in producing fertility in many birds. Here the incubation period for the young is short and a plentiful supply of food awaits the parents and young when it is needed. In other words, the factors which induce the onset of the reproductive state are correlated with the conditions which enhance the end to be achieved, namely, the production of a new individual.

Let us consider next the internal factors which induce the breeding state in the female mammal. The commonly held theory regarding the pituitaryovarian relationship governing the control of the reproductive periods in the mammal which ovulates spontaneously is as follows (figs. 53 and 59) :

( 1 ) FSH of the pituitary gland stimulates later follicular growth. This factor probably is aided by small amounts of the luteinizing factor, LH, to effect an increased production by the ovarian tissues of the estrogenic hormone. Early follicle growth probably occurs without FSH.

(2) Estrogen output by the ovary rises steadily during the period previous to ovulation.

(3) Old corpora lutea or other ovarian tissue possibly secrete minimal amounts of progesterone under the influence of lutcotrophin, LTH.

(4) As the quantity of estrogen rises in the blood stream, it inhibits the production of FSH and together with small quantities of progesterone, increases the output of LH from the pituitary gland. This combination also may cause an increased outflow of the luteotrophic factor.

(5) An increased amount of LH aids in effecting ovulation and the subsequent luteinization of the follicle. As the follicle becomes converted into the corpus luteum, the presence of the luteotrophic factor brings about the formation of increased quantities of progesterone and maintains for a time the corpus luteum and the functional luteal phase of the cycle.

(6) In those mammals possessing a scries of repeating sexual cycles, it is assumed that the fall of estrogen in the blood stream after ovulation suppresses the LH outflow and permits a fresh liberation of FSH from the anterior lobe of the pituitary gland, thus starting a new cycle. The lowering of the estrogen level may be particularly and immediately effective in forms such as the rat and mouse, which have a short metestrus or luteal phase in the estrous cycle.

e. Reproductive Cycle in Lower Vertebrate Females

While the words estrus, heat, or rut are generally applied to the mammalian groups, the recurrent periods of sexual excitement in lower vertebrates are fundamentally the same sort of reaction, although the changes in the reproductive tract associated with ovarian events are not always the same as in mammals. However, similar cyclic changes in the ovary and reproductive tract are present in the lower vertebrates, and their correlation with the activities of the pituitary gland is an established fact. Consequently, the words estrus, rut, sex excitement, and heat basically designate the same thing throughout the vertebrate series — namely, a period during which the physiology and metabolism of the parental body is prepared to undertake the reproductive functions. In this sense, the words estrus, anestrus, heat, etc. also may be applied to the male as well as to the female when the male experiences periodic expressions of the sexual state.

Although the reproductive cycle in all vertebrates represents basically a periodic development of the reproductive functions, there is a marked difference between the estrous cycle in the female mammal and the reproductive cycle in most of the other female vertebrates with the exception of viviparous forms among the snakes, lizards, and certain fishes. This difference is due to the absence of a true luteal phase in the cycle. The follicular phase and elaboration of estrogen appears to be much the same in birds, amphibia, and fishes as in the mammals, but the phase of the cycle governed by progesterone secretion, associated with a gestational condition in the accessory reproductive organs, is found only among those vertebrates which give birth to their young alive.

The reproductive cycles in certain vertebrates may be changed by selective breeding and domestication. For example, the domestic hen is derived from the wild jungle fowl. The jungle fowl conform to the general stimuli of nature as do most wild birds, and the reproductive cycle is associated with a particular season of the year. However, domestication and selection by man of certain laying strains have altered the original hereditary pattern of seasonal laying. Consequently, good layers will lay eggs over an extended period of the year, although there is a strong tendency to follow the ancestral plan by laying most of the eggs during the spring and summer months; during the fall and winter months, a smaller number of eggs are laid. Some of the varieties of the domestic hen conform more closely to the ancestral condition than do other strains. Similar changes may be produced in the buffalo, which in nature breeds in middle to late summer but in captivity has estrous periods three weeks apart throughout the year (Asdell, ’46).

G. Role of the Ovary in Gestation (Pregnancy)

1. Control of Implantation and the Maintenance of Pregnancy in Mammals

The ruling power of the ovary over the processes involved in pregnancy is absolute, particularly during its earlier phases. In the first place, the corpusluteum hormone, progesterone, is necessary to change the uterus already conditioned by the estrogenic hormone into a functionally active state. The latter condition is necessary for the nutrition and care of the embryo. A second change which the gestational hormone imposes upon the genital tract of the female is to quiet the active, irritable condition aroused by the estrogenic factor. Progesterone thus serves to neutralize or antagonize the effects of the estrogenic hormone. A placid condition of the uterus must be maintained during the period immediately following copulation if the fertilized egg is to be cared for within the uterine structure. Large doses of estrogens injected into mammals shortly after copulation prevent implantation of the embryo in all species thus far studied. (See Selye, ’48, p. 822.)

A third effect of the presence of progesterone is the inhibition of the copulatory responses. Immediately following estrus and ovulation, the female dog will fight off the aggressiveness of the male — an aggressiveness which she invited a day or two previously. This change in behavior is introduced by the development of the corpora lutea and the initiation of the luteal phase of the reproductive cycle. Similar anaphrodisiac changes are sometimes mentioned in the behavior of the human female during the luteal phase of the cycle. Progesterone injections also inhibit the copulatory responses in the ferret (Marshall and Hammond, ’44). All of the above-mentioned activities of progesterone thus inhibit or antagonize the condition aroused by estrogenic stimulation.

However, aside from these immediate metestrous and post-ovulatory changes in behavior induced by progesterone, one of its most essential aetivities is concerned with the maintenance of gestation or pregnancy. Ovariectomy or the removal of the ovaries at any time during the gestational period in the rat, mouse, and goat results in death and abortion of the embryo. During the first part of pregnancy in the rabbit, the ovaries must be left intact but may be removed in the closing phase without endangering the gestational process. In the human female, and also in the mare, cat, dog, guinea pig, and monkey, the ovaries may be removed during the latter half of pregnancy without danger to the offspring. However, ovariectomy performed in the early stages of pregnancy in these animals, as well as in all other mammals thus far studied, produces abortion (Pincus, ’36; Selye, ’48, p. 820). The corpus luteum hormone, therefore, is essential in the early phases of gestation in all mammals, and it appears to be necessary during most of the pregnant period in many other mammals.

It is highly probable that the placenta takes over the elaboration of progesterone in those mammals where ovariectomy is possible after the first part of pregnancy has elapsed. In the human female the corpus luteum normally involutes at about the third month of pregnancy, but progesterone may be extracted from the placenta after this period.

Although certain effects of the estrogenic hormone appear to be neutralized (or antagonized) by progesterone during the early phases of reproduction, other effects of estrogen in relation to progesterone are important for the maintenance of the pregnant condition. In this connection the estrogenic hormone appears to suppress some of the growth-promoting effects of progesterone. The two hormones thus work together to promote a gradual development of the uterine tissue and maintain a regulated, balanced condition throughout pregnancy. The placenta, through its ability to elaborate progesterone and estrogen during the latter phases of pregnancy, is an important feature regulating pregnancy in some mammals.

It should be emphasized in connection with the above statements that the presence of the fertilized egg and its subsequent development in some manner affects the maintenance of the corpus luteum. The mechanism by which this influence is conveyed to the ovary is unknown.

2. Gestation Periods, in Days, of Some Common Mammals*

• Adapted from Asdell, ’46; Cahalane, ’47; Kenneth, ’43.

Armadillo (Dasypus novemcinctus)

150

Bear, black (Ursiis americanus)

210

Bear, polar (Thalarctos maritimus)

240

Beaver, Canadian (Castor canadensis)

94-100

Bison (Bison bison)

276

Cat, domestic (Felis catus)

60

Cattle (Bos taurus)

282

Chimpanzee (Pan satyrus)

250

Deer, Virginian (Odocoileus virginianus)

160-200

Dog, domestic (Canis familiaris)

58-65

Donkey, domestic (Eqiius asinus)

365-380

Elephant (Elephas africanus)

641

Elephant (Elephas indicus)

607-641

Elk (A Ices alces)

250

Ferret (Putorius faro)

42

Fox, arctic (Alopex lagopus)

60

Fox, red (Vulpes vulpes and V. fulva)

52-63

Giraffe (Giraffa Camelopardalis)

450

Goat, domestic (Capra hircus)

140-160

Guinea pig (Cavia porcellus)

68-71

Horse (Equus cabaltus)

330-380

Man (Homo sapiens)

270-295

Lion (Felis leo)

106

Lynx (Lynx canadensis)

63

Marten, American (Martes americana)

267-280

Mink (Mustela vison)

42-76

Mole (Talpa europaea)

30

Monkey, macaque (Macaca mulato)

160-179

Mouse, house (Mas rnusculus)

20-21

Opossum (Didelphis virginiana)

13

Pig (Sus scrofa)

115-120

Rabbit (Lepus; Sylvilagus; Oryctolagus)

30-43

Rats (Various species)

21-25

Seal, fur (Callorhinus sp.)

340-350

Sheep, domestic (Ovis aries)

144-160

Skunk, common (Mephitis mephitis)

63

Squirrel, red (Tamiasciurus sp.) 30-40

Tiger (Felis tigris) 106

Whale (Various species) 334-365

Wolf (Canis lupus) 63

Woodchuck (Marmota monax) 35-42

Zebra, mountain (Equus zebra) 300-345

3. Maintenance of Pregnancy in Reptiles and Other Vertebrates

In certain viviparous species of the genera Storeria, Matrix and Thamnophis, Clausen (’40) reports that ovariectomy during gestation results in resorption of the embryo when performed during the earlier phases of gestation and abortion during the middle of gestation, but during the terminal portion of pregnancy the process is unaffected and the young are born normally. These results are similar to those obtained from the rabbit as noted previously.

While experimental evidence is lacking in other vertebrate groups which give birth to the young alive, the evidence obtained from reptilian and mammalian studies suggests that hormones are responsible for the maintenance of pregnancy. In harmony with this statement, it may be pointed out that in the viviparous elasmobranch fishes (e.g., sharks) corpora lutea are developed in the ovaries.

H. Role of the Ovary in Parturition or Birth of the Young

The real factors bringing about parturition are not known, and any explanation of the matter largely is theoretical. However, certain aspects of the subject have been explored. For example, it was observed above that progesterone appears to antagonize the action of estrogen with the result that the uterus stimulated to irritability and contractility under the influence of estrogen is made placid by the action of progesterone. In harmony with this action studies have shown that estrogen tends to increase during the final stages of normal gestation, while progesterone appears to decrease, accompanied by an involution of the corpora lutea. Consequently, the foregoing facts have suggested the “estrogen theory,” which postulates that activities of the uterine musculature are increased by the added amounts of estrogen in the presence of decreasing amounts of progesterone during the latter phases of pregnancy. In confirmation of this theory, it has been shown that progesterone injected into a pregnant rabbit near the end of the gestation period will tend to prolong gestation. A second theory of parturitional behavior assumes that the posterior lobe of the pituitary gland elaborates oxytocin which induces increased uterine activity, resulting in birth contractions (Waring and Landgrebe in Pincus and Thimann, ’50). Again, a third concept emphasizes Ihe possibility that the placenta may produce substances which bring about contractions necessary for the expulsion of the young (Turner, ’48, p. 428). Oxytocic substances have been extracted from the placenta, which suggests the validity of this theory.

Fig. 54. Changes occurring in the reproductive organs and mammary glands of the bitch during the reproductive cycle. The student is referred to Asdell (’46), pp. 150-156 and Dukes (’43), pp. 678-682, for detailed description and references pertaining to the data supporting this chart. The gestation period is based upon data supplied by Kenneth (’43) and the author’s personal experience with dogs.

Fig. 55. Reproductive and pregnancy cycles in the sow. (Modified from data supplied by Corner, Carnegie Inst., Washington, pub. 276, Contrib. to Embryol., 13; the parturition data derived from Kenneth, ’43.)

The specific functions of the ovary in parturition probably are more pronounced in those forms where it is essential throughout most of the gestational period, such as the viviparous snakes, and among the mammals, such forms as the opossum, rat, mouse, and rabbit. The waning of corpus-luteum activity in these species may serve to lower the level of progesterone in the body and thus permit some of the other factors, such as estrogen or the pituitary principle, to activate the uterus.

Another factor associated with the ovary and parturition is the hormone relaxin. This substance was first reported by Hisaw and further studied by this investigator and his associates (Hisaw, ’25, ’29; and Hisaw, et al., ’44).

Fig. 56. Reproductive and pregnancy cycles in the mare. (Parturition period based upon data supplied by Kenneth (’43); other data supplied by Asdell (’46) and Dukes (’43).) It is to be noted that the first corpus luteum of pregnancy degenerates after about 35 days; the second “crop of corpora lutea” (Asdell) degenerate by 150 days. The ovaries may be removed after 200 days of pregnancy without causing abortion of young.

Relaxin aids in the production of a relaxed condition of the pelvic girdle, a necessity for the formation of a normal birth passageway for the young. Relaxin somehow is associated in its formation with the presence of progesterone in the blood stream and also with the intact reproductive system. Relaxin together with estrogen and progesterone establishes a relaxed condition of the tissues in the pubic area of the pelvic girdle.

I. Importance of the Ovary in Mammary-Gland Development and Lactation

Estrogen and progesterone together with the lactogenic hormone, luteotrophin, of the pituitary gland are necessary in mammary-gland development. The entire story of the relationship of these and of other factors in all mammals or in any particular mammal is not known. However, according to one theory of mammary-gland development and function, the suggestive roles played by these hormones presumably are as follows (fig. 58): Estradiol and other estrogens bring about the development of the mammary-gland ducts; as a result a tree-like branching of the ducts is effected from a simple im

Fig. 57. Reproductive and pregnancy cycles in the cow. (Parturition period based upon data supplied by Kenneth (’43), also by Asdell (’46), Other data for chart derived from Asdell (’46).

Three main characteristics of heat or estrous period are evident: (1) A duration of heat of only about 10 to 18 hours; (2) abundant secretion during heat of a “stringy mucus,’’ derived from mucoid epithelium of vagina and from sealing plug of cervix when cow not in estrus (Asdell); and (3) ovulation occurs from 13Vi to 15Vi hours after termination of estrus (Asdell), Variation in time of ovulation may be considerable, from 2 hours before end of estrus to 26 hours after (Asdell).

mature pattern established during earlier development (fig. 5 8 A, A', B). The male mammary gland may remain similar to the condition shown in fig. 58A. The maturing of the egg follicles within the ovary and the concomitant formation of estrogen which accompanies sexual maturity is linked with the more complex state of the mammary-gland system shown in fig. 58B.

The next step of mammary-gland development is carried out under the influence of progesterone. Progesterone is necessary for the development of the terminal glandular tissue or alveoli associated with these ducts (fig. 58C, D). Finally, the pituitary lactogenic hormone (luteotrophin [LTH]; prolactin) stimulates the actual secretion of milk (fig. 58E). Recent research also has shown that the lactogenic hormone collaborates in some way with estrogen and progesterone in the development of the mammary-gland tissue.

Fig. 58. Mammary gland changes in relation to reproduction. (Figures are a modification of a figure by Corner: Hormones in Human Reproduction, Princeton, Princeton University Press. The figure in the latter work was based on a figure by C. D. Turner: Chap. XI of Sex and Internal Secretions, by Allen, et al., Baltimore, Williams & Wilkins, 1939.) Factors involved in mammary gland development and secretion are somewhat as follows: (A, A') Condition of the young, infantile gland. (B) Development from a simple, branched, tubular gland of the immature animal (A') into a compound tubular gland presumably under the direct stimulation of estrogen, according to one theory, or by the action of estrogen upon the pituitary gland which then releases mammogen I, producing these changes, according to Turner, et al.: Chap. XI, Sex and Internal Secretions, by Allen, et al., Baltimore, Williams & Wilkins. (C) Transformation of the compound tubular gland into a compound tubulo-alveolar gland under the influence of progesterone, during the first part of pregnancy, or, according to Turner, et al., by the influence of estrogen plus progesterone which causes the pituitary to release a second mammogen which produces the alveolar transformation. (D) Effect of the latter part of pregnancy is to bring about a development of the cells of the acini of the acinous or alveolar system. The unit shown in (D) represents a simple, branched, acinous gland, in which there are six alveoli or acini associated with the duct. (E) Affect of parturition is to release the lactogenic hormone (prolactin; luteotrophin) from the pituitary gland which brings about milk secretion. During pregnancy the high levels of estrogen presumably inhibit milk secretion. However, following pregnancy the level of estrogen is lowered permitting lactogenic-hormone action upon the alveoli of the gland.

The removal of the placenta and embryo at any time during gestation permits milk flow, provided the mammary glands are sufficiently developed. In the human, any remains of the placenta after birth inhibit milk secretion, probably because the estrogenic hormone is elaborated by the placental remnants. (See Selye, ’48, p. 829.)

In the rabbit, estrogen and progesterone are necessary for the elaboration of the duct and secretory acini; in the guinea pig and goat, and to some extent in the primates, including the human female, estrogen alone is capable of producing the development of the entire duct and acinous system. (See Turner, ’48, p. 430.)

During pregnancy, the actual secretion of milk is inhibited by the estrogenic hormone produced by the ovary and the placenta. The role of estrogen as an inhibitor of lactation is suggested by the fact that, after lactation has started following normal parturition, it is possible in the cow and human to suppress milk flow by the administration of estrogens. After parturition, however, estrogen is no longer present in^sufflcient amounts to suppress the secretion of milk, and the mammary gland begins to function. (In the fur seal a postpartum estrus with ovulation follows a short time after parturition. However, the amount of estrogen produced by this reproductive cycle is not sufficient to curb lactation.) The neurohumoral reflex, or “suckling reflex,” produced by the sucking young appears to maintain the flow of milk over a period of time. Probably this reflex causes a continuous discharge of the lactogenic hormone from the anterior lobe of the hypophysis.

Another theory of mammary-gland development maintains that estrogen stimulates the anterior pituitary gland to release mammogen, which causes development of the duct system, and estrogen plus progesterone induce a second mammogen which stimulates lobule-alveolar development. The lactogenic hormone produces the actual secretion of milk. The ovary thus assumes considerable importance in controlling the (morphological) development of the mammary glands in mammals, particularly in those forms in which the functional condition of the ovary is maintained throughout most of the gestational period, e.g., rat, rabbit, dog, etc. In other species, such as the human, mare, etc., the placenta through its ability to duplicate the production of the ovarian hormones, assumes a role during the latter phase of pregnancy. (For further details, consult Folley and Malpress in Pincus and Thimann, ’48; Selye, ’48, pp. 828-832; and Turner, ’48, pp. 428-448.)

Fig. 59. Stages in the reproductive cycle of the human female and its pituitary-ovarianendometrial relationships (Cf. fig. 53). (Compiled from various sources in the literature.) (a) As shown at the extreme right of the figure, a fall in the level of estrogen and progesterone in the blood stream, either or both, is associated with endometrial necrosis, bleeding, and discharge (menstruation), (b) The lowering of the estrogen level is associated with a new outflow of the follicle-stimulating hormone (FSH), as shown at the right of the figure, (c) In the left side of the figure, the influence of FSH induces egg follicles, probably several, to grow. Antral spaces appear and enlarge. The presence of a small amount of the luteinizing hormone (LH) together with FSH stimulates the secretion of estrogen by the ovarian tissues, possibly by the follicles and interstitial tissue between the follicles, (d) In consequence, the estrogen level rises in the blood stream, and menstruation subsides by the fourth day. (e) The continued influence of estrogen produces endometrial growth, and probably increases the outflow of LH from the pituitary (fig. 53). It is probable, also, that the increased estrogen level stimulates a release of the luteotrophic hormone from the pituitary, which in turn stimulates the formation of a small quantity of progesterone by either the interstitial tissue of the ovary or in old corpora lutea. (f) Some of the developing egg follicles degenerate, while one continues to develop, (g) The elevation of estrogen suppresses the outflow of FSH as indicated by the heavy broken line to the left, (h) The elevated level of estrogen together possibly with small amounts of progesterone evokes an increased outflow of LH and LTH as indicated by the heavy broken line to the right, (i) LH and FSH bring about ovulation at about the fourteenth day. (j) LH causes development of corpus luteum. (k) LTH elicits secretion of progesterone by corpus luteum. Possibly some estrogen is secreted also by corpus luteum. (1) Progesterone and estrogen stimulate added development of endometrium, (m) In the absence of fertilization of the egg, the corpus luteum regresses, with a subsequent fall of progesterone and estrogen levels in the blood stream, terminating the cycle and permitting a new menstrual procedure.

In the dog or opossum during each reproductive cycle, the mammary glands are stimulated to grow and may even secrete milk (dog). These changes closely parallel the ovarian activities, particularly the luteal phase of the cycle. In the human, functional growth changes occur in pregnancy, but, pending the events of the ordinary cycle, alterations in the duct system are slight although the breasts may be turgid due to increased blood flow and connectivetissue development.

J. Other Possible Developmental Functions Produced by the Ovary

As the eggs of the opossum and rabbit travel through the uterine (Fallopian) tube toward the uterus, they are coated with an albuminous, jelly-like coating. Similar jelly coatings are added to the eggs of the bird, reptile, frog, toad, and salamander. These coatings or membranes added to the egg as it travels through the oviduct are known as tertiary egg membranes.

In the toad, the secretion of the protective jelly by the oviduct can be elicited by the lactogenic hormone present in beef pituitary glands. The secretion of the albuminous jelly coatings around the eggs of frogs, salamanders, reptiles, and birds may be related to this hormone. The formation of the crop milk of pigeons has been shown by Riddle and Bates (’39) to be dependent upon the presence of the lactogenic hormone.

The function of the ovary in influencing the outflow of the lactogenic hormone from the pituitary, if present in the above cases of glandular secretion, must be an indirect one. Evans and Simpson in Pincus and Thimann (’50) ascribe the outflow of the “lactogenic hormone (luteotrophic hormone)” of the mammalian pituitary to estrin produced by the ovary. It is possible that in the salamanders, frogs, toads, and the birds an indirect ovarian influence may similarly induce secretion of the lactogenic hormone which in turn governs the elaboration of the albuminous jelly deposited around the egg in transit through the oviduct.

K. Determinative Tests for Pregnancy

Various tests have been used to determine the probability of pregnancy in the human female. These tests are discussed in Chapter 22.

Bibliography

Arai, H. 1920a. On the postnatal development of the ovary (albino rat) with especial reference to the number of ova. Am. J. Anat. 27:405.

. 1920b. On the cause of hypertrophy of the surviving ovary after semispraying (albino rat) and the number of ova in it. Am. J. Anat. 28:59.

Aronson, L. R. and Holz-Tucker, M. 1949. Ovulation in the mouthbreeding cichlid fish, Tilapid macrocephala (Bleeker). Anat. Rec. 105:568.

Asdell, S. A. 1946. Patterns of Mammalian Reproduction. Comstock Publishing Co., Inc., Ithaca, New York.

Blandau, R. J. and Young, W. C. 1939. The effects of delayed fertilization on the development of the guinea pig ovum. Am. J. Anat. 64:303.

Brambell, F. W. R. 1930. The Development of Sex in Vertebrates. The Macmillan Co., New York.

Burns, R. K., Jr. 1931. The process of sex transformation in parabiotic Amblystoma. II. Transformation from male to female. J. Exper. Zool. 60:339.

Burr, H. S., Hill, R. T., and Allen, E. 1935. Detection of ovulation in the intact rabbit. Proc. Soc. Exper. Biol. & Med. 33:109.

Cahalane, V. H. 1947. Mammals of North America. The Macmillan Co., New York.

Charlton, H. H. 1917. The fate of the unfertilized egg in the white mouse. Biol. Bull. 33:321.

Clausen, H. J. 1940, Studies on the effect of ovariotomy and hypophysectomy on gestation in snakes. Endocrinology. 27:700.

Cole, F. J. 1930. Early Theories of Sexual Generation. Oxford University Press, The Clarendon Press, New York.

Cole, H. H., Howell, C. E., and Hart, G. H. 1931. The changes occurring in the ovary of the mare during pregnancy. Anat. Rec. 49:199.

Corner, G. W. 1943. The Hormones in Human Reproduction. Princeton University Press, Princeton, New Jersey.

Dukes, H. H. 1943. The Physiology of Domestic Animals. Comstock Publishing Co., Inc., Ithaca, New York.

Enders, R. K., Pearson, O. P., and Pearson,

A. K. 1946. Certain aspects of reproduction in the fur seal. Anat. Rec. 94:213,

Evans, H. M. and Simpson, M. E. 1950. Chap. VI. Physiology of the gonadotrophins in The Hormones, Vol. II., by Pincus and Thimann. Academic Press, Inc., New York.

Folley, S. J. and Malpress, F. H. 1948. Chaps. 15, 16. Hormonal control of mammary growth and lactation in The Hormones, Vol. 1., by Pincus and Thimann. Academic Press, Inc., New York. Fraps, R. M., Olsen, M. W., and Neher,

B. H. 1942. Forced ovulation of normal ovarian follicles in the domestic fowl. Proc. Soc. Exper. Biol. & Med. 50:308.

Geist, S. H. and Spielman, F. 1943. Endocrine tumors of the ovary. J. Clin. Endocrinol. 3:281.

Haggstrom, P. 1921. Zahlenmassige Analyse der Ovarien eines 22-jahrigen gesunden Weibes. Upsala Lakaref. Forh. 26:1.

Hammond, J. and Marshall, F. H. A. 1925. Reproduction in the Rabbit. Oliver & Boyd, Ltd., Edinburgh.

Hartman, C. G. 1929. How large is the mammalian egg? Quart. Rev. Biol. 4:373.

Heape, W. 1905. Ovulation and degeneration of ova in the rabbit, Proc. Roy. Soc. London, s.B. 76:260,

Herrick, E. H. 1944. Some influences of stilbestrol, estrone and testosterone propionate on the genital tract of young female fowls. Poul. Sc. 23:65.

Hill, R. T., Allen, E., and Kramer, T. C. 1935. Cinemicrographic studies of rabbit ovulation. Anat. Rec. 63:239.

Hisaw, F. L. 1925. The influence of the ovary on the resorption of the pubic bones of the pocket gopher, G corny s bursarius (Shaw). J. Exper. Zool. 42:411.

. 1929. The corpus luteum hormone. I. Experimental relaxation of the pelvic ligaments of the guinea pig. Physiol. Zool. 2:59.

, Zarrow, M. X., Money, W. L.,

Talmadge, R. V. N., and Abramowitz, A. A. 1944. Importance of the female reproductive tract in the formation of relaxin. Endocrinology. 34:122. Humphrey, R. R. 1929. Studies on sex reversal in Amblystoma. I. Bisexuality and sex reversal in larval males uninfluenced by ovarian hormones. Anat. Rec. 42:119.

Kenneth, J. H. 1943. Gestation Periods.

Oliver & Boyd, Ltd., Edinburgh.

Long, J. A. 1912. The living eggs of rats and mice with a description of apparatus for obtaining and observing them. Lfniv. California Publ., Zool. 9(3): 105. Marshall, F. H. A. and Hammond, J., Jr. 1944. Experimental control by hormone action of the oestrous cycle in the ferret. J. Endocrinol. 4:159.

Mason, K. E. 1939. Chapter 22 in Allen, et al.. Sex and Internal Secretions. 2d ed., The Williams & Wilkins Co., Baltimore.

Meher, B. H. and Fraps, R. M. 1950. The addition of eggs to the hen’s clutch by repeated injections of ovulation-inducing hormones. Endocrinology. 46:482.

• 4elsen, O. E. and Maxwell, N. 1942. The

structure and function of the urogenital region in the female opossum compared with the same region in other marsupials. J. Morphol. 71:463.

and White, E. L. 1941. A method

for inducing ovulation in the anoestrous opossum (Didelphys virginiana). Anat. Rec. 81:529.

earl, R. and Boring, A. M. 1918. The corpus luteum in the ovary of the domestic fowl. Am. J. Anat. 23:1. hillips, R. E. and Warren, D. C. 1937. Observations concerning the mechanics of ovulation in the fowl. J. Exper. Zool. 76:117.

incus, G. 1936. The Eggs of Mammals. The Macmillan Co., New York.

. 1950. The Physiology of Ovarian

Hormones, Chap. I. The Hormones, Vol. II, in Pincus and Thimann, Academic Press, Inc., New York.

and Thimann, K. V. 1948. The

Hormones. Vol. I. Academic Press, Inc., New York.

Rahn, H. 1939. Structure and function of placenta and corpus luteum in viviparous snakes. Proc. Soc. Exper. Biol. & Med. 40:381.

Riddle, O. 1938. Prolactin, a product of the anterior pituitary, and the part it plays in vital processes. Scient. Monthly. 47:97.

and Bates, R. W. 193^. Chap. 20.

The preparation, assay and actions of the lactogenic hormone in Allen, et al., Sex and Internal Secretions. 2d ed., The Williams & Wilkins Co., Baltimore.

Romanoff, A. L. and Romanoff, A. J. 1949. The Avian Egg. John Wiley & Sons, Inc., New York.

Rothchild, 1. and Fraps, R. M. 1944. On the function of the ruptured ovarian follicle of the domestic fowl. Proc. Soc. Exper, Biol. & Med. 56:79.

Rugh, R. 1935a. Ovulation in the frog. I. Pituitary relations in induced ovulation. J. Exper. Zool. 71:149.

. 1935b. Ovulation in the frog.

II. Follicular rupture to fertilization. J. Exper. Zool. 71:163.

Ryder, J. A. 1885. On the development of viviparous osseous fishes. Proc. U. S. Nat. Mus. 8: No. 9, 128.

Selye, H. 1948. Textbook of Endocrinology. Acta Endocrinologica. Universite de Montreal, Montreal.

Smith, B, G. 1916. The process of ovulation in Amphibia. Michigan Acad. Sc., 18th Ann. Rep. p. 102.

Smith, P. E. 1939. Chap. XVI. The effect on the gonads of ablation and implantation of the hypophysis and the potency of the hypophysis under various conditions. Allen, et al.. Sex and Internal Secretions. 2d ed.. The Williams & Wilkins Co., Baltimore.

Strauss, F. 1939. Die Befruchtung und der Vorgang der Ovulation bei Ericulus aus der Familie der Centetiden. Biomorphosis. 1:281.

Turner, C. D. 1948. General Endocrinology. W. B. Saunders Co., Philadelphia.

Walton, A. and Hammond, J. 1928. Observations on ovulation in the rabbit. British J. Exper. Biol. 6:190.

Waring, H. and Landgrebe, F. W. 1950. Chap. VIII. Hormones of the posterior pituitary in The Hormones, Vol. II, by Pincus and Thimann. Academic Press, Inc., New York.

Wright, P. A. 1945. Factors affecting in vitro ovulation in the frog. J. Exper. Zool. 100:565.

and Hisaw, F. L. 1946. Effect of mammalian pituitary gonadotrophins on ovulation in the frog, Rana pipiens. Endocrinology. 39:247.

The Development of the Gametes or Sex Cells

A. General considerations

B. Controversy regarding germ-cell origin

C. Maturation (differentiation) of the gametes

1. General considerations

2. Basic structure of the definitive sex cell as it starts to mature or differentiate into the male meiocyte (i.e., the spermatocyte) or the female meiocyte (i.e., the oocyte)

3. Nuclear maturation of the gametes

a. General description of chromatin behavior during somatic and meiotic mitoses

b. Reductional and equational meiotic divisions and the phenomenon of crossing over

c. Stages of chromatin behavior during the meiotic prophase in greater detail

1) Leptotene (leptonema) stage

2) Zygotene or synaptene (zygonema) stage

3) Pachytene (pachynema) stage

4) Diplotene (diplonema) stage

5) Diakinesis

d. Peculiarities of nuclear behavior in the oocyte during meiosis; the germinal vesicle

e. Character of the meiotic (maturation) divisions in the spermatocyte compared with those of the oocyte

1) Dependent nature of the maturation divisions in the female meiocyte

2) Inequality of cytoplasmic division in the oocyte

f. Resume of the significance of the meiotic phenomena

4. Cytosomal (Cytoplasmic) maturation of the gametes

a. General aspects of the cytoplasmic maturation of the gametes

b. Morphogenesis (spermiogenesis) ( sperm ioteleosis) of the sperm

1) Types of sperm

2) Structure of a flagellate sperm

a) Head

b) Neck

c) Connecting body or middle piece

d) Flagellum

3) Spermiogenesis or the differentiation of the spermatid into the morphologically differentiated sperm

a) Golgi substance and acroblast; formation of the acrosome

b) Formation of the post-nuclear cap

c) Formation of the proximal and distal centrioles; axial filament

d) Mitochondrial material and formation of the middle piece of the sperm

e) The cytoplasm, axial filament, mitochondria, and tail formation

c. Cytoplasmic differentiation of the egg

1) Types of chordate eggs

a) Homolecithal (isolecithal) eggs

b) Telolecithal eggs

2) Formation of the deutoplasm

3) Invisible morphogenetic organization within the cytoplasm of the egg

4) Polarity of the egg and its relation to body organization and bilateral symmetry of the mature egg

5) Membranes developed in relation to the oocyte; their possible sources of origin

a) Chorion in St ye la

b) Egg membranes of Amphioxus

c) Vitelline membrane and zona radiata of elasmobranch fishes

d) Zona radiata of teleost fishes

e) Vitelline membrane (zona radiata) in amphibia

f) Zona radiata (zona pellucida) of the reptile oocyte

g) Vitelline membrane (zona radiata) of the hen’s egg

h) Membranes of the mammalian oocyte

5. Physiological maturation of the gametes

a. Physiological differentiation of the sperm

b. Physiological ripening of the female gamete

D. Summary of egg and sperm development

A. General Considerations

In the two preceding chapters the conditions which prepare the male and female parents for their reproductive responsibilities are considered. This chapter is devoted to changes which the male and female germ cells must experience to enable them to take part in the processes involved in the reproduction of a new individual.

The gamete is a highly specialized sex cell or protoplasmic entity so differentiated that it is capable of union (fertilization; syngamy) with a sex cell 6T the opposite sex to form the zygote from which the new individual arises. TE^ process of differentiation whereby the primitive germ cell is converted into the mature gamete is called the maturation of the germ cell.

The main events which culminate in the fully-developed germ cell are possible only after the primitive or undifferentiated germ cell has reached a certain condition known as the definitive state. When this stage is reached, the germ cell has acquired the requisite qualities which make it possible for it to differentiate into a mature gamete. Before the definitive state is reached, germ cells pass through an eventful history which involves:

( 1 ) their so-called “origin” or first detectable appearance among the other

cells of the developing body, and

(2) their migration to the site of the future ovary or testis.

After entering the developing substance of the sex gland, the primitive germ cells experience a period of multiplication. If the sex gland is that of the male, these undifferentiated sex cells are called spermatogonia; if female, they are known as oogonia.

B. Controversy Regarding Germ-cell Origin

The problems of germ-cell origin in the individual organism and of the continuity of the germ plasm from one generation to the next have long been matters of controversy. Great interest in these problems was aroused by the ideas set forth by Waldeyer, Nussbaum, and Weismann during the latter part of the nineteenth century. Waldeyer, 1870, as a result of his studies on the chick, presented the “germinal epithelium” hypothesis, which maintains that the germ cells arise from the coelomic epithelium covering the gonad. Nussbaum, 1880, championed the concept of the extra-gonadal origin of the germ cells. According to this view, derived from his studies on frog and trout development, the germ cells arise at an early period of embryonic development outside the germ-gland area and migrate to the site and into the substance of the germ gland.

At about this time the speculative writings of August Weismann aroused great interest. In 1885 and 1892 Weismann rejected the popular Darwinian theory of pangenesis, which held that representative heredity particles or “gemmules” passed from the body cells (i.e., soma cells) to the germ cells and were there stored in the germ cells to develop in the next generation (Weismann, 1893). In contrast to this hypothesis he emphasized a complete independence of the germ plasm from the somatoplasm. He further suggested that the soma did not produce the germ plasm as implied in the pangenesis theory, but, on the contrary, the soma resulted from a differentiation of the germ plasm.

According to the Weismannian view, the germ plasm is localized in the chromosomal material of the nucleus. During development this germ plasm is segregated qualitatively during successive cell divisions with the result that the cells of different organs possess different determiners. However, the nuclear germ plasm (Keimplasma) is not so dispersed or segregated in those cells which are to become the primitive sex cells; they receive the full complement of the hereditary determiners for the various cells and organs characteristic of the species. Thus, it did not matter whether the germ cells were segregated early in development or later, so long as the nucleus containing all of the determinants for the species was kept intact. In this manner the germ plasm, an immortal substance, passed from one generation to the next via the nuclear germ plasm of the sex or germ cells. This continuity of the nuclear germ plasm from the egg to the adult individual and from thence through the germ cells to the fertilized egg of the next generation, constituted the Weismann “Keimbahn” or germ-track theory. The soma or body of any particular generation is thus the “trustee” for the germ plasm of future generations.

The Weismannian idea, relative to the qualitative segregation of the chromatin materials, is not tenable for experimental and cytological evidence suggests that all cells of the body contain the same chromosomal materials. However, it should be pointed out that Weismann was one of the first to suggest that the chromosome complex of the nucleus acts as a repository for all of the hereditary characteristics of the species. This suggestion relative to the role of the nucleus has proved to be one of the main contributions to biological theory in modern times.

Fig. 60. Representation of the concept of the early embryonic origin of the primordial germ cells and their migration into the site of the developing germ gland. (A-C are adapted from the work of Allen, Anat. Anz. 29, on germ cell origin in Chrysemys; D-F are diagrams based on the works of Dustin, Swift, and Dantschakoff, etc., referred to in the table of germ-cell origins included in the text.) (A~C) Germ cells arising within the primitive entoderm and migrating through the dorsal mesentery to the site of the primitive gonad, shown in (D), where they become associated in or near the germinal epithelium overlying the internal mesenchyme of the gonad. (E, F) Increase of the primitive gonia within the developing germ gland, with a subsequent migration into the substance of the germ gland of many germ cells during the differentiation of sex.

Fig. 61 Diagrammatic representation of the process of chromatin diminution in the nematode worm, Ascaris cquorum (A. megalocephala), and of the “Keimbahn” (in black, E). One daughter cell shown by the four black dots of each division of the germcell line (i.e., the stem-cell line) is destined to undergo chromatin diminution up to the 16-cell stage. At the 16-cell stage, the germ-cell line ceases to be a stem cell (e.g., P 4 ), and in the future gives origin only to sperm cells (E). (A-D, copied from King and Beams (’38); E, greatly modified from Durkin (’32).)

Animal pole of the cleaving egg (A) is toward the top of the page. (B) Metaphase conditions of the second cleavage. Observe the differences in the cleavage planes of the prosomatic cell, Si, and that of the stem cell, P,. (C) Anaphase of the second cleavage of Si. Observe that the ends of the chromosomes in this cleaving cell are left behind on the spindle. (D) It is to be noted that the ends of the chromosomes are not included in the reforming nuclei of the two daughter cells of S,. thus effecting a diminution of the chromatin substance. In P„ P., and E.M. ST. of (D), the chromosomes are intact. E.M. ST. = second prosomatic cell. MST = mesoderm-stomodaeal cell.

A second contributory concept to the germ-cell (germ-plasm) theory was made by Nussbaum, 1880; Boveri, 1892, ’10, a and b, and others. These investigators emphasized the possibility that a germinal cytoplasm also is important in establishing the germ plasm of the individual. A considerable body of observational and experimental evidence derived from embryological studies substantiates this suggestion. Consequently, the modern view of the germ cell (germ plasm) embodies the concept that the germ cell is composed of the nucleus as a carrier of the hereditary substances or genes and a peculiar, specialized, germinal cytoplasm. The character of the cytoplasm of the germ cell is the main factor distinguishing a germ cell from other soma cells.

The matter of a germinal cytoplasm suggests the necessity for a segregation of the germinal plasm in the form of specific germ cells during the early development of the new individual. As a result, great interest, as well as controversy, has accumulated concerning this aspect of the germ-cell problem: namely, is there a separate germinal plasm set apart in the early embryo which later gives origin to the primordial germ cells, and the latter, after migration (fig. 60), to the definitive gonia; or according to an alternative view, do some or all of the definitive germ cells arise from differentiated or relatively undifferentiated soma cells? The phrase primary primordial germ cells often is used to refer to those germ cells which possibly segregate early in the embryo, and the term secondary primordial germ cells is employed occasionally to designate those which may arise later in development.

The dispute regarding an early origin or segregation of the germinal plasm in the vertebrates also occurs relative to their origin in certain invertebrate groups, particularly in the Coelenterata and the Annelida (Berrill and Liu, ’48). In other fnvertebrata, such as the dipterous insects and in the ascarid worms, the case for an early segregation is beyond argument. An actual demonstration of the continuity of the Keimbahn from generation to generation is found in Ascaris megalocephala described by Boveri in 1887. (See Hegner, ’14, Chap. 6.) In this form the chromatin of the somatic cells of the body undergoes a diminution and fragmentation, whereas the stem cells, from which the germ cells are ultimately segregated at the 16-cell to 32-cell stage, retain the full complement of chromatin material (fig. 61). Thus, one cell of the 16-cell stage retains the intact chromosomes and becomes the progenitor of the germ cells. The other 15 cells will develop the somatic tissues of the body. The diminution of the chromatin material in this particular species has been shown to be dependent upon a certain cytoplasmic substance (King and Beams, ’38).

In some insects the Keimbahn also can be demonstrated from the earliest stages of embryonic development. In these forms a peculiar polar plasm within the egg containing the so-called “Keimbahn determinants’’ (Hegner, ’14, Chap. 5) always passes into the primordial germ cells. That is, the ultimate formation and segregation of the primordial germ cells are the result of nuclear migration into this polar plasm and the later formation of definite cells from this plasm (fig. 62). The cells containing this polar plasm are destined thus to be germ cells, for they later migrate into the site of the developing germ glands and give origin to the definitive germ cells.

Many investigators of the problem of germ-cell origin in the vertebrate group of animals have, after careful histological observation, described the germ cells as taking their origin from among the early entodermal cells (see table, pp. 121-124). On the other hand, other students have described the origin of the germ cells from mesodermal tissue — some during the early period of embryonic development, while others suggest that the primordial germ cells arise from peritoneal (mesodermal) tissue at a much later time.

In more recent years much discussion has been aroused relative to the origin of the definitive germ cells in mammals, particularly in the female. According to one view the definitive germ cells which differentiate into the mature gametes of the ovary arise from the germinal epithelium (peritoneal covering) of the ovary during each estrous cycle (figs. 39A, 63, 64). For example, Evans and Swezy (’31) reached the conclusion that all germ cells in the ovaries of the cat and dog between the various reproductive periods degenerate excepting those which take part in the ovulatory phenomena. Accordingly, the new germ cells for each cycle arise from the germinal epithelium. A similar belief of a periodic proliferation of new germ cells by the germinal epithelium has been espoused by various authors. (See Moore and Wang, ’47; and Pincus, ’36, Chap. II.) More recent papers have presented views which are somewhat conflicting. Vincent and Dornfeld, ’48 (fig. 63) concluded that there is a proliferation of germ cells from the germinal epithelium of the young rat ovary, while Jones (’49), using carbon granules as a vital-marking technic, found no evidence of the production of ova from the germinal epithelium in rat ovaries from 23 days until puberty. In the adult rat, she concedes that a segregation of a moderate number of oogonia from the germinal epithelium is possible.

Fio. 62. Early development of the fly, Miflj/o/’. {A) Miastor metraloas. (B) Miastor americana. In (A) the division figures I and III (II not shown) are undergoing chromatin diminution, while nucleus IV divides as usual. In (B) one segregated germ is shown at the pole of the egg. This cell will give origin to the germ cells. Other division figures experiencing chromatin diminution.

Fig. 63. Cells proliferating inward from germinal epithelium of the ovary of a oneday-old rat. Observe cords of cells (Pfliiger’s cords) projecting into the ovarian substance. Within these cords of cells are young oogonia. (After Vincent and Dornfeld, ’48.)

Fig. 64. Cellular condition near the surface of the ovary of a young female opossum. This section of the ovary is near the hilar regions, i.e., near the mesovarium. Observe young oocytes and forming Graafian follicles. Primitive germ cells may be seen near the germinal epithelium.

Aside from the above studies of carefully-made, histological preparations relative to the time and place of origin of the primordial and definitive germ cells, many experimental attacks have been made upon the problem. Using an x-ray-sterilization approach, Parkes (’27); Brambell, Parkes and Fielding (’27, a and b), found that the oogonia and oocytes of x-rayed ovaries of the mouse were destroyed. In these cases new germ cells were not produced from the germinal epithelium. Brambell (’30) believed that the destruction of the primitive oogonia was responsible for the lack of oogenesis in these x-rayed ovaries. However, this evidence is not conclusive, for one does not know what injurious effects the x-rays may produce upon the ability of the various cells of the germinal epithelium to differentiate.

An experimental study of the early, developing, amphibian embryo relative to the origin of the primordial germ cells also has been made by various investigators. Bounoure (’39) applied a vital-staining technic to certain anuran embryos. The results indicate that the germinal plasm in these forms is associated with the early, entodermal, organ-forming area located at the vegetal pole of the cleaving egg. This germinal plasm later becomes segregated into definite cells which are associated with the primitive entoderm. At a later period these cells migrate into the developing germ gland or gonad. On the other hand, experimental studies of the urodele embryo indicate that the early germinal plasm is associated with the mesoderm (Humphrey, ’25, ’27; Nieuwkoop, ’49). Existence of an early germinal plasm associated with the entoderm in the Anura and with the mesoderm in the Urodela thus appears to be well established for the amphibia.

The evidence derived from amphibian studies together with the observations upon the fish group presented in the table (see pp. 121-124) strongly suggests that an early segregation of a germinal plasm (germ cells) occurs in these two major vertebrate groups. Also, in birds, the experimental evidence presented by Benoit (’30), Goldsmith (’35), and Willier (’37) weighs the balance toward the conclusion that there is an early segregation of germ cells from the entoderm. Similar conditions presumably are present in reptiles. In many vertebrates, therefore, an early segregation of primordial germ cells and their ultimate migration by: (1) active ameboid movement, (2) by the shifting of tissues, or (3) through the blood stream (see table, pp. 121-124) to the site of the developing gonad appears to be well substantiated.

The question relative to the origin of the definitive ova in the mammalian ovary is still in a confused state as indicated by the evidence presented above and in the table on pp. 121-124. Much more evidence is needed before one can rule out the probability that the primordial germ cells are the progenitors of the definitive germ cells in the mammals. To admit the early origin of primordial germ cells on the one hand, and to maintain that they later disappear to be replaced by a secondary origin of primitive germ cells from the germinal epithelium has little merit unless one can disprove the following position, to wit: that, while some of the primordial germ cells undoubtedly do degenerate, others divide into smaller cells which become sequestered within or immediately below the germinal epithelium of the ovary and within the germinal epithelium of the seminiferous tubules of the testis, where they give origin by division to other gonial cells. Ultimately some of these primitive gonia pass on to become definitive germ cells.

However, aside from the controversy whether or not the primordial germ cells give origin to definitive germ cells, another aspect of the germ-cell problem emphasizing the importance of the primitive germ cells is posed by the following question: Will the gonad develop into a functional structure without the presence of the primordial germ cells? Experiments performed by Humphrey (’27) on Ambystoma, and the above-mentioned workers — Benoit (’30), Goldsmith (’35), and Willier (’37) — on the chick, suggest that only sterile gonads develop without the presence of the primordial germ cells.

Finally, another facet of the germ-cell problem is this; Are germ cells completely self differentiating? That is, do they have the capacity to develop by themselves; or, are the germ cells dependent upon surrounding gonadal tissues for the influences which bring about their differentiation? All of the data on sex reversal in animals, normal and experimental (Witschi, in Allen, Danforth, and Doisy, ’39), and of other experiments on the development of the early embryonic sex glands (Nieuwkoop, ’49) suggest that the germ cells are not self differentiating but are dependent upon the surrounding tissues for the specific influences which cause their development. Furthermore, the data on sex reversal shows plainly that the specific chromosome complex (i.e., male or female) within the germ cell does not determine the differentiation into the male gamete or the female gamete, but rather, that the influences of the cortex (in the female) and the medulla (in the male) determine the specific type of gametogenesis.

The table given on pp. 121-124 summarizes the conclusions which some authors have reached concerning germ-cell origin in many vertebrates. It is not complete; for more extensive reviews of the subject see Everett (’45), Heys (’31 ), and Nieuwkoop (’47, ’49).

Species

Place of Origin, etc.

A uthor

Entospheniis wihieri (brook lamprey)

Germ cells segregate early in the embryo; definitive germ cells derived from “no other source”

Okkelberg. 1921.

J. Morphol. 35

Petromyzon marinus unicolor (lake lamprey)

Definitive germ cells derive from: a) early segregated cells, primordial germ cells, and b) later from coelomic epithelium. Suggests that primordial germ cells may induce germ-cell formation in peritoneal epithelium

Butcher. 1929.

Biol. Bull. 56

Squalus acanthias

Germ cells segregate from primitive entoderm; migrate via the mesoderm into site of the developing gonad

Woods. 1902.

Am. J. Anat. 1

Amia and

Lepidosteiis

Germ cells segregate early from entoderm; continue distinct and migrate into the developing gonad via the mesoderm (see fig. 60)

Allen. 1911.

J. Morphol. 22

Species

Place of Origin, etc.

Author

Lophius piscatorius

Germ cells segregate from primitive entoderm; migrate through mesoderm to site of gonad; migration part passive and part active

Dodds. 1910.

J. Morphol. 21

Fundulus

heteroclitus

Germ cells segregate from peripheral entoderm lateral to posterior half of body; migrate through entoderm and mesoderm to the site of the developing gonad

Richards and Thompson. 1921.

Biol. Bull. 40

Cottus bairdii

Primordial germ cells derive from giant cells, in the primitive entoderm; migrate through the lateral mesoderm into the site of the developing gonad

Hann. 1927.

J. Morphol. 43

Lebistes reticulatus (guppy)

Germ cells segregate early in development; first seen in the entodermmesoderm area; migrate into the sites of the developing ovary and testis, giving origin to the definitive germ cells

Goodrich, Dee, Flynn, and Mercer. 1939. Biol. Bull. 67

Rana tempo raria

Germ cells segregate from primitive entoderm; migrate into developing genital glands

Witschi. 1914.

Arch. f. mikr. Anat. 85

Rana temporaria

Primordial germ cells from entoderm discharged at first spawning. Later, the definitive germs cells of adults originate from peritoneal cells

Gatenby. 1916.

Quart. J. Micr. Sc. 61

Rana catesbiana

Primordial germ cells segregate from primitive entoderm; definitive germ cell derives from primordial cells according to author’s view but admits possibility of germinal epithelium origin

Swingle. 1921.

J. Exper. Zool. 32

Rana temporaria, Triton alpestris, Bufo vulgaris

Primordial germ cells segregate from entoderm

Bounoure. 1924. Compt. rend. Acad. d. Sc. 178, 179

Rana sylvatica

Primordial germ cells originate from entoderm and migrate into the developing gonads. They give origin to the definitive sex cells

Witschi. 1929.

J. Exper. Zool. 52

H emidactylium scutatum

Primordial germ cells arise in mesoderm between somite and lateral plate; move to site of gonad by shifting of tissues

Humphrey. 1925.

J. Morphol. 41

Species

Place of Origin, etc.

Author

Ambystorna

maculatiirn

Most germ cells somatic in origin from germinal epithelium, although a few may come from primordial germ cells of entodermal origin

McCosh. 1930.

J. Morphol. 50

Triton, and Ambystorna mexicanum

Germ cells differentiate from lateral plate mesoderm

Nieuwkoop. 1946.

Arch. Neerl. de zool. 7

Chrysemys

marginata

(turtle)

Primordial germ cells from entoderm; most of definitive germ cells arise from peritoneal cells

Dustin. 1910.

Arch, biol., Paris. 25

Sternotherus

odoratus (turtle)

Primordial cells segregate early from entoderm; later definitive cells derive from these and from peritoneal epithelium

Risley. 1934.

J. Morphol. 56

Callus ( domesticus) gallus (chick)

Germ cells arise from primitive cells in entoderm of proamnion area and migrate by means of the blood vessels to the site of the developing gonad. Definitive germ cells of sex cords and later seminiferous tubules derive from primordial germ cells

Swift. 1914, 1916.

Am. J. Anat. 15, 20

Callus (domesticus) gallus (chick)

Primordial germ cells arise from enlodermal cells

Dantschakoff. 1931. Zeit. f. Zellforsch., mikr. Anat. 15

Chick and albino rat

Early primordial cells degenerate; definitive cells from peritoneal epithelium

Firket. 1920.

Anat. Rec. 18

Didelpliys

virginiana

(opossum)

Germ cells arise from germinal epithelium

Nelsen and Swain. 1942.

J. Morphol. 71

Mils musculiis (mouse)

Oogonia derived from primordial germ cells; spermatogonia from epithelial cells of testis cords

Kirkham. 1916.

Anat. Rec. 10

Mus musculus (mouse)

Primordial germ cells of ovary arise from germinal epithelium during development of the gonads. These presumably give origin to the definitive sex cells

Brambell. 1927.

Proc. Roy. Soc. London, s.B. 101

Felis dornestica (cat)

Primordial cells segregate early but do not give origin to definitive germ cells which derive from germinal epithelium

de Winiwarter and Sainmont. 1909. Arch, biol., Paris. 24

Species

Place of Origin, etc.

A uthor

Felis dome Stic a (cat)

Definitive ova derived from germinal epithelium of the ovary at an early stage of gonad development

Kingsbury. 1938.

Am. J. Anat. 15

Cavia porcellus (guinea pig)

Primordial germ cells from entodermal origin degenerate; the primordial germ cells derived from the germinal epithelium give rise to the definitive germ cells in the testis

Bookkout. 1937.

Zeit. f. Zellforsch, mikr. Anat. 25

Homo sapiens (man)

Primordial germ cells found in entoderm of yolk sac; migrate by ameboid movement into developing gonad

Witschi. 1948. Carnegie Inst., Washington Publ. 575. Contrib. to Embryol.' 32

C. Maturation (Differentiation) of the Gametes

1. General Considerations

Regardless of their exact origin definitive germ cells as primitive oogonia or very young oocytes are to be found in or near the germinal epithelium in the ovaries of all vertebrates in the functional condition (figs. 39B, 64). In the testis, the primitive spermatogonia are located within the seminiferous tubules as the germinal epithelium, in intimate association with the basement membrane of the tubule (figs. 65, 66).

The period of coming into maturity (maturation) of the gametes is a complicated affair. It involves profound transformations of the cytoplasm, as well as the nucleus. Moreover, a process of ripening or physiological maturing is necessary, as well as a morphological transformation. The phrase “maturation of the germ cells” has been used extensively to denote nuclear changes. However, as the entire gamete undergoes morphological and physiological change, the terms nuclear maturation, cytosomal maturation, and physiological maturation are used in the following pages to designate the various aspects of gametic development.

One of the most characteristic changes which the germ cell experiences during its maturation into a mature gamete is a reduction of chromatin material. Because of this, the germ cell which begins the maturing process is called a meiocyte. This word literally means a cell undergoing diminution and it is applied to the germ cell during meiosis or the period in which a reduction in the number of chromosomes occurs. The word haplosis is a technical name designating this reduction process.

The word meiocyte thus is a general term applicable to both the developing

MATURATION OF GAMETES

125

Fig. 65. Semidiagrammatic representation of a part of the seminiferous tubule of the

cat testis.

male and female germ cells. On the other hand, the word spermatocyte is given to the developing male gamete during the period of chromosome diminution, whereas the word oocyte is applied to the female gamete in the same period. When, however, the period of chromosome diminution is completed and the chromosome number is reduced to the haploid condition, the developing male gamete is called a spermatid while the female gamete is referred to as an ootid or an egg. {Note: the word egg is applied often to the female gamete during the various stages of the oocyte condition as well as after the maturation divisions have been accomplished.)

The reduction of chromatin material is not the only effect which the meiotic process has upon the chromatin material, or possibly upon the developing cytosomal structures as well. This fact will become evident during the descriptions below concerning the meiotic procedures.

Another prominent feature of the gametes during the meiocyte period is their growth or increase in size. This growth occurs during the first part of the meiotic process when the nucleus is in the prophase condition and it involves both nucleus and cytoplasm. The growth phenomena are much more pronounced in the oocyte than in the spermatocyte. Due to this feature of growth, the oocyte and spermatocyte also are regarded as auxocytes, that is growing cells, a name introduced by Lee, 1897. The words meiocyte and auxocyte thus refer to two different aspects of the development of the oocyte and the spermatocyte.

126

THE DEVELOPMENT OF THE GAMETES OR SEX CELLS

2. Basic Structure of the Definitive Sex Cell as It Starts to Mature or Differentiate into the Male Meiocyte (i.e., the Spermatocyte) or the Female Meiocyte (i.e., the Oocyte)

The definitive sex cells of both sexes have a similar cytological structure. The component parts are (fig. 68):

(1) nucleus,

(2) investing cytoplasm,

(3) idiosome,

(4) Golgi substance, and

(5) chondriosomes.

The nucleus is vesicular and enlarged, and the nuclear network of chromatin may appear reticulated. A large nucleolus also may be visible. The investing cytoplasm is clearer and less condensed in appearance than that of ordinary cells. The idiosome (idiozome) is a rounded body of cytoplasm which, in many animal species, takes the cytoplasmic stain more intensely than the surrounding cytoplasm. Within the idiosome it is possible to demonstrate the centrioles as paired granules in some species. Surrounding the idiosome are various elements of the Golgi substance, and near both the idiosome and Golgi elements, is a mass of chondriosomes (mitochondria) of various sizes and shapes. The idiosome and its relationship with the Golgi material, the mitochondria, and the centrioles varies considerably in different species of animals.

Much discussion has occurred concerning the exact nature of the idiosome. Some investigators have been inclined to regard the surrounding Golgi substance as a part of the idiosome, although the central mass of cytoplasm containing the centrioles is the “idiosome proper” of many authors (Bowen, ’22). Again, when the maturation divisions of the spermatocyte occur, the idiosome and surrounding Golgi elements are broken up into small fragments. However, in the spermatids the Golgi pieces (dictyosomes) are brought together once more to form a new idiosome-like structure, with the difference that the latter “seems never to contain the centrioles” (Bowen, ’22). It is, therefore, advisable to regard the idiosome as being separated into its various components during the maturation divisions of the spermatocyte and to view the reassemblage of Golgi (dictyosomal) material in the spermatid as a different structure entirely. This new structure of the spermatid is called the acroblast (Bowen, ’22; Leuchtenberger and Schrader, ’50). (See fig. 68B.) A similar breaking up of the idiosome occurs in oogenesis (fig. 68F, G). However, all meiocytes do not possess a typical idiosome. This fact is demonstrated in insect spermatocytes, where the idiosomal material is present as scattered masses to each of which some Golgi substance is attached.

The various features which enter into the structure of the definitive germ

MATURATION OF GAMETES

127

cell do not behave in the same way in each sex during gametic differentiation. While the behaviors of the chromatin material in the male and female germ cells closely parallel each other (fig. 67), the other cytosomal features follow widely divergent pathways, resulting in two enormously different gametic entities (fig. 68A-H).

Fig. 66. Section of part of a seminiferous tubule of human testis. (Redrawn from Gatenby

and Beams, ’35.)

SECOND M E lOT I C DIVISION

SPERMATID OR EGG

Fig. 67. Diagrammatic representation of the nuclear changes occurring during meiosis in spermatocyte and oocyte. Six chromosomes, representing three homologous pairs, are used. Observe the effects of the crossing over of parts of chromatids. The diplotene condition of oocyte depicted by arrows and the enlarged nucleus. The haploid condition is shown in each of the spermatids or in the egg and its three polocytes.

128

Fig. 68. Possible fate of the primitive meiocyte and its cytoplasmic inclusions when exposed to testicular or ovarian influences. Particular attention is given to the idiosome. Under male-forming influences the idiosome components are dispersed during the maturation divisions and are reassembled into three separate component structures, namely, (1) Acroblast of Golgi substance, (2) centriolar bodies, and (3) mitochondrial bodies (see B). Each of these structures, together with the post-nuclear granules of uncertain origin, play roles in spermatogenesis as shown. Under ovarian influences the idiosome is dispersed before the maturation divisions. The Golgi substance and mitochondria play (according to theory, see text) their roles in the formation of the deutoplasm.

129

130

THE DEVELOPMENT OF THE GAMETES OR SEX CELLS

3. Nuclear Maturation of the Gametes

Most of our information concerning the maturation of the nucleus pertains to certain aspects of chromosome behavior involved in meiosis, particularly the reduction of the chromosome number together with some activities of “crossing over” of materials from one chromosome to another. But our information is vague relative to other aspects of nuclear development. For example, we know little about the meaning of growth and enlargement of the nucleus as a whole during meiosis, an activity most pronounced in the oocyte. Nor do we know the significance of nuclear contraction or condensation in the male gamete after meiosis is completed. Therefore, when one considers the nuclear maturation of the gametes, it is necessary at this stage of our knowledge to be content mainly with observations of chromosomal behavior.

a. General Description of the Chromatin Behavior During Somatic and

Meiotic Mitosis

As the maturation behavior of the chromatin components in the spermatocyte and oocyte are similar, a general description of these activities is given in the following paragraphs. Before considering the general features and details of the actions of the chromosomes during meiosis, it is best to recall some of the activities which these structures exhibit during ordinary somatic and gonial mitoses.

Cytological studies have shown that the chromosomes, in most instances, are present in the nucleus in pairs, each member of a pair being the homologue or mate of the other. Homologous chromosomes, therefore, are chromosomal pairs or mates. During the prophase condition in ordinary somatic and gonial mitoses, the various chromosomal mates do not show an attraction for each other. A second feature of the prophase stage of ordinary cell division is that each chromosome appears as two chromosomes. That is, each chromosome is divided longitudinally and equationally into two chromosomes. At the time when the metaphase condition is reached and the chromosomes become arranged upon the metaphase plate, the two halves or daughter chromosomes of each original chromosome are still loosely attached to each other. However, during anaphase, the two daughter chromosomes of each pair are separated and each of the two daughter nuclei receives one of the daughter chromosomes. Reproduction of the chromatin material and equational distribution of this material into the two daughter cells during anaphase is a fundamental feature of the ordinary type of somatic and gonial mitoses. The two daughter nuclei are thus equivalent to each other and to the parent nucleus. In this way, chromosomal equivalence is passed on ad infinitum through successive cell generations.

On the other hand, a different kind of chromosomal behavior is found during meiosis, which essentially is a specialized type of mitosis, known as a meiotic mitosis. In one sense it is two mitoses or mitotic divisions with only

Fig. 69. Steps in spermatogenesis in the grasshopper. In the center of the chart is represented a longitudinal section of one of the follicles of a grasshoper testis with its various regions of spermatogenic activity. In the upper right of the chart the apical-cell complex is depicted with its central apical cell, spermatogonia, and surrounding epithelial cells. The primary spermatogonia lie enmeshed between the extensions of the apical cell and the associations of these extensions with the surrounding epithelial elements of the complex. (Also see Wenrich, 1916, Bull. Mus. Comp. Zool. Harvard College, 60.)

131

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THE DEVELOPMENT OF THE GAMETES OR SEX CELLS

one prophase; that is, two metaphase-anaphase separations of chromosomes preceded by a single, peculiar prophase. The peculiarities of this meiotic prophase may be described as follows: As the prophase condition of the nucleus is initiated, an odd type of behavior of the chromosomes becomes evident — a behavior which is entirely absent from ordinary somatic mitosis: namely, the homologous pairs or mates begin to show an attraction for each other and they approach and form an intimate association. This association is called synapsis (figs. 67, 69, zygotene stage). As a result, the two homologous chromosomes appear as one structure. As the homologous chromosomes are now paired together and superficially appear as one chromosome, the number of “chromosomes” visible at this time is reduced to one-half of the ordinary somatic or diploid number. However, each “chromosome” is in reality two chromosomes and, therefore, is called a bivalent or twin chromosome.

While the homologous chromosomes are intimately associated, each mate reproduces itself longitudinally just as it would during an ordinary mitosis (fig. 67, pachytene stage). (The possibility remains that this reproduction of chromatin material may have occurred even before the synaptic union.) Hence, each bivalent chromosome becomes transformed into four potential chromosomes, each one of which is called a chromatid. This group of chromatids is, collectively speaking, a tetrad chromosome. (As described below, interchange of material or crossing over from one chromatid to another may take place at this time.) As a result of these changes, the nucleus now contains the haploid number of chromosomes, (i.e., half of the normal, diploid number) in the form of tetrads (fig. 67, pachytene stage). However, as each tetrad represents four chromosomes, actually there is at this time twice the normal number of chromosomes present in the nucleus (fig. 67; compare leptotene, pachytene, diplotene and diakinesis).

The next step in meiosis brings about the separation of the tetrad chromosome into its respective chromatids and it involves two divisions of the cell. These divisions are known as meiotic divisions. As the first of these two divisions begins, the tetrad chromosomes become arranged in the mid- or metaphase plane of the spindle. After this initial step, the first division of the cell occurs, and half of each tetrad (i.e., a dyad) passes to each pole of the mitotic spindle (fig. 67, first meiotic division). Each daughter cell (i.e., secondary spermatocyte or oocyte) resulting from the first maturation (meiotic) division thus contains the haploid or reduced number of chromosomes in the dyad condition, each dyad being composed of two chromatids. A resting or interphase nuclear condition occurs in most spermatocytes, following the first maturation division, but in the oocyte it usually does not occur (fig. 69, interkinesis).

As the second maturation division is initiated, the dyads become arranged on the metaphase plate of the mitotic spindle. As division of the cell proceeds, half of each dyad (i.e., a monad) passes to the respective poles of the spindle

MATURATION OF GAMETES

133

(fig. 67, second meiotic division; fig. 69). As a result of these two divisions, each daughter cell thus contains the haploid or reduced number of chromosomes in the monad (monoploid) condition (fig. 67, spermatid or egg). Meiosis or chromatin diminution is now an accomplished fact.

It is to be observed, therefore, that the meiotic phenomena differ from those of ordinary mitosis by two fundamental features:

(1) In meiosis there is a conjugation (synapsis) of homologous chromosomes during the prophase stage, and while synapsed together each of the homologues divides equationally; and

(2) following this single prophase of peculiar character, two divisions follow each other, separating the associated chromatin threads.

While the meiotic prophase is described above as a single prophase preceding two metaphase-anaphase chromosome separations, it is essentially a double prophase in which the process of synapsis acts to suppress one of the equational divisions normally present in a mitotic division; a synapsed or double chromosome, therefore, is substituted for one of the longitudinal, equational divisions which normally appears during a somatic prophase. It is this substitution which forms the basis for the reduction process, for two mitotic divisions follow one after the other, preceded by but one equational splitting, whereas in ordinary mitosis, one equational splitting of the chromosomes always precedes each mitotic division.

b. Reductional and Equational Meiotic Divisions and the Phenomenon of

Crossing Over

In the first meiotic division (i.e., the first maturation division), if the two chromatids which are derived from one homologous mate of the tetrad are separated from the two chromatids derived from the other homologous mate the division is spoken of as reductional or disjunctional. In this case the two associated chromatids of each dyad represent the original chromosome which synapsed at the beginning of meiotic prophase (fig. 67, tetrads B and C, first meiotic division). If, however, the separation occurs not in the synaptic plane but in the equational plane, then the two associated chromatids of each dyad come, one from one synaptic mate and one from the other; such a division is spoken of as an equational division (fig. 67, tetrad A, first meiotic division). There appears to be no fixity of procedure relative to the separation of the tetrads, and great variability occurs. However this may be, one of the two meiotic divisions as far as any particular tetrad is concerned is disjunctional (reductional) and the other is equational, at least in the region of the kinetochore (see p. 135 and fig. 70). If the first division is reductional, the second is equational and vice versa. Disjunction in the first maturation division is often referred to as pre-reduction, while that in the second maturation division is called post-reduction.

Fig. 70. Some of the various possibilities which may occur as a result of the exchanges of parts of chromatids during the crossing-over phenomena associated with meiosis. Two chiasmata (singular, chiasma) are shown in (A), (C), (E). Observe that homologous chromosome A has split equationally into chromatids A and A', while homologous chromosome B has divided equationally into B and B'. The resulting interchanges between respective chromatids of the original homologous chromosomes are shown in (B), (D), (F). The kinetochore (place of spindle-fiber attachment) is indicated by the oval or circular area to the left of the chromatids. (Modified from White: Animal Cytology and Evolution, London, Cambridge University Press, 1943.)

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MATURATION OF GAMETES

135

The foregoing statement regarding disjunctional and equational divisions should be considered in the light of the phenomenon of crossing over. In the latter process, a gene or groups of genes may pass from one chromatid to the other and vice versa during their association at the four strand stage (fig. 70). In the region of the centromere or kinetochore (i.e., the point) of the achromatic, spindle-fiber attachment) and nearby regions, cross overs are thought not to occur (fig. 70, kinetochore). Consequently, in the regions of the kinetochore, the statements above regarding disjunctional and equational divisions of the chromosomes appear to be correct. However, the terms disjunctional and equational may mean little in other regions of the chromosomes of a tetrad during the meiotic divisions. For example, let us assume as in fig. 70 (see also fig. 67), that we have chromatids A and A', B and B', A and B representing the original homologues or synaptic chromosomes which have divided into these chromatids respectively. Then during the tetrad stage of association or slightly before, let us assume that there has been a crossing over of genes from chromatid A to chromatid B and from chromatid B to chromatid A in a particular area (fig. 70A). (It is to be observed that chromatids A' and B' are not involved in this particular instance.) Further, let us assume that AA' and BB' as a whole are separated at the first maturation division, the kinetochore and immediate regions would represent a disjunctional division, but for the particular area where crossing over is accomplished, the division would be equational (fig. 70A, B; central portions of chromatids A and B in fig. 70B). Thus, it would be for other regions where cross overs may have occurred. Other cross-over possibilities are shown in fig. 70C-F.

c. Stages of Chromatin Behavior During the Meiotic Prophase in Greater Detail

The following five stages of chromatin behavior within the prophase nucleus during meiosis are now in common usage. They are based on the stages originally described by H. von Winiwarter, ’00. The substantive form is presented in parentheses.

1) Leptotene (Leptonema) Stage. The leptotene stage (figs. 69, 71) represents the initial stage of the meiotic process and is seen especially well in the spermatocyte. At this time the nucleus of the differentiating germ cell begins to enlarge, and the diploid number of very long, slender chromatin threads make their appearance. (Compare “resting” and leptotene nuclei in figs. 69, 71.) The chromatin threads may lie at random in the nucleus or they may be directed toward one side, forming the so-called “bouquet” condition (fig. 69, leptotene stage) . The nucleolus is evident at this time (fig. 7 IB) .

2) Zygotene or Synaptene (Zygonema) Stage. The zygotene stage (figs. 69, 71, 85) is characterized by a synapsis of the chromatin threads. This synapsis or conjugation occurs between the homologous chromosomes, that is, the chromosomes which have a similar genic constitution. Synapsis appears to

o*

A. RESTING NUCLEUS

CHROMOSOMES THREAO'LIKE \ AND SCATTERED THROU6H1 OUT NUCLEUS, NOT VISIBLE AS DISTINCT CHROMOSOMES

8. LEPTOTENE NUCLEUS

CHROMOSOMES BECOME EVIDENT AS DISTINCT STRUCTURES

C. ZYGOTENE (SYNAPTENE) NUCLEUS

HOMOLOGOUS CHROMOSOMES BEGIN TO SYNAPSE, I.E., UNITE IN PAIRS

D. PACHYTENE NXLEUS

CHROMOSOME PAIRS BECOME CLOSELY ASSOCIATED AND EACH PAIR SHORTENS AND APPEARS MUCH THICKER. EACH "CHROMOSOME" IS MADE UP OF FOUR CHROMATIDS, I E.. IT C5 A TETRAD DUE TO THE FACT THAT EACH HOMOLOGOUS CHROMOSOME WHICH ENTERED INTO THE ORIGINAL UNION HAS DIVIDED LONGITUDINALLY INTO TWO CHROMATIDS

F. GERMINAL VESICLE

YOUNG GERMINAL VESICLE OF CAT OOCYTE IN TRANSITION FROM THE MORE TYPICAL DIPLOTENE CONDITION INTO THE MATURE GERMINAL VESICLE SHOWN BELOW

':^G. GERMINAL VESICLE

THE GERMINAL VESICLE STAGE OF THE OOCYTE NUCLEUS OF THE CAT IN WHICH THE DIPLOTENE CHROMATIDS ARE ELONGATED AND DO NOT TAKE THE BASIC STAINS READILY

â–  It

H, GERMINAL VESICLE

GERMINAL VESICLE OF DEVELOPING SHARK OOCYTE SHOWING MANY NUCLEOLI AND "lAMP-BRUSH"CHROMOSOMES

E. DIPLOTENE NUCLEUS

THE CHROMATIDS OF EACH TETRAD SHOW A REPULSION FOR EACH other and THE TETRAD AS A WHOLE BEGINS TO OPEN UP THE CHROMATIDS ALSO BECOME MORE ATTENDANT) PARTICULARLY IN THE OOCYTE WHERE THE NUCLEUS ENLARGES AND FORMS THE GERMINAL VESICLE IN WHICH THERE IS A LARGE AMOUNT

OF nuclear sap. the

CHROMATIDS BECOME VERY LONG AND MAY SHOW LATERAL LOOPS

I. GERMINAL VESICLE

germinal VESICLE OF AMPHIOXUS OOCYTE WITH LARGE NUCLEOLUS!?)

Fig. 71. Certain aspects of the oocyte nucleus during the meiotic prophase. (A-G) Chromatin and nuclear changes in the oocyte of the cat up to the diplotene condition when the germinal vesicle is fully developed. (After de Winiwarter and Sainmont, Arch, biol., Paris, 24.) (H, I) Germinal vesicle in the dogfish, Scy Ilium canicula, and in

Amphioxus. (After Marechal, La Cellule, 24.) Observe the typical “lamp-brush” chromosome conditions in the germinal vesicle of the shark oocyte. These lamp-brush chromosomes are developed during the diplotene stage of meiosis by great attenuation of the chromosomes and the formation of lateral extensions or loops from the sides of the chromosomes.

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MATURATION OF GAMETES

137

begin most often at the ends of the threads and progresses toward the middle (fig. 67, zygotene). At this stage the chromatin threads may show a strong tendency to collapse and shrink into a mass toward one end of the nucleus (fig. 85C, D). This collapsed condition, when present, is called synizesis. The zygotene stage gradually passes into the pachytene condition.

3) Pachytene (Pachynema) Stage. Gradually, the synapsis of the homologous chromosomes becomes more complete, and the threads appear shorter and thicker. The contracted threads in this condition are referred to as pachynema (figs. 69, 71, 85E). The nucleus in this manner comes to contain a number of bivalent chromosomes, each of which is made up of two homologous mates arranged side by side in synaptic union, known technically as parasynapsis. (Telosynapsis probably is not a normal condition.) Consequently, the number of chromosomes now appears to be haploid. Each pachytene chromosome (i.e., each of the pair of homologous chromosomes) gradually divides equationally into two daughter thread-like structures, generally referred to as chromatids. The exact time at which division occurs during meiosis is questionable. The entire group of four chromatids which arise from the splitting of the synapsed homologues is called a tetrad.

4) Diplotene (Diplonema) Stage. In the diplotene stage (figs. 67, 69, 71, 85F, G), two of the chromatids tend to separate from the other two. (See fig. 70A, C, E.) The four chromatids in each tetrad may now be observed more readily, at least in some species, because the various chromatids of each tetrad show a repulsion for one another, and the chromatids move apart in certain areas along their length. This condition is shown in both the male and female meiocyte, but in the latter, the repulsion or moving apart is carried to a considerable degree and is associated with a great lengthening and attenuation of the chromatids. (See fig. 67.) In the female meiocyte at this stage, the chromosomes become very diffuse and are scattered throughout the nucleus, somewhat resembling the non-mitotic condition (figs. 71F-T, 72B-E). The peculiar behavior of the chromosomes and nucleus of the oocyte in the diplotene stage of meiosis is described more in detail on p. 141.

Although there is a tendency for the chromatids to widen out or separate from each other at this time, they do remain associated in one or more regions. In these regions of contact, the paired chromatids appear to exchange partners. This point of contact is called a chiasma (plural, chiasmata). Hence, a chiasma is the general region where the chromatids appear to have exchanged partners when the tetrad threads move apart in the diplotene state. (See fig. 70, chiasmata.)

5) Diakinesis. The diplotene stage gradually transforms into the diakinesis state (figs. 67, 69, 72F, 85H) by a process of marked chromosomal contraction. There also may be an opening up of the tetrads due to a separation of the homologous mates in the more central portions of the tetrad, with the result that only the terminal parts of the chromatids remain in contact. This

Fig. 72. Growth of the nucleus during meiosis in the amphibian egg, showing the enlarged germinal vesicle and diplotene lamp-brush chromosomes with lateral loops. (A) Early diplotene nucleus of the frog. (B, C, E) Different phases of the diplotene nucleus in this form. These figures are based upon data provided by Duryee (’50) and sections of the frog ovary. (D) Drawing of the unfixed germinal vesicle of Triturus. Some aspects of the attenuate chromatin threads with lateral loops are shown. The nucleoli are numerous and occupy the peripheral region of the germinal vesicle. (F) Semidiagrammatic drawing of the later phases of the developing frog egg. It shows the germinal vesicle assuming a polar condition, with the initial appearance of germinal vesicle shrinkage before the final dissolution of the nuclear membrane. Observe that the chromosomes are contracting and now occupy the center of the germinal vesicle.

138

MATURATION OT GAMETES

latter process is called “terminalization.” Coincident with this partial separation, a further contraction of the tetrads may occur. As a result, at the end of diakinesis the tetrads may assume such curious shapes as loops, crosses, rings, etc., scattered within the nucleus of the female and male meiocyte (fig. 69, diakinesis). The nuclear membrane eventually undergoes dissolution, and

Fig. 73. Various aspects of Sertoli-cell conditions in the fowl. (Redrawn from Zlotnick, Quart. J. Micr. Sc., 88.) (A) Resting Sertoli cell, showing mitochondria. (B) Sertoli

element at the beginning of cytoplasmic elongation. (C) Sertoli cell with associated late spermatids.

Fig. 74. Types of chordate sperm. All the chordate sperm belong to the flagellate variety. (A) Amphioxus (protochordate). (B) Salmo (teleost). (C) Perea (teleost). (D) Petromyzon (cyclostome). (E) Raja (elasmobranch). (F) Bufo (anuran). (G) Rana (anuran). (H) Salamandra (urodele). (I) Anguis (lizard). (J) Crex (bird). (K) Fringilla (bird). (L) Turdus (bird). (M) Echidna (monotrematous mammal). (N) Mus (eutherian mammal). (O, P) Man (full view and side view, respectively).

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MATURATION OF GAMETES

141

the tetrads become arranged on the metaphase plate of the first maturation division. (See figs. 69, first maturation division; 72F, 119A, B.) This division is described on pp. 132 and 133.

d. Peculiarities of Nuclear Behavior in the Oocyte During Meiosis; the Germinal Vesicle

Although the movements of the chromosomes during meiosis in the developing male and female gamete appear to follow the same general behavior

Fig. 75. Non-flagellate sperm. (A-C) Ameboid sperm of Polyphemus. (After Zacharias.) (D) Lobster, Homarus. (After Herrick.) (E) Decapod Crustacea, Galathea (Anomura). (After Koltzoff.) (F) Nematode woim, Ascaris.

Fig. 76. Conjugate sperm of grasshopper associated temporarily to form the “sperm boat.”

pattern (fig. 67), some differences do occur. For example, in the female when the diplotene stage is reached, the repulsion of the tetrad threads is greater (figs. 67, $ and 9 \ 12). Furthermore, the chromatids elongate and become very attenuate although they appear to retain their contacts or chiasmata (fig. 72). Side loops and extensions from the chromatids also may occur, especially in those vertebrates with large-yolked eggs (e.g., amphibia, fishes, etc.). (See figs. 71H, 72B-D.) When these lateral extensions are present, the chromosomes appear diffuse and fuzzy, taking on the characteristics which

142

THE DEVELOPMENT OF THE GAMETES OR SEX CELLS

ACR OSOME VACUOLE NUCLEU S POS T UCLEAR CAP

CK GRANULE ANTE RIO R

CENT RIOLE

CELL MEMBRANE

mitochondrial sheath

POSTERIOR OR R ING CE NTRI OLE

AXIAL FILAMEN T OF TAIL

Fig. 77. Spatula-type sperm of various mammals. (Compiled from Bowen; Gatenby and Beams; Gatenby and Woodger; see references in bibliography.) Observe the vacuole inside the head of the sperm. Gatenby and Beams found that this vacuole, in some instances, stains similar to a nucleolus, but suggest it may be a hydrostatic organ, or respiratory structure. (P. 20, Quart. J. Micr. Sc., 78.)

suggest their description as “lamp-brush” chromosomes. Another difference of chromatic behavior is manifested by the fact that the chromosomes in the developing female gamete during the diplotene stage are not easily stained by the ordinary nuclear stains, whereas the chromosomes in the spermatocyte stain readily.

Aside from the differences in chromosomal behavior, great discrepancies in the amount of growth of the nucleus occur in the two gametes during meiosis. The nucleus of the oocyte greatly increases in size and a large quantity of nuclear fluid or sap comes to surround the chromosomes (figs. 7 IF, G; 72C, ^ F). Correlated with this increase in nuclear size, the egg grows rapidly.

MATURATION OF GAMETES

143

and deutoplasmic substance is deposited in the cytoplasm (fig. 68F-H). As differentiation of the oocyte advances, the enlarged nucleus or germinal vesicle assumes a polar position in the egg (figs. 68H, 70F). When the oocyte has finished its growth and approaches the end of its differentiation, the

A

Fig. 78. Different shapes and positions of the acrosome. (A) Type of acrosome found in Mollusca, Echinodermata, and Annelida. (B) Reptilia, Aves, and Amphibia. (C) Lepidoptera. (D) Mammalia. (E) Many Hemiptera and Coleoptera. (After Bowen, Anat. Rec., 28.) (F) Sperm of certain birds, i.e., finches. (After Retzius, Biol.

Untersuchungen, New Series 17, Stockholm, Jena.) Observe the well-developed acrosome in the form of a perforatorium. The spiral twist of the acrosome shown in this drawing is characteristic of passerine birds.

Fig. 79. Sperm of urodele amphibia. (After Meves, 1897, Arch. f. mikr. Anat. u. Entwichlingsgesch., 50; McGregor, 1899, J. Morphol., 15. (A~E) Stages in the morphogenesis of the sperm of Salamandra. (F) Diagram of head, middle piece, etc. of the sperm of the urodele.

chromosomes within the germinal vesicle condense once again, decrease in length (fig. 72F), and assume conditions more typical of the diakinesis stage (figs. 67; 1 19A). The tetrad chromosomes now become visible. Following the latter chromosomal changes, the nuclear membrane breaks down (fig. 119A), and the chromatin elements pass onto the spindle of the first maturation division (fig. 119B). The nuclear sap, membrane, nucleolus, and general framework pass into the surrounding cytoplasmic substance (figs. 119A; 132A-C). This nuclear contribution to the cytoplasm appears to play an important part in fertilization and development, at least in some species (fig. 132C; the clear protoplasm is derived from the nuclear plasm).

e. Character of the Meiotic (Maturation) Divisions in the Spermatocyte Compared with Those of the Oocyte

1) Dependent Nature of the Maturation Divisions in the Female Meiocyte.

The maturation divisions in the developing male gamete occur spontaneously and in sequence in all known forms. But in most oocytes, either one or both of the maturation divisions are dependent upon sperm entrance. For example, in Ascaris, a nematode worm (fig. 133), and in Nereis, a marine annelid worm (fig. 130), both maturation divisions occur after the sperm has entered and are dependent upon factors associated with sperm entrance. A similar condition is found in the dog (van der Stricht, ’23; fig. 115) and in the fox (Asdell, ’46). In the urochordate, Styela, the germinal vesicle breaks down, the nuclear sap and nucleolus move into the surrounding protoplasm, and the first maturation spindle is formed as the egg is discharged into the sea water (fig. 116A, B). Further development of the egg, however, awaits the entrance of the sperm (fig. 116C--F). Somewhat similar conditions are found in other Urochordata. In the cephalochordate, Amphioxus, and in the vertebrate group as a whole (with certain exceptions) the first polar body is formed and the spindle for the second maturation division is elaborated before normal sperm entrance (figs. 117C, D; 119D). The second maturation division in the latter instances is dependent upon the activities aroused by sperm contact with the oocyte. In the sea urchin, sperm can penetrate the egg before the maturation divisions occur; but, under these conditions, normal development of the egg does not occur. Normally in this species both maturation divisions are effected before sperm entrance, while the egg is still in the ovary. When the egg is discharged into the sea water, the sperm enters the egg, and this event affords the necessary stimulus for further development (fig. 131).

2) Inequality of Cytoplasmic Division in the Oocyte. When the first maturation division occurs, the two resulting cells are called secondary spermatocytes in the male and secondary oocytes in the female (figs. 67, 69). The secondary spermatocytes are smaller both in nuclear and cytoplasmic volume. They also form a definite nuclear membrane. Each secondary spermatocyte then divides and forms two equal spermatids. In contrast to this condition

MATURATION OF GAMETES

145

of equality in the daughter cells of the developing male gamete during and following the maturation divisions, an entirely different condition is found in the developing female gamete. In the latter, one of the secondary oocytes is practically as large as the primary oocyte, while the other or first polar body (polocyte) is extremely small in cytoplasmic content although the nuclear material is the same (fig. 117D). During the next division the secondary oocyte behaves in a manner similar to that of the primary oocyte, and a small second polocyte is given off, while the egg remains large (fig. 117E, F). Unlike the secondary spermatocyte, the secondary oocyte does not form a nuclear membrane. The polar body first formed may undergo a division, resulting in a total of three polar bodies (polocytes) and one egg (ootid).

/. Resume of the Significance of the Meiotic Phenomena

In view of the foregoing data with regard to the behavior of the male and female gametes during meiosis, the significant results of this process may be summarized as follows:

(1) There is a mixing or scrambling of the chromatin material brought about by the crossing over of genic materials from one chromatid to another.

(2) Much chromatin material with various genic combinations is discarded during the maturation divisions in the oocyte. In the latter, two polar bodies are ejected with their chromatin material as described above. The egg thus retains one set of the four genic combinations which were present at the end of the primary oocyte stage; the others are lost. (A process of discarding of chromatin material occurs in the male line also. For although four spermatids and sperm normally develop from one primary spermatocyte, great quantities of sperm never reach an egg to fertilize it, and much of the chromatin material is lost by the wayside.)

(3) A reduction of the number of chromosomes from the diploid to the haploid number is a significant procedure of all true meiotic behavior.

(For more detailed discussions and descriptions of meiosis, see De Robertis, et al., ’48; Sharp, ’34, ’43; Snyder, ’45; White, ’45.)

4. Cytosomal (Cytoplasmic) Maturation of the Gametes a. General Aspects of Cytoplasmic Maturation of the Gametes

During the period when the meiotic prophase changes occur in the nucleus of the oocyte, the cytoplasm increases greatly and various aspects of cytoplasmic differentiation are effected. That is, differentiation of both nuclear and cytoplasmic materials tend to occur synchronously in the developing

PROACROSOMIC SUBSTANCE

Fig. 80. Morphogenesis of guinea-pig and human sperm. (A) Spermatocyte of guinea pig before first maturation division. The Golgi complex with included proacrosomic granules and centrioles is shown. (After Gatenby and Woodger, ’21.) (B) Young sister

spermatids of guinea pig. (C) Later spermatid of guinea pig showing acroblast with proacrosomic granules. (D) Young human spermatocyte, showing Golgi apparatus with proacrosomic granules similar to that shown in (A). (After Gatenby and Beams, ’35.) (E) Spermatid of guinea pig later than that shown in (C), showing acroblast with Golgi substance being discarded from around the acroblast. (F) Later human spermatid, showing Golgi substance surrounding acroblast with acrosome bead. (After Gatenby and Beams, ’35.) (G) Later human spermatid, showing acroblast, with acrosome bead

within, surrounded by a vacuole. (After Gatenby and Beams, ’35.) (H) Later spermatid

of guinea pig, showing outer and inner zones of the acrosome. The inner zone corresponds somewhat to the acrosome bead shown in (G) of the human spermatid. (After Gatenby and Wigoder, Proc. Roy. Soc., London, s.B., 104.)

female gamete. In the male gamete, on the other hand, the meiotic processes are completed before morphological differentiation of the cytoplasm is initiated.

Another distinguishing feature in the morphogenesis of the sperm relative to that of the egg is that the cytoplasmic differentiation of the sperm entails a discarding of cytoplasm and contained cytoplasmic structures, whereas the oocyte conserves and increases its cytoplasmic substance (fig. 68). In regard

MATURATION OF GAMETES

147

to the behavior of the cytoplasms of the two developing gametes, it is interesting to observe that the idiosome-Golgi-mitochondrial complex behaves very differently in the two gametes (fig. 68).

A third condition of egg and sperm differentiation involves the possible function of the “nurse cells.” In the vertebrate ovary the follicle cells which surround the egg have much to do with the conditions necessary for the differentiation of the oocyte. The latter cannot carry the processes of differentiation to completion without contact with the surrounding follicle cells. Spermiogenesis also depends upon the presence of a nurse cell. In the vertebrate seminiferous tubule, the Sertoli cell is intimately concerned with the transformation of the spermatid into the morphologically adult sperm, and a close contact exists between the developing sperm element and the Sertoli cell during this period (figs. 65, 66, 73). In the discharge of the formed sperm elements into the lumen of the tubule, the Sertoli cell also is concerned (Chap. 1).

b. Morphogenesis (Spermiogenesis; Spermioteleosis) of the Sperm

1) Types of Sperm. There are two main types of sperm to be found in animals, namely, flagellate and non-flagellate sperm (figs. 74, 75). Flagellate sperm possess a flagellum or tail-like organelle; non-flagellate sperm lack this structure. The flagellate type of sperm is found quite universally among animals; non-flagellate sperm occur in certain invertebrate groups, particularly in the nematode worms, such as Ascaris, and in various Crustacea, notably the lobster, crab, etc. (fig. 75). Flagellate sperm may be either uniflagellate or biflagellate. Single flagellate sperm occur in the majority of animals, while a biflagellate form is found in the platode, Procerodes. However, biflagellate sperm may be found as abnormal specimens among animals normally producing uniflagellate sperm.

Conjugate sperm are produced in certain animal species. For example, two sperm heads adhere closely together in the opossum (fig. 125), also in the beetle, Dytiscus, and in the gastropod, Turritella. Many sperm heads become intimately associated in the grasshopper to form the so-called “sperm boat” (fig. 76). However, all conjugate sperm normally separate from each other in the female genital tract.

2) Structure of a Flagellate Sperm. The flagellate sperm from different species of animals vary considerably in size, shape, and morphological details. Some possess long, spear-shaped heads, some have heads resembling a hatchet, in others the head appears more or less cigar-shaped, while still others possess a head which resembles a spatula (fig. 74). The spatula-shaped head is found in the sperm of the bull, opossum, man, etc. The description given below refers particularly to the spatula-shaped variety. Although all flagellate sperm resemble one another, diversity in various details is the rule,

Fig. 81. Later stages of human spermatogenesis. (Redrawn from Gatenby and Beams,

1935.)

Fig. 82. Stages of guinea-pig spermatogenesis. Observe dual nature of the acrosome; also, middle-piece bead (kinoplasmic droplet). (A-C redrawn from Gatenby and Beams, 1935; D redrawn from Gatenby and Woodger, ’21.)

and the description given below should be regarded as being true of one type of sperm only and should not be applied to all flagellate sperm.

A fully differentiated spatulate sperm of the mammals possesses the following structural parts (fig. 77).

a) Head. Around the head of the sperm there is a thin, enveloping layer of cytoplasm. This cytoplasmic layer continues posteriad into the neck, middle piece, and tail. Within the cytoplasm of the head is the oval-shaped nucleus. Over the anterior half of the nucleus the apical body or acrosome is to be found, forming, apparently, a cephalic covering and skeletal shield for the

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nucleus. The caudal half of the nucleus is covered by the post-nuclear cap. This also appears to be a skeletal structure supporting this area of the nucleus; moreover, it affords a place of attachment for the anterior centrosome and the anterior end of the axial filament.

In human and bull sperm the acrosome is a thin cap, but in some mammalian sperm it is developed more elaborately. In the guinea pig it assumes the shape of an elongated, shovel-shaped affair (fig. 82), while in the mouse and rat it is hatchet or lance shaped (fig. 74N). In passerine birds the acrosome is a pointed, spiral structure often called the perforatorium (fig. 78). On the other hand, in other birds, reptiles, and amphibia it may be a simple, pointed perforatorial structure (figs. 74, 78, 79). In certain invertebrate species, it is located at the caudal or lateral aspect of the nucleus (figs. 75, 78).

b) Neck. The neck is a constricted area immediately caudal to the posterior nuclear cap and between it and the middle piece. Within it are found the anterior centriole and the anterior end of the axial filament. In this particular region may also be found the so-called neck granule.

c) Connecting Body or Middle Piece. This region is an important portion of the sperm. One of its conspicuous structures is the central core composed of the axial filament and its surrounding cytoplasmic sheath. At the distal end of the middle piece, the central core is circumscribed by the distal, or ring centriole. Investing the central core of the middle piece is the mitochondrial' sheath. The enveloping cytoplasm is thicker to some degree in this area of the sperm than that surrounding the head.

d) Flagellum. The flagellum forms the tail or swimming organ of the sperm. It is composed of two general regions, an anterior principal or chief piece and a posterior end piece. The greater part of the axial filament and its sheath is found in the flagellum. A relatively thick layer of cytoplasm surrounds the filament and its sheath in the chief-piece region of the flagellum, but, in the caudal tip or end piece, the axial filament seems to be almost devoid of enveloping cytoplasm. The end piece often is referred to as the naked portion of the flagellum.

In figure 79 is shown a diagrammatic representation of a urodele amphibian sperm. Two important differences from the mammalian sperm described above are to be observed, namely, the middle piece is devoid of mitochondria and is composed largely of centrioles 1 and 2, and the tail has an elaborate undulating or vibratile filament associated with the chief piece.

3) Spermiogenesis or the Differentiation of the Spermatid into the Morphologically Differentiated Sperm. The differentiation of the spermatid into the fully metamorphosed sperm is an ingenious and striking process. It involves changes in the nucleus, during which the latter as a whole contracts and in some forms becomes greatly elongated into an attenuant structure. (See figs. 79B-F; 85L-P.) It also is concerned with profound modifications of the cytoplasm and its constituents; the latter changes transform the inconspicuous

Spermatid into a most complicated structure. Some of these changes are outlined below.

a) Golgi Substance and Acroblast; Formation of the Acrosome. The Golgi substance or parts thereof previously associated with the idiosome of the spermatocyte (fig. 80A) proceeds to form the acrosome of the developing spermatid as follows: In the differentiating human sperm, the Golgi substance of the spermatocyte (fig. SOD) becomes aggregated at the future anterior end of the nucleus, as shown in fig. 80F, where it forms an acroblast within a capsule of Golgi substance. This acroblast later forms a large vacuole within which is the acrosomal “bead” (figs. 68B; 80G). The acrosomal bead proceeds to form the acrosomal cap, shown in figure 81 A, and the latter grows downward over the anterior pole of the nucleus (fig. 81 A, B). Most of the Golgi substance in the meantime is discarded (fig. 81 A, B). (Sec Gatenby and Beams, ’35.)

In the guinea pig the acroblast together with other Golgi substance, migrates around the nucleus toward the future anterior pole of the latter where the acroblast takes up its new position (fig. 80B, C, E). (See Gatenby and Woodger, ’21.) As shown in figure 80E, the acroblast is composed of inner and outer acrosomal substances. These inner and outer areas of the acroblast give origin respectively to the inner and outer zones of the acrosome (fig. 82). The peripheral or surrounding Golgi material of the acroblast detaches itself meanwhile from the developing acrosome (fig. 80E, H) and drifts downward toward the posterior end of the sperm. Eventually it is discarded with the excess cytoplasm and some mitochondrial material. In some animal species (e.g., grasshopper) the acrosomal substance arises from a multiple type of acroblast (Bowen, ’22). (See fig. 83.) Nevertheless, the general process of acrosome formation is similar to that outlined above.

b) Formation of the Post-nuclear Cap. All spatulate sperm of mam

Fig. 83. Formation of the acrosome from a multiple acroblast in the grasshopper. (After

Bowen, Anat. Rec., 24.)

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Fig. 84. The mitochondrial nebenkern and its elaborate development in Brachynema. (After Bowen, J, Morphol., 37 and Biol. Bull., 42.) (B-I) Division of the nebenkern

(A) and its elaboration into two attenuant strands extending posteriad into the flagellum.

mals possess a nucleus which has an acrosomal cap over its anterior aspect and a post-nuclear cap covering its posterior area. Both of these caps tend to meet near the equator of the nucleus (fig. 77).

The exact origin of the post-nuclear cap is difficult to ascertain. In the human sperm it appears to arise from a thickened membrane in association with centriole 1 (fig. 80G, post-nuclear membrane). This membrane grows anteriad to meet the acrosomal cap (fig. 81A-C). In the sperm of the guinea pig, a series of post-nuclear granules in the early spermatid appear to coalesce to form the post-nuclear cap (fig. 82A-C).

c) Formation of the Proximal and Distal Centrioles; Axial Filament. While the above changes in the formation of the acrosome are progressing, the centriole (or centrioles) of the idiosome move to the opposite side of the nucleus from that occupied by the forming acrosome, and here in this position the proximal and distal centrioles of the future sperm arise. In this area the neck granules also make their appearance (figs. 68B; 80F-H). The axial filament arises at this time and it probably is derived from the two centrioles simultaneously (fig. 80F, H). The centrioles soon become displaced along the axial filament, the caudal end of which projects from the surface of the cell membrane (fig. 80F-H). The axial filament grows outward posteriorly from the cell membrane in line with the two centrioles and the acrosome-forming material. The anterior-posterior elongation of the sperm thus begins to make its appearance (fig. 80H). The anterior centriole retains a position close to the nuclear membrane, but the posterior or ring centriole moves gradually posteriad toward the cell surface (figs. 81, 82A-C).

d) Mitochondrial Material and Formation of the Middle Piece OF THE Sperm. The behavior of the mitochondria in the formation of sperm varies greatly. In the spatulate sperm described above, a portion of the mitochondrial substance becomes aggregated around the axial filament in the middle-piece area (figs. 77, 82D). In certain amphibian sperm the middle piece appears to be formed mainly by centrioles 1 and 2 (fig. 79D-F). In certain insects the mitochondrial body or nebenkern, divides into two masses which become extended into elongated bodies associated with the flagellum (fig. 84). Some of the mitochondrial substance is discarded with the Golgi substance and excess cytoplasmic materials.

e) The Cytoplasm, Axial Filament, Mitochondria, and Tail Formation. Synchronized with the above events, the cytoplasm becomes drawn out in the posterior direction, forming a thin cytoplasmic layer over the sperm head, and from thence posteriad over the middle piece and the chief piece of the flagellum. However, the end piece of the flagellum may be devoid of investing cytoplasm (fig. 77). As the cytoplasm is elongating posteriorly over the contained essential structures of the forming sperm, much of the cytoplasm and Golgi substance and some mitochondria are discarded and lost from the sperm body. It may be that these discarded bodies form a part of the essential substances of the spermatic (seminal) fluid. (See Chap. 1.) (See figs. 66; 68B-E; 81; 82; 85M-0.)

The centralized core of the tail is the axial filament which arises in relation to centrioles 1 and 2 and grows posteriad through the middle piece and tail

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(figs. 80F-H; 81A-C; 82A-~C; 85M-P). A considerable amount of mitochondrial material may also enter into the formation of tail (fig. 84).

A peculiar, highly specialized characteristic of many sperm tails is the development of a vibratile membrane associated with the axial filament (fig. 79E, F). Its origin is not clear, but it probably involves certain relationships with the mitochondrial material as well as the cytoplasm and axial filament.

In the formation of the human and guinea-pig sperm, the nucleus experiences only slight changes in shape from that of the spermatid. However, in many animal species, spermiogenesis involves considerable nuclear metamorphosis as well as cytoplasmic change (figs. 69, 79, 85).

In summary it may be stated that while the various shapes and sizes of mature flagellate sperm in many animal species, vertebrate and invertebrate,

Fig. 85. Spermatogenesis in the common fowl. Observe extreme nuclear metamorphosis. (After Miller, Anat. Rec., 70.) (A) Resting spermatocyte. (B) Early leptotene stage. (C, D) Synaptene stage. (E) Pachytene stage. (F, G) Diplotene stage. (H) Diakinesis. (I) First division, primary sperm. (J-P) Metamorphosing sperm.

are numerous, there is a strong tendency for spermiogenesis to follow similar lines of development. Deviations occur, but the following comparisons between mammalian and insect spermiogenesis, somewhat modified from Bowen (’22), illustrate the uniformity of transformation of the basic structures of the primitive meiocyte:

Mammalian Sperm

Insect Sperm

Nucleus — head

Nucleus — head

Centrioles — originally double and arranged in a proximal-distal formation. The axial filament arises from both centrioles

Centrioles — same as in mammals

Mitochondria — form an elaborate sheath for the anterior portion of the axial filament

Mitochondria — form a somewhat similar sheath for the axial filament

Idiosome and Golgi apparatus (acroblast portion) — gives origin to a vesicle which contains a granule, the acrosome granule, which is involved in the production of the acrosome

Idiosome and Golgi apparatus — much the same as in mammals

Excess Golgi substance — cast off with excess cytoplasm

Excess Golgi substance — cast off with excess cytoplasm

Excess cytoplasm — cast off — may be part of seminal fluid or possibly may be engulfed by Sertoli cells

Excess cytoplasm — cast off — may be part of seminal fluid or possibly may be engulfed by epithelial cells of the sperm cyst wall

c. Cytoplasmic Differentiation of the Egg

The cytoplasmic differentiation of the egg involves many problems. These problems may be classified under three general headings, viz.:

(1) Formation of the deutoplasm composed of fats, carbohydrates and proteins,

(2) development of the invisible organization within the true protoplasm or hyaloplasm, and finally,

(3) formation of the vitelline or egg membrane or membranes.

In view of the complexity of these three problems and of their importance to the egg in the development of the new individual, the mature oocyte or egg is in a sense no longer a single cell. Rather, it is a differentiated mass of protoplasm which is capable, after proper stimulation, to give origin to a new individual composed of many billions of cells. As such, the differentiation of the oocyte within the ovary represents a relatively unknown period of embryological development.

.CENTROSPHERE

Fig. 86. Young oogonia of the fowl entering the growth (oocyte) stage. (A) Idiosome from which the Golgi substance has been removed and stained to show the centrosphere (archoplasm). The centrosome has two centrioles. (B) Idiosome with surrounding Golgi substance. The mitochondria surround the Golgi substance and the nucleus. (After Brambell, ’25.)

Fig. 87. The so-called mitochondrial yolk body in the developing egg of the fowl. (A) Oocyte from 11 -week-old chick, showing mitochondrial cloud and Golgi substances I and II. (B) Oocyte from ovary of adult fowl, showing both types of Golgi substance and mitochondrial cloud. (C) Oocyte from ovary of adult fowl, showing the appearance of the mitochondrial yolk body within the mitochondrial cloud. (D) Oocyte from ovary of adult fowl, showing fragmentation of Golgi substance 1 and the association of the resulting Golgi granules around the mitochondrial yolk body. (After Brambell, ’25.)

Fig. 88. Portion of follicle and periphery of oocyte from ovary of the adult bird, showing the mitochondria and their transformation into the M-yolk spheres of Brambell. (After Brambell, ’25.)

Before considering the various aspects of cytoplasmic differentiation of the oocyte, it is best for us to review the types of vertebrate and other chordate eggs in order to be able to visualize the various goals toward which the developing oocyte must proceed.

1) Types of Chordate Eggs. Eggs may be classified according to the amount of deutoplasm (yolk, etc.) present in the cytoplasm as follows:

a) Homolecithal (Isolecithal) Eggs. True homolecithal eggs in the phylum Chordata are found only in the mammals, exclusive of the Prototheria. Here the deutoplasm is small in amount, and is present chiefly in the form of fat droplets and small yolk spherules, distributed in the cytoplasm of the egg (figs. 118A, B; 147A).

b) Telolecithal Eggs. In the telolecithal egg the yolk is present in considerable amounts and concentrated at one pole. Telolecithality of the egg in the phylum Chordata exists in various degrees. We shall arrange them in sequence starting with slight and ending with very marked telolecithality as follows:

(1) Amphioxus and Styela. In Amphioxus and Styela from the subphyla Cephalochordata and Urochordata, respectively, the yolk present is centrally located in the egg before fertilization but becomes concentrated at one pole at the time of the first cleavage where it is contained for the most part within the future entoderm cells (figs. 132D, 167A).

(2) In many Amphibia, such as the frogs and toads, and also in the Petromyzontidae or fresh-water lampreys among the cyclostome fishes, the yolk present is greater in amount than in the preceding eggs. As such, it is concentrated at one pole, the future entodermal or vegetal

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pole, and a greater degree of telolecithality is attained than in the eggs of Amphioxus or Styela (fig. 141 A).

(3) In many Amphibia, such as Necturus, also in Neoceratodus and Lepidosiren among the lung fishes, and in the cartilaginous ganoid fish, Acipenser, yolk is present in considerable amounts, and the cytoplasm of the animal pole is smaller in comparison to the yolk or vegetal pole (figs. 150, 151, 152).

(4) In the bony ganoid fishes, Amia and Lepisosteus, as well as in the Gyrnnophiona (legless Amphibia) the yolk is situated at one pole and is large in quantity (figs. 153B-F; 154).

(5) Lastly, in a large portion of the vertebrate group, namely, in reptiles, birds, prototherian mammals, teleost and elasmobranch fishes, and in the marine lampreys, the deutoplasm is massive and the protoplasm which takes part in the early cleavages is small in comparison. In these eggs the yolk is never cleaved by the cleavage processes, and development of the embryo is confined to the animal pole cytoplasm (figs. 46, 47).

2) Formation of the Deutoplasm. The cytoplasm of the young oocyte is small in quantity, with a clear homogeneous texture (figs. 68A; 86A, B). As the oocyte develops, the cytoplasmic and nuclear volumes increase (fig. 68F), and the homogeneity of the cytoplasm is soon lost by the appearance of deutoplasmic substances (fig. 68G, H). In the oocyte of the frog, for example, lipid droplets begin to appear when the oocyte is about 50 /x in diameter (fig. 72A). (See Brachet, ’50, p. 53.) A little later glycogen makes its appearance, and finally yolk protein arises.

The origin of fat droplets and yolk spherules has been ascribed variously to the activities of chondriosomes (mitochondria and other similar bodies), Golgi substance, and of certain vacuoles. Most observers place emphasis upon the presence of a so-called “yolk nucleus” or “yolk-attraction sphere” situated near the nucleus of many oocytes as a structure associated with fat and yolk formation. In general, two types of yolk bodies have been described. One is the yolk nucleus of Balbiani and the other the mitochondrial yolk body of Brambell. The yolk nucleus of Balbiani (fig. 86A, B) consists of the following:

( 1 ) a central body, the centrosphere or archoplasmic sphere within which one or more centriole-like bodies are found, and

(2) surrounding this central body, a layer of Golgi substances and chondriosomes (i.e., mitochondria, etc.).

This cytoplasmic structure probably is related to the idiosome of the oogonia (fig. 68A).

The formation of the deutoplasm, according to the theory associated with the Balbiani type of yolk nucleus is as follows: The surrounding pallial layer of Golgi substance and mitochondria moves away from the central portion (i.e., away from the centrosphere) of the yolk nucleus and becomes scattered and dispersed as small fragments within the cytosome (fig. 68F, G). The yolk nucleus as an entity thus disappears, and its fragments become immersed within the substance of the cytoplasm. Coincident with this dispersion of yolk nuclear material, rapid formation of small yolk spherules and fat droplets occur (fig. 68H). It appears thus that the formation of the deutoplasm composed of fat droplets and yolk spherules is directly related to the activities of the Golgi substance and chondriosomes.

Fig. 89. (A) Cytoplasm of oocyte, showing formation of a second kind of yolk (the M-C-yolk) in a vacuole surrounding the M-yolk sphere. (After Brambell, ’25.) (B)

Passing of Golgi substance from the follicle cells into the ooplasm of developing oocyte of the fowl. (After Brambell, ’25.)

A

I A

Fig. 90. Diagrams showing contrasting theories explaining the organization of polarity of the cytoplasm of the fully developing egg or oocyte. Diagram at left shows polarity explained according to quantitative differences, while the diagram to the right shows qualitative differences. A = animal pole; V = vegetal pole. E represents a substance or a factor, while EN-1, EN-2, etc., represent different quantities of substance E distributed from pole to pole. SEC, SEN and SM are different chemical substances assumed to be responsible for the determination of the ectoderm, entoderm, and mesoderm of the developing embryo. (After Barth: Embryology, New York, Dryden Press.)

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On the other hand, the interpretation and description of the yolk body and its subsequent activities given by Brambell (’25) present a different view. According to the latter author, the yolk body is composed entirely of mitochondria; the Golgi substance and centrosphere are absent. Yolk formation proceeds as follows: As the young oocyte grows, the mitochondria increase in number and form the mitochondrial cloud (fig. 87A, B). The transitory mitochondrial yolk body differentiates within this cloud (fig. 87C). The mitochondrial yolk body ultimately breaks up into a mass of mitochondria, and the latter becomes dispersed in the cytoplasm of the oocyte (figs. 68F, G; 87D). Some of these dispersed mitochondria transform directly into yolk spheres (figs. 68H, 88, 89). Following this, another kind of yolk is formed in vacuoles surrounding these original yolk spheres (figs. 68H, 89 A, yolk spheres plus vacuoles). The fat droplets (C-yolk) within the ooplasm are formed according to Brambell “possibly under the influence of Golgi elements” (fig. 68H, fat droplets). Relative to the function of the yolk nucleus and its mitochondria, Brachet (’50), p. 57, considers it significant at the beginning, but its real importance is still to be understood.

The relationship, if any, of the oocyte nucleus to the deposition of yolk materials is not apparent. One must not overlook the real probability that the germinal vesicle (i.e,, the enlarged nucleus of the oocyte) may be related to the increase and growth of the cytoplasm and to yolk formation, for it is at this time that the chromatin threads surrender their normal diplotene appearance and become diffusely placed in the germinal vesicle. They also lose much of their basic chromatin-staining affinities while the Feulgen reaction is diminished (Brachet, ’50, p. 63). With regard to the possible function of the germinal vesicle in yolk synthesis, the following quotation is taken from a publication by Brachet (’47):

It is well worth pointing out that Puspiva (1942), using a very delicate and precise technique, found no correlation between the dipeptidase content of the nucleus and the onset of vitellus synthesis: such a correlation exists, however, in the case of the cytoplasm where dipcptidase increases markedly when the first yolk granules make their appearance. These results suggest that there is not evidence that the nucleus is the site of an especially active metabolism; cytoplasmic dipeptidase probably plays a part in yolk protein synthesis; if the nucleus controls such a synthesis, it works in a very delicate and still unknown way.

However, the means by which protein synthesis is effected still is a problem which awaits explanation (Northrop, ’50). (The interested student should consult Brachet, ’50, Chap. Ill, for a detailed discussion of the cytochemistry of yolk formation.)

Another aspect of the problem of cytoplasmic growth and differentiation of the oocyte presents itself for further study. Brambell (’25) concluded from his observations that Golgi substance passes from the follicle cells into the ooplasm of the growing bird oocyte and contributes to the substance of the peripheral layer (fig. 89B). Palade and Claude (’49) suggest that at least some of the Golgi substance be identified as myelin figures which develop “at the expense of lipid inclusions.” Thus it may be that the Golgi substance which Brambell observed (fig. 89B) passing from the follicle cells to the oocyte represents lipid substance. In the growing oocyte of the rat, Leblond (’50) demonstrated the presence of small amounts of polysaccharides in the cytoplasm of the oocyte, while the surrounding zona pellucida and follicle cells contained considerable quantities. These considerations suggest that the blood stream using the surrounding follicle cells as an intermediary may contribute food materials of a complex nature to the growing cytoplasm of the oocyte.

The localization of the yolk toward one pole of the egg is one of the movements which occurs during fertilization in many teleost fishes. In these forms, the deutoplasmic materials are laid down centrally in the egg during oogenesis, but move poleward at fertilization (fig. 122). A similar phenomenon occurs also during fertilization in Amphioxus and Styela among the protochordates. In many other fishes and in the amphibia, reptiles, birds, and monotrematous mammals, the yolk becomes deposited or polarized toward one pole of the oocyte during the later stages of oocyte formation, as the cytoplasm and the germinal vesicle move toward the other pole (figs. 68H, 72F). The polarization of the deutoplasmic substances thus is a general feature of the organization of the chordate egg.

3) Invisible Morphogenetic Organization Within the Cytoplasm of the Egg.

Two general categories of substances are developed within the cytoplasm of the oocyte during its development within the ovary, viz.:

(1) the visible or formed cytoplasmic inclusions, and

(2) an invisible morphogenetic ground substance.

The former group comprises the yolk spherules, fats, and other visible, often pigmented bodies which can be seen with the naked eye or by means of the microscope. The morphogenetic ground substance probably is composed of enzymes, hormones, and various nucleocytoplasmic derivatives enmeshed within the living cytoplasm. However, although we may assume that the basic, morphogenetic ground substance is composed of enzymes, hormones, etc., the exact nature of the basic substance or its precise relationship to the various formed inclusions of the cytoplasm is quite unknown (see Fankhauser, ’48, for discussion). More recent experiments demonstrate that the yolk or deutoplasmic material not only serves as a reservoir of energy for embryonic development but also is in some way connected with the essential, basic organization of the egg.

Although we know little concerning the exact nature of the morphogenetic organization of the egg or how it forms, studies of embryological development force upon us but one conclusion, to wit, that, during the period when the oocyte develops in the ovary, basic conditions are elaborated from which the

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future individual arises (Fankhauser, ’48). Within the cytoplasm of the mature egg of many chordates, this inherent organization is revealed at the time of fertilization by the appearance of definite areas of presumptive organ-forming substances. For example, in the egg of the frog and other amphibia, the yolk pole is the stuff from which the future entodermal structures take their origin; the darkly pigmented animal or nuclear pole will eventually give origin to epidermal and neural tissues; and from the zone between these two areas mesodermal and notochordal tissues will arise (fig. 119K). Similar major organ-forming areas in the recently fertilized egg have been demonstrated in other chordates, as in the ascidian, St ye la, and in the cephalochordate, Amphioxus. In the eggs of reptiles, birds, and teleost and elasmobranch fishes, while the relationship to the yolk is somewhat different, major organ-forming areas of a similar character have been demonstrated at a later period of development (Chaps. 6-9). This suggests that these eggs also possess a fundamental organization similar, although not identical, to that in the amphibian egg.

4) Polarity of the Egg and Its Relation to Body Organization and Bilateral Symmetry of the Mature Egg. One of the characteristic features of the terminal phase of egg differentiation in the chordate group is the migration of the gerrninal vesicle toward the animal pole of the egg (figs. 72F, 119A). As stated aboveTTn many vertebrate eggs the deutoplasmic jnaterial becomes situated at the opposite pole, known as the vegetal (vegetative) or yolk pole, either before fertilizatloiToTsEortty after. The relatively yolk-free protoplasm aggregates at the animal pole. Consequently the maturation divisions of the egg occur at this pole (fig. 1 19 A, B, D). The formation of a definite polarity of the egg, therefore, is one of the main results of the differentiation of the oocyte.

Various theories have been suggested in an endeavor to explain polarity in the fully developed egg or oocyte. All these theories emphasize qualitative and quantitative differences in the cytoplasmic substances extending from one pole of the egg to the other (fig. 90).

The animal and vegetal poles of the egg have a definite relationship to the organization of the chordate embryo. In Amphioxus, the animal pole becomes the ventro-anterior part of the embryo, while in the frog the animal pole area becomes the cephalic end of the future tadpole, and the yolk pole comes to occupy the posterior aspect. In teleost and elasmobranch fishes the yolk-laden pole lies in the future ventral aspect of the embryo, and it occupies a similar position in the reptile, bird, and prototherian mammal (see fig. 215). Studies have shown that the early auxiliary or trophoblastic cells in eutherian mammals lie on the ventral aspect of the future embryo. Consequently, it is to be observed that the various substances in mature vertebrate and protochordate eggs tend to assume a polarized relationship to the future embrydnic axis and body organization.

Many vertebrate and protochordate eggs possess a bilateral symmetry which becomes evident when the fertilization processes are under way or shortly after their conclusion. The appearance of the gray crescent in the frog’s egg (fig. 119K) and in other amphibian eggs during fertilization and the similar appearance of the yellow crescent in the fertilized egg of the ascidian, Styela (fig. 132D) serve to orient the future right and left halves of the embryo. Conditions similar to that of Styela, but lacking the yellow pigment, are present in Amphioxus. Similarly, in the chick, if one holds the blunt end of the egg to the left, and the pointed end to the right, the early embryo appears most often at right angles, or nearly so, to the axis extending from the broader to the smaller end of the egg, and in the majority of cases the cephalic end of the embryo will appear toward the side away from the body of the observer. There is some evidence that the “yolk” or egg proper is slightly elongated in this axis. It appears, therefore, that the general plane of bilateral symmetry is well established in the early chick blastoderm, although the early cleavages do not occur in a manner to indicate or coincide with this plane. In prototherian mammals, a bilateral symmetry and an anteroposterior orientation is established in the germinal disc at the time of fertilization, soon after the second polar body is discharged (fig. 136).

5) Membranes Developed in Relation to the Oocyte; Their Possible Sources of Origin. A series of membranes associated with the surface of the oocyte are formed during its development within the ovary. Three general types of such membranes are elaborated which separate from the oocyte’s surface at or before fertilization, leaving a perivitelline space between the egg’s surface and the membrane. They are:

( 1 ) A true vitelline membrane which probably represents a specialization or product of the ooplasmic surface. For a time this membrane adheres closely to the outer boundary of the ooplasm, but at fertilization it separates from the surface as a distinct membrane.

(2) A second membrane in certain chordates is elaborated by the follicle cells. It is known as a chorion in lower Chordata but is called the zona pellucida in mammals.

(3) A zona radiata or a thickened, rather complex, membrane is formed in many vertebrates; it may be considered to be a product of the ooplasm or of the ooplasm and the surrounding follicle cells.

All of the above membranes serve to enclose the egg during the early phases of embryonic development and therefore may be considered as primary embryonic membranes. As such, they should be regarded as a definite part of the egg and of the egg’s differentiation in the ovary. A description of these membranes in relation to the egg and possible source of their origin in the various chordate groups is given below.

a) Chorion in Styela. A previously held view maintained that the chorion

INNER FOLLICULAR EPITHELIUM

FOAM LAYER

CHORION

TEST CELL

[yy a.

OOPLASMIC MEMBRANE

PERIVITELLINE SPACE

Fig. 91. Formation of the chorion in the egg of Styela. (A) Chorion is shown along the inner aspect of the follicular epithelium. The test cells lie in indentations of the peripheral ooplasm. (B) Optical section of an ovulated egg. (Redrawn and modified from Tucker, ’42.)

â–  cA A.

FOLLICULAR EPITHELIUM

â– VITELLINE MEMBRANE

ZONA RADIATA YOLK

^^^t^SECONDARY FOLLICULAR EPITHELIUM

FOLLICULAR E PITHELI UM

VI TELLINE MEMBRANE

YOLK

D.

Fig. 92. Developing vitelline membranes of Scyllium canicula. Observe that two membranes are present in the young egg; later these membranes fuse into one membrane. (A) Surface area of young oocyte with a vitelline membrane and zona radiata. (B) Slightly older oocyte with the radiate zone not as prominent. (C) Older oocyte with a single, relatively thick, vitelline membrane. (D) Nearly mature oocyte with a thin vitelline membrane. (After Balfour, Plate 25, The Works of Francis Maitland Balfour, ed. by Foster and Sedgwick, London, Macmillan, 1885.)

and “test” cells of the egg of Styela were ejected from the surface cytoplasm at the time of ovulation (Conklin, ’05). A recent view, however, maintains that the test cells arise from follicle cells and come to lie in indentations of the periphery of the egg outside of the thin vitelline membrane (Tucker, ’42). The chorion is formed by the inner layer of follicle cells and comes to lie between the test cells and the inner layer of follicle cells in the mature egg (fig. 91A). At ovulation the chorion moves away from the surface of the oocyte. At this time also, the test cells move outward from their indentations in the peripheral ooplasm and come to lie in the perivitelline space between the egg surface and the chorion (fig. 9 IB). An ooplasmic membrane which

FILAMENTS

-LS(?)

MEMBRANE ZONA R A DIAT A

Fig. 93. Vitelline membranes of certain teleost fishes. (After Eigenmann, 1890.) (A)

Pygosteus pungtius. Radial section through micropyle of egg about 0.4 mm. in diameter. (B) Radial section through micropyle of egg of Perea, the perch. (C) Vitelline membranes of Fundulus heteroclitus about 0.8 mm. in diameter.

MATURATION OF GAMETES

165

represents the thin surface layer of ooplasm is present. However, it does not separate from the periphery of the egg at fertilization. During its early development, the embryo remains within this chorionic shell. The chorion thus represents the primary embryonic membrane of this species.

b) Egg Membranes of Amphioxus. Two surface membranes are formed and eventually separate from the egg of Amphioxus. The outer vitelline membrane is elaborated on the surface of the egg and remains in contact with this surface until about the time of the first maturation division. It then begins to separate from the egg’s surface. (See Chap. 5.) After the sperm enters and the second maturation division occurs, a second, rather thick, vitelline membrane also separates from the egg. The first and second vitelline membranes then fuse together and become greatly expanded to form the primary embryonic membrane. (See Chap. 5.) A thin ooplasmic membrane remains at the egg’s surface.

c) Vitelline Membrane and Zona Radiata of Elasmobranch Fishes. In the egg of the shark, Scy Ilium canicula, two egg membranes are formed, an outer and an inner membrane. The outer membrane is a homogeneous vitelline membrane, while the membrane which comes to lie beneath this outer membrane has a radiate appearance and hence may be called a zona radiata. This latter membrane soon loses its radiate appearance and becomes a thin membrane along the inner aspect of the vitelline membrane (fig. 92 A, B). In the mature egg both of these membranes form a thin, composite, vitelline membrane (fig. 92C, D). At about the time of fertilization the latter membrane separates from the egg’s surface; a perivitelline space then lies between these structures and the surface ooplasm of the egg.

d) Zona Radiata of Teleost Fishes. The surface ooplasm in teleost fishes gives origin to a membrane which in many cases has a radiate appearance. In some species this membrane appears to be composed of two layers. This radiate membrane which forms at the surface of the egg of teleost fishes appears to be the product of the ooplasm, and, therefore, should be regarded as a true vitelline membrane. In the perch a true chorion also is formed as a gelatinous or filamentous layer produced external to the radiate membrane by the follicle cells (fig. 93B). In Fundulus heteroclitus there are apparently three distinct parts to the membrane which surrounds the ooplasm of the egg:

( 1 ) a zona radiata,

(2) a thin structureless membrane external to the zona, and,

(3) the filamentous layer whose filaments are joined to the thin membrane around the zona (fig. 93C).

These three layers are probably derived from the ooplasm of the egg (Eigenmann, ’90). Consequently, the filamentous chorion or gelatinous layer, if derived from the egg itself, is not a true chorion in this particular egg.

VITELLINE MEMB RANE

Fig. 94. Vitelline membrane of an almost mature egg of the frog.

FO LL I C U L A R EPITHELIUM

PRO LON GAT IONS OF FOLLICULAR CELL OUTER LAYE R INNER LAYER KN 0 B-LI KE

ENLARGEMENTS OF FOLLICULAR P R0L0N6ATI0NS

FUNDAMENTAL

SUBSTANCE

Fig. 95. Zona radiata (zona pellucida) or vitelline membrane of Chrysemys picta.

(After Thing, ’18.)

FOLLICLE CELLS

Fig. 96. Zona radiata of the egg of the fowl. (After Brambell, ’25.)

166

MATURATION OF GAMETES

167

At one end of the forming egg, a follicle cell sends an enlarged pseudopodiumlike process inward to the surface of the egg. As a result of this enlarged extension of the follicle cell to the ooplasmic surface, an enlarged pore-like opening in the zona radiata is formed. This opening persists as the micropyle after the egg leaves the ovary (fig. 93A).

As the teleost egg is spawned, the chorionic layer hardens when it comes in contact with the water. If fertilization occurs, the surface of the egg emits a fluid and shrinks inward from the zona radiata. In this manner, a perivitelline space is formed between the egg, and the zona is filled with a fluid. The egg is thus free to revolve inside of the zona (Chap. 5).

e) Vitelline Membrane (Zona Radiata) in Amphibia. In the amphibia, a vitelline membrane is formed probably by the surface ooplasm, although there may be contributions by the follicle cells of the ovary (Noble, ’31, p. 281). This membrane separates from the egg at the time of fertilization, forming a perivitelline space (fig. 94). The latter space is filled with fluid. Later the vitelline membrane expands greatly to accommodate the developing embryo. A delicate surface layer or membrane forms the outer portion of the ooplasm below the vitelline membrane. In some amphibia the vitelline membrane may have a radiate appearance.

f) Zona Radiata (Zona Pellucida) of the Reptile Oocyte. In the turtle group, the development of the zona radiata (pellucida) appears to be the product of the follicle cells (Thing, ’18). Filamentous prolongations of the follicle cells extend to the surface ooplasm of the developing egg (fig. 95). A homogeneous substance produced by the follicle cells then fills the spaces between these prolongations. The filamentous extensions of the follicle cells in this way produce a radiating system of canals passing through the homogeneous substance; hence the name, zona radiata. Bhattacharya describes Golgi substance as passing from the follicle cells through the canals of the zona radiata into the egg’s ooplasm in the developing eggs of Testudo graeca and Uromastix hardwicki. (See Brambell, ’25, p. 147.)

In contradistinction to the above interpretation, Retzius (’12) describes the homogeneous substance which forms the zona radiata of the lizard, Lacerta viridis, as originating from the ooplasm of the egg.

g) Vitelline Membrane (Zona Radiata) of the Hen’s Egg. The vitelline membrane, as in the turtle groups, appears to form about the young oocyte as a result of contributions from the surrounding follicle cells although the superficial ooplasm of the oocyte may contribute some substance. This occurs before the rapid deposition of yolk within the developing oocyte. It is probable that the follicle cells send small pseudopodium-like strands of cytoplasm through the numerous perforations of the very thin vitelline membrane around the oocyte’s surface into the superficial ooplasm in a similar manner to that which occurs in reptiles. The vitelline membrane (zona radiata) thus assumes a radiate appearance as it increases in thickness (figs. 47, 96).

Fig. 97. Kinoplasmic bead or droplet upon the middle piece of mammalian sperm.

(A) Pig sperm. (After Retzius, Biol. Untersuchungen, New Serfes, 10; Stockholm: Jena.)

(B) Cat sperm. (After Retzius, Biol. Untersuchungen, New Series, 10; Stockholm: Jena.) (C-D) Dog sperm. (C) Upper part of epididymis. (D) Lower or caudal part of epididymis.

When the vitelline membrane thickens, the loci where the cytoplasmic strands from the follicle cells pass through the membrane become little canals or canaliculi. As the oocyte increases in size, a thin space forms between the vitelline membrane or zona radiata and the follicle cells; it is filled with fluid and forms the follicular space. The egg now is free to rotate within the follicle. In consequence, the pole of the egg containing the blastodisc always appears uppermost. Due to the increasing pendency of the egg follicle as the egg matures, the blastodisc comes to rest, a short while previous to ovulation, at the base of the pedicle where the blood vessels are most abundant (fig. 47B). During the latter phases of oocyte development, the vitelline membrane constitutes an osmotic membrane through which all nourishment must pass to the oocyte, particularly in its later stages of growth. Tlie surface ooplasm forms a delicate surface membrane beneath the zona radiata.

h) Membranes of the Mammalian Oocyte. All mammalian oocytes possess a membrane known as the zona pellucida. It is a homogeneous layer interposed between the ooplasm and the follicle cells. By some investigators it is regarded as a product of the oocyte, while others regard it as a contri

MATURATION OF GAMETES

169

bution of the ooplasm and follicle cells. The majority opinion, however, derives the zona pellucida from the follicle cells. In addition to the zona pellucida, the oocyte of the prototherian mammals has a striate layer lying close to the surface of the oocyte. This striated layer probably is derived from the surface ooplasm. This membrane later disappears, and a perivitelline space occupies the general area between the surface of the oocyte and the zona pellucida (fig. 46; Chap. 5). The zona pellucida separates from the egg surface after sperm contact.

5. Physiological Maturation of the Gametes a. Physiological Differentiation of the Sperm

Added to the nuclear and cytoplasmic transformations of the sperm described above, a further process of sperm ripening or maturing appears to be necessary. In the mammal, for example, the sperm cell must pass through the epididymis to achieve the ability to fertilize the egg. This is shown by the fact that sperm taken from the seminiferous tubules will not fertilize, although, morphologically, two sperm, one from the testis and one from the epididymis cannot be distinguished other than by the presence in some mammals of the so-called “kinoplasmic droplet” (figs. 82D, 97). These droplets do not appear in great numbers upon ejaculated sperm but are found on sperm, particularly in epididymides. It is possible that these droplets may arise from a secretion from the epididymal cells (CoIIery, ’44). In the dog, these droplets are attached to the neck of the sperm in the caput epididymidis but are found at the posterior end of the middle piece of the sperm in the cauda epididymidis and vas deferens and are probably lost at the time of ejaculation (Collery, ’44). Investigators differ greatly in interpreting the significance of this body. However, these droplets do seem in some way to be directly or indirectly concerned with the physiological maturing of the sperm. In this connection Collery (’44) notes that sperm are probably motile on leaving the seminiferous tubules, but active forward movement is not seen until the bead has reached the junction of middle piece and tail.

In the fowl, Domm (’30, p. 318) suggests the probability that the sperm may undergo an aging or ripening process essential for reproduction somewhere in the reproductive system other than the seminiferous tubules. The work of Lipsett quoted in Humphrey (’45) suggests that the accessory reproductive system also is necessary for a ripening process of the sperm in urodele amphibia.

On the other hand, in the frog, sperm taken from the testis have the ability to fertilize eggs. In this case, the sperm probably undergo a physiological ripening in the testis along with morphological differentiation.

The foregoing considerations suggest that a physiological maturation of the sperm is necessary to enable the sperm to take part in the fertilization process.

b. Physiological Ripening of the Female Gamete

The physiological maturing of the oocyte is linked to factors which influence the developing egg at about the time the maturation divisions occur. Sea-urchin sperm may penetrate the egg before the maturation divisions occur (Chap. 5). However, development does not take place in such instances. On the other hand, sperm entrance after both maturation divisions are completed initiates normal development. In the protochordate, Styela, marked cortical changes transpire at about the time the egg leaves the ovary, and as it reaches the sea water, the germinal vesicle begins to break down. The oocyte becomes fertilizable at about this time. In Amphioxus, although the first polar body is given off within the adult body, the egg apparently is not fertilizable until it reaches the external salt-water environment. The secondary oocyte of the frog presumably must remain within the uterus for a time to ripen in order that ensuing development may be normal. These and other instances suggest that physiological changes — changes which are imperative for the normal development of the egg — are effected at about the time that the maturation divisions occur.

D. Summary of Egg and Sperm Development

From the foregoing it may be seen that the development of the gametes in either sex involves a process of maturation. This maturation entails changes in the structure and constitution of the nucleus and cytoplasm, and, further, a functional or physiological ripening must occur. The comparative maturation events in the egg and sperm may be summarized as follows:

Egg ( in Oogenesis)

Sperm (in Spermatogenesis)

1. Nuclear maturation

a. Homologous chromosomes synapse and undergo profound changes during which parts of homologous chromosomes may be interchanged; ultimately, the chromosome number is reduced to the haploid number

b. Nucleus enlarges, and contained nuclear fluid increases greatly; ultimately the nuclear fluid is contributed to cytoplasm upon germinal vesicle break down

c. Nuclear maturation occurs simultaneously with cytoplasmic differentiation

1. Nuclear maturation

a. (Similar to the female)

b. Nucleus remains relatively small and enlargement is slight; nuclear fluid small in amount; during spermiogenesis the nucleus may contract into a compact mass; considerable elongation of nucleus occurs in many species

c. Nuclear maturation occurs before spermiogenesis or cytoplasmic differentiation

SUMMARY OF EGG AND SPERM DEVELOPMENT

Egg ( in Oogenesis)

Sperm (in Spermatogenesis)

2. Cytoplasmic maturation

This involves:

2. Cytoplasmic maturation

This involves:

a. Polarization of cytoplasmic materials and the nucleus in relation to the future maturation phenomena; the nucleus becomes displaced toward one pole, the animal pole, and the yolk, and other cytoplasmic materials; in many eggs becomes displaced toward the opposite or vegetal pole

a. Polarization of nucleus and cytoplasmic materials along an elongated antero-posterior axis, with the head, neck, middle piece, and tail occupying specific regions along this axis. The nucleus occupies a considerable portion of the anterior region or head

b. Formation of deutoplasm or stored food material, varying greatly in amount in different animal species. The deutoplasm is composed of fats, carbohydrates, and protein substances

b. Little food substances stored within cytoplasm; food reserve in seminal fluid

c. Cytoplasm increased in amount; formation of basic organ-forming areas or cytoplasmic stuffs from which the future embryo arises

c. Discarding of a considerable amount of cytoplasm, some Golgi elements and mitochondria. Retention of some Golgi elements, centrioles, mitochondria, etc.

d. Formation of primary embryonic membranes

d. No specific membranes formed around sperm, although elaborate membranes for motile purposes are formed in some sperm

3. Physiological maturation or the development of a fertilizable stage

This involves:

a. Formation of an organization which when stimulated by external influences initiates and carries on the processes necessary for normal embryonic development

3. Physiological maturation or the development of the ability to contact and fertilize the egg

This involves:

a. Development of an organization which, when stimulated by proper external substances, responds by a directed movement resulting in locomotion; also capable of being attracted by egg substances

b. Acquisition of ability to enter into a developmental union with a sperm

b. Acquisition of ability to fertilize, i.e., to enter into a developmental union with an egg or oocyte

c. Development of ability to form and secrete gynogamic substances which aid in the fertilization process. (See Chap. 5)

c. Acquisition of ability to produce and secrete androgamic substances which aid in the fertilization process

d. Assumption of an inhibited or dormant condition during which metabolic processes proceed slowly in anticipation of the fertilization event

d. Assumption of an active metabolic state

Bibliography

Asdell, S. A. 1946. Patterns of Mammalian Reproduction. Comstock Publishing Co., Inc., Ithaca, New York.

Benoit, J. 1930. Destruction des gonocytes primaires dans le blastoderme du poulet par les rayons ultra-violets, aux premiers stades du developpement embryonnaire. Proc. 2nd Internal. Cong, for Sex Research. p. 162.

Berrill, N. J. and Liu, C. K. 1948. Germplasm, Weismann, and Hydrozoa. Quart. Rev. Biol. 23:124.

Bolles, Lee A. 1897. Les cineses spermatogenetiques chez V Helix pomatia. La Cellule. 13:199.

Bounoure, L. 1939. L’origine des Cellules Reproductrices et le Probleme de la Lignee Germinale Gauthier Villars, Paris.

Boveri, T. 1887. Ober differenzierung der Zellkerne wahrend der Furchung des Eies von Ascaris rnegalocephala. Anat. Anz. 2:688.

. 1892. Ober die Entstehung des

Gegensatzes zwischen den Geschlechtszellen und den somatischen Zellen bei Ascaris rnegalocephala. Sitz. d. Gesellsch. f. Morph, u. Physiol. Munchen. 8:114.

. 1910a. Ober die Teilung centrifu gierter Eier von Ascaris rnegalocephala. Arch. f. Entwicklungsmech der Organ. 30(2): 101.

. 1910b. Die Potenzen der Ascaris Blastomeren bei abgeanderter Furchung. Festschrift fur R. Hertwig. 3:131.

Bowen, R. H. 1922. On the idiosome, Golgi apparatus, and acrosome in the male germ cells. Anat. Rec. 24:159.

. 1922. Studies on insect spermatogenesis. J. Morphol. 37:79.

Brachet, J. 1947. Nucleic acids in the cell and the embryo, in Symposia of the Society for Experimental Biology, No. 1, Nucleic Acid. p. 207.

. 1950. Chemical Embryology. Interscience Publishers, Inc., New York.

Brambell, F. W. R. 1925. The oogenesis of the fowl (Callus bankiva). Philos. Tr. Roy. Soc., London, s.B. 214:113.

. 1930. The Development of Sex in

Vertebrates. The Macmillan Co., New York.

, Parkes, A. S., and Fielding, V.

1927a. Changes in the ovary of the mouse following exposures to x-rays. I. Irradiation at three weeks old. Proc. Roy. Soc., London, s.B. 101:29.

, , and . 1927b.

Changes in the ovary of the mouse following exposures to x-rays. 11. Irradiation at or before birth. Proc. Roy. Soc., London, s.B. 101:95.

Brown, G. L., Callan, H. G., and Leaf, G. 1950. Chemical nature of the nuclear sap. Nature, London. 165:600.

Collery, L. 1944. Note on the physiology of the mammalian epididymis and spermatozoon. Proc. Roy. Irish Acad. 49: Section B, 213.

Conklin, E. G. 1905. The organization and cell-lineage of the ascidian egg. J. Acad. Nat. Sc. Philadelphia. 13:5.

. 1932. The embryology of Amphi oxus. J. Morphol. 54:69.

De Robertis, E. D. P., Nowinski, W. W., and Saez, F. A. 1948. General Cytology. W. B. Saunders Co., Philadelphia.

Domm, L. V. 1930. Artificial insemination with motile sperm from ovariectomized fowl. Proc. Soc. Exper. Biol. & Med. 28:316.

Durkin, B. 1932. Experimental Analysis of Development. W. W. Norton & Co., Inc., New York.

Duryee, W. R. 1950. Chromosomal physiology in relation to nuclear structure. Ann. New York Acad. Sc. 50:920.

Eigenmann, C. H. 1890. On the egg membranes and micropyle of some osseous fishes. Bull. Mus. Comp. Zool. at Harvard College. 19:129.

Evans, H. M. and Swezy, O. 1931. Ovogenesis and the normal follicular cycle in adult mammalia. Memoirs. The University of California Press. 9: No. 3.

Everett, N. B. 1945. The present status of the germ-cell problem in vertebrates. Biol. Rev. 20:45.

Fankhauser, G. 1948. The organization of the amphibian egg during fertilization and cleavage. Ann. New York Acad. Sc. 49:684.

Flynn, T. T. and Hill, J. P. 1939. The development of the Monotremata, Part IV. Growth of the ovarian ovum, maturation, fertilization and early cleavage. Tr. Zool. Soc. London. 24: Part 6, 445.

Gatenby, J. B. and Beams, H. W. 1935. The cytoplasmic inclusions in the spermatogenesis of man. Quart. J. Micr. Sc. 78:1.

and Woodger, J. H. 1921. The

cytoplasmic inclusions of the germ cells. Part IX. On the origin of the Golgi apparatus on the middle-piece of the ripe sperm of Cavia, and the development of the acrosome. Quart. J. Micr. Sc. 65:265.

Goldsmith, J. B. 1935. The primordial germ cells of the chick. 1. The effect on the gonad of complete and partial removal of the “germinal” crescent and of removal of other parts of the blastodisc. J. Morphol. 58:537.

Hegner, R. W. 1914. The germ cell cycle in animals. The Macmillan Co., New York.

Heys, F. 1931. The problem of the origin of germ cells. Quart. Rev. Biol. 6:1.

Humphrey, R. R. 1925. The primordial germ cells of Hemidactylium and other Amphibia. J. Morphol. & Physiol. 41:1.

. 1927. Extirpation of the primordial germ cells of Amblystoma; its effect upon the development of the gonad. J. Exper. Zool. 49:363.

. 1945. Sex determination in A ruby stomid salamanders. Am. J. Anat, 76:33.

Jones, R. M. 1949. The use of vital staining in the study of the origin of germ cells in the female rat, Mas norwegicus. J. Morphol. 84:293.

King, R. N. and Beams, H. W. 1938. An experimental study of chromatin diminution in Ascaris. J. Exper. Zool. 77:425.

Leblond, C. P. 1950. Distribution of periodic acid-reactive carbohydrates in the adult rat. Am. J. Anat. 86:1.

Leuchtenberger, C. and Schrader, F. 1950. The chemical nature of the acrosome in the male germ celt^. Proc. Nat. Acad. Sc. 36:677.

Marechal, J. 1907. Sur I’ovogenese des selaciens et de quelques autres chordates. La Cellule. 24:15.

Moore, C. R. and Wang, H. 1947. Ovarian activity in mammals subsequent to chemical injury of the cortex. Physiol. Zool. 20:300.

Nieuwkoop, P. D. 1947. Experimental investigations on the origin and determination of the germ cells, and on the development of the lateral plates and germ ridges in urodeles. Arch, neerl. de zool. 8 : 1 .

. 1949. The present state of the

problem of the “Keimbahn” in the vertebrates. Experientia. 5:308.

Noble, G. K. 1931. Biology of the Amphibia. McGraw-Hill Book Co., Inc., New York.

Northrop, J. H. 1949. Chap. I. Enzymes and the synthesis of proteins in The Chemistry and Physiology of Growth. Edited by Parpart, A. K. Princeton University Press, Princeton, New Jersey.

Nussbaum, M. 1880. Zur Differenzirung des Geschlcchts im Thierreich. Arch. f. mikr. anat. 18:1.

Palade, G. E. and Claude, A. 1949, The nature of the Golgi apparatus. 1. Parallelism between intercellular myelin figures and Golgi apparatus in somatic cells. II. Identification of the Golgi apparatus with a complex of myelin figures. J. Morphol. 85:35, 71.

Parkes, A. S. 1927. On the occurrence of the oestrous cycle after x-ray sterilization. Part III. The periodicity of oestrus after sterilization of the adult. Proc. Roy. Soc. London, s.B. 101:421.

Pincus, G. 1936. The Eggs of Mammals. The Macmillan Co., New York.

Retzius, G. 1912. Zur Kenntnis der Hiillen und besonders des Follikelepithelels an den Eiern der Wirbeltiere. Biol. Untersuch. N.F. 17:1.

Sharp, L. W. 1934. Introduction to Cytology. 3d ed., 441-445. McGraw-Hill Book Co., Inc., New York. .

1943. Fundamentals of Cytology. 1st ed. McGraw-Hill Book Co., Inc., New York.

Snyder, L. H. 1945. The Principles of Heredity. 3d ed. D. C. Heath and Co., New York, Boston, London.

Thing, A. 1918. The formation and structure of the zona pellucida in the ovarian eggs of turtles. Am. J. Anat. 23:237.

Tucker, G. H. 1942. The histology of the gonads and development of the egg envelopes in an ascidian (Styela plicata Lesueur). J. Morphol. 70:81.

van der Stricht, O. 1923. Etude comparee des ovules des mammiferes aux differentes periodes de I’ovogenese. Arch, biol., Paris. 33:229.

Vincent, W. S. and Dornfeld, E. J. 1948. Localization and role of nucleic acids in the developing rat ovary. Am. J. Anat. 83:437.

Waldeyer, W. 1870. Eierstock und Ei. Wilhelm Engelmann, Leipzig.

Weismann, A. 1893. The Germ-plasm — A Theory of Heredity. English translation by W. N. Parker and H. Ronnfeldt. Walter Scott, Ltd., London.

White, M. J. D. 1945, Animal Cytology and Evolution. Cambridge University Press, London.

Willier, B. H. 1937. Experimentally produced sterile gonads and the problem of the origin of germ cells in the chick embryo. Anat. Rec. 70:89.

Wilson, E. B. 1925. The Cell in Development and Heredity. 3d ed., p. 310. The Macmillan Co., New York.

Winiwarter, H. von. 1900. Recherches sur I’ovogenese et I’organogenese de I’ovaire des mammiferes (lapin et homme). Arch, biol., Paris. 17:3.

Witschi, E. 1939. Chap. IV in Allen, et al.. Sex and Internal Secretions. 2d ed.. The Williams and Wilkins Co., Baltimore.

. 1948. Migration of the germ cells of human embryos from the yolk sac to the primitive gonadal folds. Carnegie Inst., Washington Publ. 575. Contrib. Embryol. 32:67.