Book - The Frog Its Reproduction and Development 3

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Chapter 3 - Reproductive System of the Adult Frog Rana pipiens=

The Male

Secondary Sexual Characters Primary Sexual Characters The Testes Spermatogenesis Reproductive Behavior Accessory Structures

The Female

Secondary Sexual Characters

Primary Sexual Characters The Ovaries

The Body Cavity and the Oviducts Oogenesis — Maturation of the Egg

The Male

Secondary Sexual Characters

The mature male frog is generally smaller than the female, ranging from 60 to 110 mm. in length from snout to anus. The identifying features which distinguish it from the female are a darkened thumb pad which changes thickness and color intensity as the breeding season approaches; a distinct low, guttural croaking sound with the accompanying swelling by air of the lateral vocal sacs located between the tympanum and the forearm; a more slender and streamlined body than that of the female; and the absence of coelomic cilia except in the peritoneal funnels on the ventral face of the kidneys. Males of many species carry additional features such as brilliant colors on the ventral aspects of the legs {R. sylvatica), black chin {B. fowleri), or the size and color of the tympanic membrane.

Primary Sexual Characters

The Testes

The testes of the frog are paired and internal organs and are suspended to the dorsally placed kidneys by a double fold of peritoneum known as the mesorchium. This mesentery surrounds each testis and is continuous with the peritoneal epithelium which covers the ventral face of each kidney and lines the entire body cavity.

The testes are whitish and ovoid bodies lying ventral to and near the anterior end of each kidney. The vasa efferentia, ducts from the testes, pass between the folds of the mesorchium and into the mesial margin of the adjacent kidney. During the breeding season, or after slight compression of the testis of the hibernating frog, these ducts become the more apparent due to the presence in them of whitish masses of spermatozoa in suspension. The ducts are very small in diameter, tough walled, and interbranching. They are lined with closely packed cuboidal cells. Each duct is connected directly with a number (8 to 12) of Malpighian corpuscles of the kidneys, by way of the Bowman's capsules. These connections are permanent so that many of the anterior uriniferous tubules of the frog kidney will contain spermatozoa during the breeding season. The presence of spermatozoa in the kidney also can be achieved artificially by injecting the male frog with the anterior pituitary sex-stimulating hormone. Since these anterior Malpighian corpuscles carry both spermatozoa (during the breeding season) and excretory fluids (at all times), they are truly urogenital ducts having a dual function. This situation does not hold for higher vertebrates.

The spermatozoa are produced in subdivisions of the testes known as seminiferous tubules. These are closely packed, oval-shaped sacs, which are separated from each other by thin partitions (septula) of supporting (connective) tissue known as interstitial tissue. This tissue presumably has some endocrine function. The thickness of this tissue is much reduced immediately after breeding or pituitary stimulation. The interstitial tissue is continuous with the covering of the testes known as the tunica albuginea, and the whole testis is enclosed in the thin peritoneal epithelium.


Shortly after the normal breeding season in the spring for Rana pipiens, the spermatogonium, which has ceased all mitotic activity, enters upon a period of rest but not inactivity. During this period the nucleus passes through a sequence of complex changes which represent an extended prophase. This is in anticipation of the two maturation divisions that finally produce the haploid spermatid which metamorphoses into a spermatozoon.

The nucleus of the spermatogonium contains chromatin which appears as relatively coarse lumps distributed widely over an achro

Primordiol germ cell - 2N

Primary oocyte {2N)



First polar body (Meiotic •Reductionol division)

®N-Secondary spermatocyte (2N / (Meiotic = Reductional division)

d) j-Spermatid (N) (Metamorphosis)

Spermatozoon (N)

Frog's egg remains at metoptiase of second maturation division until it IS fertilized or dies

The maturation process. Schematized drawings. The post-reductional division is shown. In many forms the first division is reductional and the second is equational. The end result is the haploid gamete, in either instance.

matic reticulum. Both the cytoplasm and the nucleus grow and the chromatic granules become finely divided and arranged into contiguous rows, bound by an achromatic thread, together known as chromosomes. This is the leptotene stage of spermatogenesis. Shortly the chromosomes become arranged in pairs which converge toward that side of the nucleus where the centrosome is found. The opposite ends of the paired chromosomes merge into the general reticulum. This is the synaptene stage. The chromatin granules become telescoped together on the filaments so that the aggregated granules, known as chromosomes, appear much shorter and thicker. Pairs of chromosomes become intertwined and the loose terminal ends become coiled and tangled together. This is the contraction or synizesis stage. Then the members of the various pairs become laterally (parabiotically) fused. While there is no actual reduction in total chromatin, there is a temporary and an apparent (but not real) reduction in the total number of chromosomes to the haploid condition, because of this fusion. There is no actual reduction in the total amount of chromatin

Prophases of the heterotype division in the male Axolotl. (1) Nucleus of spermogonium or young spermocyte. (2) Early leptotene. (3) Transition to synaptene. (4) Synaptene with the double filaments converging toward the centrosome. (5) Contraction figure. (6, 7) Pachytene. (8) Early diplotene. (9) Later diplotene. (10) The heterotypic double chromosomes; the nuclear membrane is disappearing. (Courtesy, Jenkinson: "Vertebrate Embryology," Oxford, The Clarendon Press.)

material, nor is there any permanent reduction at this stage in the number of chromosomes. Their identity is lost only temporarily. This is known as the pachytene stage. The members of each pair then separate again. It must be remembered, however, that (a) the separation need not be along the original line of fusion and that (b) an exchange of homologous sections of the chromosomes may occur without any cytological evidence. In any case, the diploid number of chromosomes reappears and this is then known as the diplotene stage.

During these changes in the chromatin material of the nucleus, the volume of the nucleus and the cytoplasm are considerably increased, the nuclear membrane breaks down, and the chromosomes assume bizarre shapes and various sizes. They may be paired, curved, or straight; "V" and "C" and reversed "L" shapes, figure 8's, and grouped as tetrads. This is known as the diakinesis stage. The chromosomes are then lined up on a spindle in anticipation of the first of the two maturation divisions.

Spermatogenesis in the frog is seasonal and is completed within the testes. The walls of the seminiferous tubules produce spermatogonia which go through mitotic divisions and then the series of nuclear changes (described above) without mitosis. This results in the appearance, toward the lumen of each tubule, of clusters of mature spermatozoa. By the time of hibernation (October) all the spermatozoa that are to become available for the following spring breeding season will have matured. At this time the testis will exhibit only these spermatozoa and relatively few spermatogonia, without the intervening maturation stages. The spermatogonia are found close to the basement membrane of the seminiferous tubule. These then await their turn to undergo the maturation changes necessary for the production of spermatozoa which will be ready for the breeding season a year and a half thereafter. The elongated and filamentous


May June July August September October November Dec- Mor.

Normal cyclic changes in the primary and secondary sexual characters of the frog, Rana pipiens. (From Glass and Rugh, 1944, J. MorphoL, 74:409.)

Spermatozoa -l*^**^.. « // /^^-^

Spermatogonia^^** "^^Jf j-*t •^•' ' % .

Sperm bundle Sertoli cell nucleus Interstitial tissue,

[^'spermatocytes \ Tunica olbuginea Spermatozoa Low cuboidal epithelium;


jLumen\ Sertoli cell nuclei \ Spermatids \ Seminiferous

Spermatids Spermatogonia ^'spermatocyte tubules


Collecting tubule

Spermatogenesis in the frog: Rana pipiens. (A) Testis of a recently metamorphosed frog. (B) Similar to "A" except that the frog was previously treated with the anterior pituitary hormone to evacuate the seminiferous tubules, leaving only

{Continued on facing page.)

Primary spermatocyte

Secondary spermatocytes

Sertoli cell


Second maturation division figures


Lumen of tubule

Heads of

mature spermatozoa

Spermatogenetic stages in the seminiterous tubule of the frog testis.

tails of the clustered mature spermatozoa project into the lumen of each seminiferous tubule.

If one studies the July or August testis, which organ is then in

its height of spermatogenetic activity, he can find all the stages of

(Continued from opposite page.) the interstitial tissue, spermatogonia, and a few primary spermatocytes. The post-breeding condition. (C) High-power magnification of "A." (D) High-power magnification of "B." (E) Testis of an August frog, showing all stages of spermatogenesis. (F) Partially (pituitary) activated testis, similar to "E," showing released spermatozoa within the lumen, and all stages of spermatogenesis around the periphery of the seminiferous tubule. (G) Testis of a hibernating, pre-breeding frog. Note the clusters of mature spermatozoa attached to single Sertoli cells. The lumen is filled with tails, few spermatogonia, and primary spermatocytes around the periphery of the seminiferous tubule. (H) Testis of the male during amplexus, showing spermatozoa liberated into the lumen of the seminiferous tubule. (I) Collecting tubule of the frog testis full of mature spermatozoa, showing their origin from the connecting seminiferous tubules. Collecting tubules are lined with low cuboidal epithelium and are continuous with the vasa efferentia and the Malpighian corpuscles of the kidney.

maturation from the spermatogonium to the spermatozoon. The spermatogonia are always located around the periphery of the seminiferous tubule and are small, closely packed cells, each with a granular, oval nucleus. In between the spermatogonia may be found occasional very large cells, the primary spermatocytes. These tend to be irregularly spherical, possessing large and vesicular nuclei. The cells are so large that they may be seen under low power magnification (X 100) of the microscope. Apparently they divide to form secondary spermatocytes almost immediately, for they are so few and far between. The secondary spermatocytes (which develop as the result of the first division) are about half the size of the primaries, and lie toward the lumen of the tubule. They generally have a darkly staining nucleus, and the cytoplasm may be tapered toward one side. The spermatid, following another division, is even smaller and possesses a condensed nucleus of irregular shape. Clusters of spermatids appear as clusters of granules, the dark nucleus being almost as small as the cross section of a sperm head. The metamorphic stages from spermatid to spermatozoon are difficult to identify with ordinary magnification, and are often confused with the spermatids themselves. During this change the inner of two spermatid centrioles passes into the nucleus while the outer one gives rise to the tail-like flagellum.

The mature spermatozoon averages about 0.03 mm. in length. It has an elongated, solid-staining head (nucleus) with an anterior acrosome, pointing outwardly toward the periphery of the seminiferous tubule. The short middle piece generally is not visible but the tail appears as a gray filamentous extension into the lumen, about four or more times the length of the sperm head.

In any cross section of the testis, bundles of sperm heads or tails may be cut at right angles or tangentially, giving misleading suggestions of structure. The mature spermatozoon is dependent upon external sources of nutrition so that it joins from 25 to 40 other spermatozoa, all of whose heads may be seen converging into the cytoplasm of a relatively large, columnar-type basal cell known as the Sertoli cell. This is functionally a nurse cell, supplying nutriment to the clusters of mature spermatozoa until such

Frog spermatozoon. Total length 0.03 to 0.04 mm.

Kidney of the male frog during amplexus showing spermatozoa in the kidney tubules and Malpighian corpuscles, en route to the uro-(mesonephric) -genital (vasa efferentia) duct.

time as they may be liberated through the genital tract to function in fertilization.

In observing a section of the summer testis of the frog under low power magnification, it is readily apparent that each seminiferous tubule may contain all the stages of maturation and that each stage is found in a cluster or group within the tubule. Each group of similar cells is derived presumably from a single original spermatogonium, by the processes of mitosis and meiosis. This is reminiscent of the condition found in the grasshopper (Rhomaleum) testis. Maturation of the germ cells occurs in groups so that when the spermatid stage is reached, the tips of the metamorphosing spermatozoon heads are all gathered together into the cytoplasm of the Sertoli cell. Spermatozoa may remain thus throughout the entire period of hibernation only to be liberated under the influence of sex-stimulating hormones during the early spring. These spermatozoa are functionally mature, as can be demonstrated by dissecting them from the testes and using them to fertihze frogs' eggs artificially at any time from late in August until the normal breeding season in April or May.

tectum inferior colliculus ^thalamus

nucleus N.m

nucleus N.iz

tegmentum -- pg^s dorsalis

tiypothaJamus l olfactory bulb cerebral hemisphere preoptic area

Diagrammatic sagittal section through the brain of Rami pipiens indicating the regions of the brain that were found to be of primary importance for the mediation of each of four phases of sexual behavior. (From Aronson, 1945, Bull. Am. Mas. Nat. Hist., 86:89.)

Reproductive Behavior

It has been proved definitely that the anterior pituitary hormone causes the release of the mature spermatozoa from the testis. But this hormone also releases other maturation stages. It is therefore probable that there are smooth muscle fibers, either among the interstitial cells or in the tunica albuginea of the testes, which fibers contract to force the spermatozoa from the seminiferous tubules. It would be as difficult to physiologically demonstrate the presence of these fibers in the testis as it is simple to demonstrate them in the contracting cyst wall of the ovary.

Responding to sex stimulation, the spermatozoa become free from their Sertoli cells and are forced from the lumen of the seminiferous tubule into the related collecting tubule. These collecting tubules are small and are lined with closely packed cuboidal cells. They join the vasa efferentia which leave the testis to pass between the folds of the mesorchium and thence into the Malpighian corpuscles of the kidney. From this point the spermatozoa pass by way of the excretory ducts, the uriniferous tubules, and into the mesonephric duct (ureter) which may be found attached to the lateral margin of the kidney. Within the excretory system the spermatozoa are immotile, due to the slightly acid environment. They are carried passively down the ureter to the slight dilation near the cloaca, known as the seminal vesicle. Within the vesicle the spermatozoa are stored briefly in clusters until amplexus and oviposition occur. At oviposition the male ejaculates the spermatozoa into the neutral or slightly alkaline water where they are activated and then are able to fertilize the eggs as they emerge from the cloaca of the female.

During the normal breeding season amplexus is achieved as the females reach the ponds where the males are emitting their sex calls. During amplexus there are definite muscular ejaculatory movements on the part of the male frog, coinciding with oviposition on the part of the female. Amplexus may be maintained by the male for many days, even with dead females. As soon as the eggs are laid and the male has shed his sperm, he goes through a brief weaving motion of the body and then releases his grip to swim away. The frogs completely neglect the newly laid eggs.

Accessory Structures

In the male frog the ureter is not directly connected with the bladder, as it is in higher vertebrates. It is possible that the bladder in the Anura may be an accessory respiratory and hydrating organ, particularly in the toads, where water may be stored during migrations onto land.

The male frog also has a duct, homologous to the oviduct of the female, known as the "rudimentary oviduct" or Miillerian duct. This duct normally has no lumen, and is very much reduced in size so that it may be difficult to locate. There is experimental evidence that this duct may be truly a vestigial oviduct since it responds to ovarian or female sex hormones by enlarging and acquiring a lumen.

At the anterior end of the testes of some Anura (e.g., toads) there may be found an undeveloped ovary known as Bidder's organ. This structure is said to respond to the removal of the adjacent testis or to the injection of female sex hormones by enlarging to become structurally like an ovary. Occasionally isolated ova have been found within the seminiferous tubules of an otherwise normal testis, suggesting the similar origin and the fundamental similarity of the testis and the ovary.

Urogenital system of the male frog. The Miillerian ducts (vestigial oviducts) arc seen lateral to the kidneys. They respond to female sex-stimulating hormones. They converge with the Wolffian (mesonephric) duct at the cloaca. Note that the left testis is usually smaller than the right. The vasa efferentia pass between the folds of the mesorchium into the dorsally placed kidneys where they join the uriniferous tubules at the glomeruli.

Finally, attached to the anterior end of the testis of the hibernating frog may be seen finger-like fat bodies (corpora adiposa) which represent stored nutrition for the long period of hibernation, and for the pre-breeding season when food is scarce. Under the microscope these fat bodies appear as clusters of vacuolated cells, and are not to be confused with the mesorchium. It is believed that they, as well as the gonads, arise from the genital ridges of the early embryo. The fat bodies tend to be reduced immediately after the breeding season, only to be built up again as the time for hibernation approaches.

The Female

Secondary Sexual Characters

The mature female frog is generally larger than the male of the same age and species, the Rana pipiens female measuring from 60 to 1 10 mm. in length from snout to anus. The sexually mature female has a body length of at least 70 mm. It can be identified by the absence, at any season, of the dark thumb pad; the inability to produce lateral cheek pouches resulting from the croaking reaction; a flabby and distended abdomen; and the presence of peritoneal cilia. These cilia are developed in the female in response to the prior development and secretion of ovarian hormones.

Primary Sexual Characters

The Ovaries

The ovaries of the frog are paired, multi-lobed organs, attached to the dorsal body wall by a double-layered extension of the peritoneum known as the mesovarium. This peritoneum continues around the entire ovary as the theca externa. Each lobe of the ovary is hollow and its cavity is continuous with the other 7 to 12 lobes. The ovaries of the female are found in the same relative position as the testes of the male but the peritoneum extends from the dorso-mesial wall rather than from the kidneys, as in the male.

The size of the ovary varies with the seasons more than does the size of the testis. From late summer until the spring breeding season the paired ovaries will fill the body cavity and will often distend the body wall. They may contain from 2,000 {Rana pipiens) to as many as 20,000 eggs {Rana catesbiana) , each measuring about 1.75 mm. in diameter {Rana pipiens). The mature eggs are highly pigmented on the surface of the animal pole, so that the ovary has a speckled appearance of black pigment and white yolk, representing the animal and the vegetal hemispheres of the eggs.

There is no appreciable change in the size of the ovary during hibernation, nor is there any observable cytological change in the ova. However, if a female is forced to retain her eggs beyond the normal breeding period by isolating her from males or by keeping her in a warm environment and without food, the ova will begin to deteriorate (cytolize) within the ovary. Immediately after the spring breeding season, when the female discharges thousands of mature ova, the remaining ovary with its oogonia (to be developed for the following year) is so small that it is sometimes difficult to locate. There is no pigment in the tissue of the ovary (in the stroma or in the immature ova), and each growing oocyte appears as a small white sphere of protoplasm contained within its individual follicle sac.

Late development of the frog ovary.

The histology of the ovary shows that within its outer peritoneal covering, the theca externa, are suspended thousands of individual sacs, each made up of another membrane, the theca interna or cyst wall, which contains smooth muscle fibers. This theca interna is derived from the retro-peritoneal tissue. The smooth muscle fibers can be seen histologically and can be demonstrated physiologically. The theca interna surrounds each egg except for the limited area bulging toward the body cavity, where it is covered by only the theca externa. This is the region which will be ruptured during ovulation to allow the egg to escape its follicle into the body cavity. The theca interna, plus the limited covering of the theca externa, and the follicle cells together comprise the ovarian follicle. These two membranes make up the rather limited ovarian stroma of the frog ovary, and they contain both blood vessels and nerves. Within each follicle are found follicle cells, with their oval and granular nuclei, derived originally from oogonia. These follicle cells surround the developing oocyte and are found in close association with it throughout those processes of maturation which occur within the follicle. Enclosed within the follicle cells, and closely applied to each mature egg, is the non-cellular and transparent vitelline membrane, probably derived from both the ovum and the follicle cells. This membrane is developed and applied to the egg during the maturation process so that it is not seen around the earlier or younger oogonia. Since the bulk of the egg is yolk (vitellus), this membrane is appropriately called the vitelline membrane. It is sometimes designated as the primary (of several) egg membranes. After the egg is fertilized this membrane becomes separated from the egg and the space between is then known as the perivitelline space, filled with a fluid. The fluid may be derived from the egg which would show compensatory shrinkage. As the oocyte matures and enlarges, the follicle cells and membranes are so stretched and flattened that they are not easily distinguished. It is therefore best to study these structures in the immature ovary.

The egg will mature in any of a variety of positions within its follicle, the exact position probably depending upon the maximum blood supply. As one examines an ovary the eggs will be seen in all possible positions, some with the animal hemisphere and others with the vegetal hemisphere toward the theca externa and body cavity. It is believed that the most vascular side of the follicle wall will tend

Small ovarian egg of the frog surrounded by its follicle (/.) and theca {th.), which is continued into the pedicle (p.). {h.v.) A blood vessel between follicle and theca. (v.m.) Vitelline membrane, (c/z.) Chromatin filaments, now achromatic. («.) Chromatic nucleoli, ejected from the nucleus (n'.) and becoming achromatic (/!".). (Courtesy, Jenkinson: "Vertebrate Embryology," Oxford, The Clarendon Press.)

Section of a mature ovarian egg to show the area of ultimate follicular rupture and the surrounding membranes of the egg.

Blood vessel

Theca externa

ThecQ interna

Theca externa ThecG interna

Follicle cells

Figure 2

Theca interna Pigment

Figure 3

Point of follicular rupture Follicle ceils | ^^/ Theca externa

Blood vessel


Point of follicular rupture

Follicle cell

Follicle cells Theca interna Theca externa Blood vessel

Figure 4

to produce the animal hemisphere of the egg, and hence give it its fundamental symmetry and polarity.

The frog's egg is essentially a large sac of yolk, the heavier and larger granules of which are concentrated at the vegetal pole. There is a thin outer layer of cytoplasm, more concentrated toward the animal hemisphere and in the vicinity of the germinal vesicle or immature nucleus. Surrounding the entire egg is a non-living surface coat, also containing pigment. This pigment is presumably a metabolic byproduct. This coat is necessary for retaining the shape of the egg and in aiding in the morphogenetic processes of cleavage and gastrulation (Holtfreter).

The Body Cavity and the Oviducts

Lateral to each ovary is a much-coiled oviduct suspended from the dorsal body wall by a double fold of peritoneum. Its anterior end

Reactions of the frog ovary to stimulation by the frog anterior pituitary hormone. (A) Ovary of a female receiving inadequate injection of the pituitary hormone, partially emptying the eggs into the body cavity. (B) Ovary of a female receiving almost enough pituitary hormone to empty all of its follicles, one lobe alone retaining some of its eggs. During the breeding season the female's own pituitary gland is sufficient to bring about complete ovulation of all eggs.

Photograph of Rana pipiens female body cavity at the height of ovulation.

is found between the heart and the lateral peritoneum, at the apex of the liver lobe. At this anterior end is a slit-like infundibulum or ostium tuba with ciliated and highly elastic walls. The body cavity of the female is almost entirely lined with cilia, each cilium having its effective beat or stroke in the general direction of one of the ostia. These cilia are produced in response to an ovarian hormone and therefore are regarded as secondary sex characters. They are found on the peritoneum covering the entire body cavity, on the liver, and on the pericardial membrane. There are no cilia on the lungs, the intestines, or the surface of the kidneys except in the ciliated peristomial (peritoneal) funnels which lead into the blood sinuses of the kidneys. The abundant supply of cilia of the female means that eggs ovulated from any surface of the ovary will be carried by constant ciliary currents anteriorly toward and into one or another of the ostia. This can be demonstrated easily by opening the body cavity of an actively ovulating frog or by excising a strip of ventral abdominal wall of the adult female, inverting it in amphibian Ringer's solution, and placing on it some of the body cavity eggs. Any object of

Deposition of jelly on the frog's egg. (Top) String of eggs removed from the oviduct to show the deposition of jelly. The jelly is swollen in water. (Bottom) Frog's eggs showing varying amounts of jelly, indicating progressive deposition along the oviduct. (A) From upper third of oviduct. (B) From middle of oviduct. (C) From body cavity (no jelly). (D) From uterus.

Recently emptied ovarian follicle of the frog.

similar size or weight, such as pellets of paraffin, will be carried along by the ciliary currents in the original direction of the ostium. These cilia function the year around, and will carry to the ostia any objects of approximately the size and weight of frog eggs that may be placed in the body cavity. One might suggest, therefore, that the oviducts may act as accessory excretory ducts, for certainly body cavity fluids must be similarly eliminated.

As the egg leaves the ovary it is nude except for the non-living, transparent, and closely applied vitelline membrane. Thus far it has been impossible to fertilize these body cavity eggs and have them develop. When they are placed in a sperm suspension some will show surface markings which resemble very closely the normal cleavage spindles and the cleavage furrows but none have developed as embryos as yet. These body cavity eggs are often quite distorted, due to the fact that the ovulation process involves a rupture of the follicle and forcing out of the egg from a very muscular follicle. The egg is

Follicular rupture and ovulation in the frog. {Top, left) Three eggs emerging simultaneously from their follicles. {Top, right) Eggs in various stages of emer (Continued on facing page.)

Photograph of the open body cavity of an actively ovulating female frog showing the entrance of an egg into the ostium.

literally squeezed from the follicle, through a small aperture. The process looks like an Amoeba crawling through an inadequate hole. Ovulation (rupture and emergence of the egg) takes several minutes at laboratory temperatures, and is not accompanied by hemorrhage. By the time the egg reaches the ostium (within 2 hours), as the result of ciliary propulsion, it is again spherical.

Ciliary currents alone force the egg into the ostium and oviduct. The ostial opening is very elastic and does not respond to the respira (Continued from opposite page.) gence and adjacerrt empty follicles. (Center) Egg about to drop free into the body cavity, showing degree of constriction by the follicular opening. (Bottom) Excised follicles of an ovulating frog continuing the process of emergence of eggs. Note the plasticity of the egg at this time.

tory or heart activity, as some have described. The eggs are simply forced into the ostium, from all angles, stretching its mouth open to accept the egg. As soon as the egg enters the oviduct and begins to acquire an albuminous (mucin-jelly) covering, it becomes fertilizable. One can remove such an egg from the oviduct by pipette or by cutting the oviduct 1 inch or more from the ostium, and can fertilize such an egg in a normal sperm suspension. The physical (or chemical) changes which occur between the time the egg is in the body cavity and the time it is removed from the oviduct, which make it fertilizable, are not yet understood.

As the egg is propelled through the oviduct by ciliary currents, it receives coatings of albumen (jelly). The initial coat is thin but of heavy consistency, and is applied closely to the egg. The egg is spiraled down the oviduct by its ciliated lining so that the application of the jelly covering is quite uniform. There are, in all, three distinct layers of jelly, the outermost one being much the greater in thickness but the less viscous. The intermediate layer is of a thin and more fluid consistency. There is hyperactivity of the glandular elements of the oviduct just before the normal breeding season, or after anterior pituitary hormone stimulation, so that the duct is enlarged several times over that of the oviduct of the hibernating female.

The presence of the jelly layers on the oviducal or the uterine egg

Distribution of coelomic cilia within the body cavity of the female frog. (Left) Schematic section through the level of the ovaries. (Right) Schematic drawing of the open body cavity. The cilia in the body cavity of the female develop in response to the elaboration of an ovarian hormone, and function in propelling the eggs to the two ostia.

is not readily apparent because it requires water before it reaches its maximum thickness. Eggs sectioned within the oviduct show the jelly as a transparent coating just outside the vitelline membrane. As soon as the egg reaches the water, however, imbibition swells the jelly until its thickness becomes greater than the diameter of the egg

The function of the jelly is to protect the egg against injury, against ingestion by larger organisms, and from fungus and other infections. Equally important, however, is the evidence that this jelly helps the egg to retain its metabolically derived heat so that the jelly can be said to act as an insulator against heat loss. Bernard and Batuschek (1891) showed that the greater the wave length of light the less heat passed through the jelly around the frog's egg, in comparison with an equivalent amount of water and under similar conditions.

Oviducal blood vessels

Passage of eggs through the oviduct. The eggs of the frog are greatly distorted as they pass down the oviduct toward the uterus. They accumulate albumen around them, but, since they spiral down the duct, the albumen jelly is evenly deposited and the eggs become spherical as the jelly swells when the eggs pass from the uterus into the water.

Oviducts of the frog under various states of sexual activity. (A) Post-ovulation condition, collapsed and dehydrated. (B) Actively ovulating condition, oviduct full of eggs, edematous. (C) Oviduct of non-ovulating, hibernating female.

Originally, and erroneously, the jelly was thought to act as a lens which would concentrate the heat rays of the sun onto the egg, but since the jelly is largely water, which is a non-conductor of heat rays, this theory is untenable. One can demonstrate that the temperature of the egg is higher than the temperature of the immediate environment, even in a totally darkened environment. So, the jelly has certain physical functions in addition to those as yet undetermined functions which aid in rendering the egg fertilizable.

The egg takes about 2 to 4 hours, at ordinary temperatures, to reach the highly elastic uterus, at the posterior end of the oviduct and adjacent to the cloaca. Each uterus has a separate opening into the cloaca, and the ovulated eggs are retained within this sac until, during amplexus (sexual embrace by the male), they are expelled into the water and are fertilized by the male. Generally the eggs are not retained within the uterus for more than a day or so. There may be quite a few hours between the time of appearance of the first and the last eggs in the uteri.

Oogenesis — Maturation of the Egg

The maturation process can best be described as it begins, immediately after the normal breeding season in the spring. At this time the ovary has been freed of its several thousand mature eggs and contains only oogonia with no pigment and little, if any, yolk. Even at this early stage each cluster of oogonia represents a future ovarian unit, consisting of many follicle cells and one ovum. There has been no way to determine which oogonium is to be selected for maturation into an ovum and which will give rise to the numerous follicle cells that act as nurse cells for the growing ovum. It is clear, however, that both follicle cells and the ovum come from original oogonia. All ova develop from oogonia which divide repeatedly. These pre-maturation germ cells divide by mitosis many times and then come to rest, during which process there is growth of some of them without nuclear division. These become ova while those that fail to grow become follicle cells. However, there are pre-prophase changes of the nucleus of the prospective ovum comparable to the pre-prophase changes in spermatogenesis. The majority of oogonia, therefore, never mature into ova, but become follicle cells.

Prophases of the heterotypic division in the female (ovary of tadpole). (1) Nucleus of oogonium. (2) Leptotene. (3) Synaptene. (4, 5) Contraction figures. (6) Pachytene. (7) Later pachytene, multiplication of nucleoli. (8, 9) Diplotene: the chromatin filaments are becoming achromatic; granules of chromatin are being deposited on the nucleoli. (Courtesy, Jenkinson: "Vertebrate Embryology," Oxford, The Clarendon Press.)

Normal nuclear growth cycle of the ovum of Rana pipiens. (Stage 1 ) Smallest follicle in which the chromosomes within the germinal vesicle can be seen. (Stage 2) The paired chromosomes are barely visible, embedded in a nucleoplasmic gel. Egg diameters less than 200 microns. (Stage 3) Eggs measuring from 200 to 500 microns in diameter, more detail visible through the transparent theca cells. Lateral loop production begins. Zone of large irregular nucleoli may be seen just beneath the nuclear membrane. (Stage 4) First development of yellowbrown color and yolk. Eggs range in size from 500 to 700 microns in diameter. Chromosomes attain length of about 450 microns. For salamanders of comparable stage chromosomes measure 700 microns in length. (Stage 5) Chromosome frame begins contraction while the nucleus continues to grow in eggs ranging in diameter from 750 to 850 microns. This is approximately half the ultimate size. Chromosomes shorten and have fewer and smaller loops. The major nucleolar production continues and sacs appear on the surface of the nuclear membrane. (Stage 6) Egg diameter about 1.8 millimeters and germinal vesicle is of maximum size. Chromosome frame now about 1/1000 of the nuclear volume, coated by a denser substance which can be coagulated by the calcium ion. Chromosomes have shortened to 40 microns or less and have lost all large and small hyaline bodies called loop fragments.

Heavy arrows indicate the mixing of nuclear material in the cytoplasm after the breakdown of the germinal vesicle. The dotted arrow indicates migration of the central chromosomal mass toward the animal pole to become the maturation spindle for the first polar body. (From W. R. Duryee, 1950, Antu N, Y, Acad. Sci., 50, Art. 8.)

The process of maturation involves contributions from the nucleus and the cytoplasm. First, chromatin nucleoli aid in the synthesis of yolk, and second, the breakdown of the germinal vesicle allows an intermingling of the nuclear and the cytoplasmic components. Only a small portion of the germinal vesicle is involved in the maturation

Ovarial wall of Rami temporaria. Note young transparent eggs (stages 1 and 2) and larger opaque eggs {stage 3). The arrow points to an isolated nucleus from another egg (stage 3) , which has floated into the field. (Courtesy, W. R. Duryee, 1950, Ann. N. Y. Acad. ScL, 50, Art. 8.)

spindle so that it may be at this time that the nucleus exerts its initial influence on the cytoplasm. All cytoplasmic differentiations must be initiated at a time when the hereditary influences of the nucleus are so intermingled with it.

Growth Period to Primary Oocyte Stage. Growth is achieved largely by the accumulation of yolk. As soon as growth begins the cell no longer divides by mitosis and is known as an oocyte rather than an oogonium. The growth process is aided by the centrosome, which is found to one side of the nucleus, and around which gather the granules or yolk platelets. The chromatin filaments become achromatic and the nucleoli increase in number, by fragmentation, and become more chromatic. Many of the nucleoli, which are concentrations of nucleo-protein, pass through the nuclear membrane into the surrounding cytoplasm during this period. It is not clear whether this occurs through further fragmentation of the nucleoli into particles of microscopic or sub-microscopic size, and then their ejection through the nuclear membrane. It may occur by the loss of identity (and chromatic properties) by possible chemical change and subsequent diffusion of the liquid form through the membrane to be

The entire set of chromosomes of Rana lemporaria. The 1 3 pairs of chromosomes in this species are remarkably like those of other species of Rana. They are seen in stage 6. (Q) Four ring-shaped pairs. (R) Four medium-sized pairs. (S) Three longest (super) pairs. (T) Two short T-shaped pairs. (Courtesy, W. R. Duryee, 1950, Ann. N. Y. Acad. ScL, 50, Art. 8.)

Similarity of frog and of salamander chromosome structure. Stage 4 chromosomes. There is apparently a single chromonema along which compound granules and chromomeres of varying shapes and sizes are firmly embedded or attached. These chromosomes were treated with 0.2 M NaHCOa to remove the lateral loops and reveal the chromonemata. Chromosome granules are attached to the paired chromonemata at homologous loci. Some matric coating is present. Mild acidification following carbonate treatment has removed most of the lateral loops. (Courtesy, W. R. Duryee, 1950, Ann. N. Y. Acad. ScL, 50, Art. 8.)

resynthesized on the cytoplasmic side of the membrane. During the growth of the oocyte, further nucleoli appear within the nucleus, only to fragment and later to pass out into the cytoplasm. The presence of chromatic nucleoli in the cytoplasm is closely associated with the accumulation (deposition) of yolk.

The granules within the cytoplasm (extruded fragments of nucleoli) function as centers of yolk accumulation and have therefore been named "yolk nuclei." This is an unfortunate name, for the structure is a nucleus only in the sense that it is a center of aggregation. It is not a true cell nucleus. The centrosome and other granular centers lose their identity and the yolk granules then become scattered throughout the cytoplasm.

The source of all yolk for the growing ova is originally the digested food of the female. This nutrition is carried to the ovary by way of the blood system and conveyed to the nurse or follicle cells and thence to the oocyte. The yolk is at first aggregated around yolk nuclei, then concentrated to one side of the nucleus. Finally it assumes a ring shape around the nucleus between an inner and an outer zone of cytoplasm. Subsequently the nucleus is pushed to one side by the ever-increasing mass of yolk so that eventually there is an axial

Lateral loops of the amphibian chromosome. The lateral loops originate from chromosomal granules and the lateral branches are not homogeneous in structure, but are made up of smaller particles embedded in a hyaline cylinder. These lateral loops occur in separable clusters of 1 to 9 loops along a single chromonema. These loops reach their greatest development at stage 4, when the chromosome frame is most expanded. They average 9.5 microns in length but may reach 24 microns. They are not resorbed back into the chromosome and the number of loops per chromosome decreases with time, although the number of chromomeres per chromosome remains constant. (Courtesy, W. R. Duryee, 1950, Ann. N. Y. Acad. ScL, 50, Art. 8.)

gradient of concentration of oval yolk platelets from one side of the egg to the other. The smaller platelets are found in the vicinity of the nucleus, in the animal hemisphere. The larger platelets are located toward the vegetal hemisphere. There is an increase averaging from 200 to 700 per cent in the total lipoid substance, neutral fat, total fatty acids, total cholesterol, ester cholesterol, free cholesterol.

Paired chromosomes showing

^'chromomeres and side loops ^^'"^, '*'"":'^*'- ^^

Chromosomes in the amphibian nucleus. Stage 5 of the growing oocyte showing the isolated nucleus and its paired chromosomes with chromomeres and side loops. (Courtesy, W. R. Duryee, Laboratory of Terrestrial Magnetism, Washington, D. C.)

and phospholipin content of the ovaries of Rana pipiens occurring during the production and growth of ova (Boyd, 1938). The primary oocyte may show a sHght flattening of the surface directly above the region of the nucleus.

These growth changes and the unequal distribution of pigment, yolk, and cytoplasm are the first indications of polarity or a gradient system within the egg. When the polarity is well established, the cytoplasm, the superficial melanin or black pigment, and the nucleus are all at the animal hemisphere (pole). The light colored yolk is more concentrated toward the vegetal pole. The egg is then regarded as a telolecithal egg. During this phase of egg maturation there is a drain on the metabolism of the frog which requires an excess of food intake because the materials for egg growth must be synthesized from nutritional elements received from the vascular system of the female. For Rana pipiens this period of most active feeding comes during the summer when the natural foods, insects, worms, etc., are the most abundant.

During the growth of the oocyte in general there are important changes occurring within the nucleus (germinal vesicle) of the egg. Thirteen pairs of chromosomes may be seen in synizesis (contraction), converging toward the centrosome at the "yolk nucleus" stage. A little later the nuclear membrane develops sac-like bulges, the nucleoli are scattered, and there is a colloidal chromosome core which almost fills the entire nucleus. The chromosomes themselves are small and almost invisible. When the primary oocyte is about half its ultimate size, there appear definite sacs on the nuclear surface. The fragmented nucleoli are located at the periphery of the lobulated nuclear membrane, and the chromosome frames have become relatively large. The chromosomes, by this time, have reached their

maximum length and possess large lateral loops. Finally, in the fully grown nucleus of the primary oocyte the nuclear sacs are very prominent, and the nucleoli appear in clusters in the center of the

Diploid metaphase chromosomes from the tail fin of a 15day-old Rana pipiens tadpole. (Courtesy, K. R. Porter, 1939, Biol. Bull., 77:233.)

egg, surrounding the chromosome frame. This frame is a gel structure which gives rise to the first maturation spindle, containing 13 pairs of slightly contracted chromosomes.

These structural features can be observed in the living germinal vesicle if it is removed from the oocyte and placed in isotonic and balanced salt medium, omitting the calcium ion. A minute amount of NaHoPO, is added to shift the pH toward the acid side, which makes the chromosomes the more visible. Or, the chromosomes may be

Isolated germinal vesicle (nucleus) of Rami pipiens. Nucleus isolated in calcium-free Ringer's solution, from stage 6, showing sac-like organelles which protrude from the nuclear membrane. The cloud of central nucleoli surrounds and obscures the chromosome frame. (Courtesy, W. R. Duryee, 1950, Ann. N. Y. Acad. Sci., 50, Art. 8.)

Stage 5 germinal vesicle of Rami catesbiana. Shows chromosome pairs with but few lateral loops and peripheral nucleoli outside of the chromosome frame. (Courtesy, W. R. Duryee, 1950, Ann. N. Y. Acad. Sci., 50, Art. 8.)

Stained with crystal violet in a calcium-free medium. Amphibian cells are among the largest in the animal kingdom and the frog's egg nucleus is large enough to see with the naked eye. It can be removed with considerable ease and examined beneath the binocular microscope.

Before the time of hibernation the eggs that are to be ovulated for the next spring are in the fully grown primary oocyte stage, having their full complement of yolk, cytoplasm, and pigment. Externally more than one-half of the egg appears densely black, due to surface pigment granules, while the rest is creamy white. The nucleus is prepared for the maturation divisions. Such an egg measures about 1.75 mm. in diameter. The surface layer of the amphibian egg is formed before fertilization and it is definitely not hyaline, as it is in some Invertebrate eggs. It contains many small yolk grains and irregular accumulations of spherical, black pigment granules. With each cleavage, subsequent to fertilization, this superficial coat is divided between the blastomeres, being an integral part of the living cell. There is no clear-cut demarcation between this surface coat and the inner cytoplasm and yolk. It is believed that these growth changes of the egg are under the influence of the basophilic cells of the anterior pituitary gland, which cells are greater in number at this time than at any other.

During the growth period the vitelline membrane appears on the surface of the oocyte as a thin, transparent, non-living, and closely adherent membrane. It is formed presumably by a secretion from the egg itself, aided by the surrounding follicle cells. It appears to be similar in all respects to the membrane of the same name found around the eggs of all vertebrates.

Ovulation and Maturation. Ovulation, or the liberation of the egg from the ovary, is brought about by a sex-stimulating hormone from the anterior pituitary gland. Just before and during the normal spring breeding period there is a temporary increase in the relative number of acidophilic cells in the anterior pituitary. It is believed that this is not coincidental but a causal factor in sex behavior in the frog. However, until such time as extracts of specific cell types can be made, this will be difficult to prove conclusively. Attempts on the part of the male to achieve amplexus are resisted by the female not sexually stimulated. However, such a female can be made to accept the male by injecting the female with whole anterior pituitary glands from other frogs. It is very probable that environmental factors such as light, temperature, and food may act through the endocrine system to prepare the frogs for breeding when they reach the swampy marshes in the early spring, after protracted hibernation. It must be pointed out, however, that there are frogs in essentially the same environments which breed in July (Rana clamitans) and August {Rana catesbiana) , so that the causal factors appear to be either complex or possibly different for different species. The pituitaries of the hibernating frogs do contain the sex-stimulating factor, but apparently to a lesser degree than the glands of frogs approaching the breeding season. The injection of 6 glands from adult female frogs will cause an adult female of Rana pipiens to ovulate as early as the last week in August, some 8 months before the normal breeding period. One or two such glands will accomplish the same results if used early in

Production of ova and the process of ovulation. (Top) Section through a primary o5cyte of Rana pipiens showing the absence of the vitelUne membrane,

{Continued on facing page.)

The maturation divisions in the female (Axolotl). (1) First polar spindle with heterotypic chromosomes. (2) Extrusion of first polar body. (3) Appearance of second polar spindle. Longitudinal division of chromosomes in egg and in first polar body. (4) Second polar spindle radial. Homoeotypic chromosomes on equator (metaphase). (5) Polar view of the same. (6) Anaphase. (7) Extrusion of second polar body. (8) Second polar body with resting nucleus. (9) Female pronucleus in resting condition, closely surrounded by yolk granules. (Courtesy, Jenkinson: "Vertebrate Embryology," Oxford, The Clarendon Press.)

[Continued from opposite page.) the presence of the follicle and cyst wall, and the predetermined area of ultimate follicular rupture. {Center) Mature ovum with axial gradient of pigment and yolk. The vitelline membrane is now present, and the cyst wall of smooth muscle cells is stretched. (Bottom) Ovarian egg partially emerged from its follicle during ovulation. Note degree of constriction, contraction of cyst wall, and fully developed vitelline membrane around the entire egg.

The position of the master endocrine gland — the anterior pituitary of Rana pipiens.

April. Another explanation for this may be offered, namely that the ovary itself may become more sensitive to such stimulation as the breeding season approaches.

The ovulation process itself consists of the rupture and the emergence of eggs from their individual follicles. The surface of the egg, separated from the body cavity by only the non-vascular theca externa, is first ruptured and then the egg slowly emerges through the small opening. Since the egg is known to contain a peptic-like enzyme, it is believed that the pituitary hormone may activate this enzyme to digest away the tight and non-vascular covering. Then by stimulation of the smooth muscle fibers of the cyst wall (theca interna) the process of emergence is completed. The relation of the pituitary to smooth muscle activity has long been established clinically.

It is true that the ovarian stroma shows undulating contractions at all seasons, irrespective of sex activity. An egg will emerge at any time from a surgically ruptured follicle. If an ovulating female is etherized and the body cavity is opened, the ovary may be removed and placed in amphibian Ringer's solution and the ovulation process may be observed directly. This will go on for several hours after all connections with the nerve and blood supply are cut off. From the initial rupture of the theca externa until the egg drops free into the body cavity there is a lapse of from 4 to 10 minutes at ordinary laboratory temperatures.

The first maturation division occurs at the time of ovulation. The heterotypic chromosomes (i.e., of bizarre shapes) are placed on the spindle of the amphiaster whose axis is at right angles to the egg surface. Movement of the chromosomes is identical with that found in ordinary mitosis. The outermost group of telophase chromosomes are pinched off, with a small amount of cytoplasm and no yolk, to comprise the first polar body. The innermost telophasic group of chromosomes remain within a clear (i.e., yolk-free) area of the egg as the nuclear mass of the secondary oocyte. These changes occur as the egg leaves the ovary and before it reaches the oviduct. Possibly the same forces which bring about follicular rupture also influence this maturation process.

The second maturation division begins without any intermediate rest period for the chromosomes, at about the time the egg enters the oviduct. There may be a variation in time up to 2 hours for eggs to reach the ostium, depending upon the region of the body cavity into which they are liberated. Thus the stage of maturation of different eggs within the oviduct may vary considerably. There is a longitudinal division of the chromosomes of the egg which are lined up in metaphase on the second maturation spindle, the axis of which is at right angles to the egg surface. Since the spindle is primarily protoplasmic, and is made up in part of fibers (which may be contractile), the space occupied by the spindle will be free of yolk. Since it is peripherally placed, and represents a slight inner movement after the elimination of the first polar body, the surface layer of the egg is slightly de-pigmented just above the spindle region. This situation is exaggerated in aged eggs, a relatively large de-pigmented area of the cortex appearing toward the center of the animal hemisphere.

Maturation is not completed until or unless the egg is activated by sperm or stimulated by parthenogenetic means. However, every egg reaching the uterus is in metaphase of the second maturation division, awaiting the stimulus of activation to complete the elimination of the second polar body.