Book - Sex and internal secretions (1961) 14

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I have decided to take early retirement in September 2020. During the many years online I have received wonderful feedback from many readers, researchers and students interested in human embryology. I especially thank my research collaborators and contributors to the site. The good news is Embryology will remain online and I will continue my association with UNSW Australia. I look forward to updating and including the many exciting new discoveries in Embryology!
Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex
Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction
The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions
Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation
Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds
Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior
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Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy


Biology of Eggs and Implantation

Richard J. Blandau, Ph.D., M.D.

Professor Of Anatomy, University Of Washington School Of Medicine, Seattle, Washington

Contents


I. Introduction

In recent years there has been much more intense research activity on the morphology, physiology, and biochemistry of spermatozoa and semen of mammals than on their eggs and the fluids forming their environment. The significant increase in the investigations of the male gametes is due largely to stimuli resulting from the necessity of perfecting techniques of artificial insemination in domestic animals and of elucidating the problems of infertility and contraception in man. A distinct advantage with respect to investigations of the male is the ready availability and large number of gametes which can be obtained from a single subject. In contrast, the mammalian egg is available in restricted numbers and then only at very specific times in the reproductive cycle. Furthermore, there are very real difficulties in maintaining mammalian eggs in a normal physiologic state after they have been removed from their usual environment.


Even though there have been notable advances in the investigations of the complicated physiologic and biochemical mechanisms which exist in the development, storage, transport, and syngamy of the gametes since Dr. Carl G. Hartman's erudite discussions of the subject in 1932 and 1939, our understanding of the fundamental problems involved in maintaining the continuous stream of life from generation to generation is still in its infancy. As we proceed 20 years later, it will be clear that the older methods of classical histology have not yet outlived their usefulness. But it will also be apparent that many of the advances which have been made, particularly in the investigation of mammalian materials, can be attributed largely to the use of new and improved techniques for the collection and study of living gametes and embryos. For this reason, the subject to which this chapter is devoted will be introduced with an enumeration and description of some of the methods which have contributed so much to the work of the last two decades. Most important of these are the methods which have been developed for recovering eggs and embryos from the oviducts and uterus, and they, therefore, will be described as a preliminary to the discussion which follows.


A. Methods for Recovering Mammalian Eggs and Embryos

1. Collecting Ova from the Oviducts

In animals such as the guinea pig, rat, mouse, and hamster, in which the oviducts are highly coiled, several procedures may be followed for obtaining the tubal eggs. The coils of oviduct can be trimmed from the mesosalpinx with iridectomy scissors. By stroking the length of the tube with a fine, curved, blunt probe, the entire contents can be expressed and the ova separated from the debris.

Another method is that of placing the oviducts in a balanced salt solution and mincing them into small pieces with a pair of fine, pointed scissors, and then searching for the ova. Both of the above methods are wasteful of time and material, because the ova may be damaged and the full number frequently is not recovered.



Fig. 14.1. Apparatus for washing ova from the oviducts of mammals.



The best method for obtaining ova from the coiled oviducts of the rat, mouse, hamster, and guinea pig is to insert a fine pipette filled with a suitable solution into the lumen of the fimbriated end. The pipette is held in place with fine watchmaker's forceps. Gentle pressure is exerted on the fluid in the pipette by a simple arrangement whereby air pressure can be controlled in the manner illustrated in Figure 14.1. If the oviducts are removed and cut just above the uterotubal junction, ova may be seen to escape slowly from the cut end. By controlling the pressure, all of the ova can be kept within a circumscribed area and any other contents of the oviduct, such as spermatozoa, can be accurately counted or evaluated (Rowlands, 1942; Simpson and Williams, 1948; Blandau and Odor, 1952; Noyes and Dickmann, 1960; Dickmann and Noyes, 1960).

2. Collecting Free Ova from the Uterus

Flushing of free ova from the uterus has been performed in the monkey (Hartman, 1944) and cow (Rowson and Dowling, 1949; Dracy and Petersen, 1951). In the monkey the uterine lumen may be entered with a hypodermic needle inserted into the uterus through the abdominal wall. The contents of the uterus are then flushed through a funnel, the stem of which has been inserted into the cervical lumen. Several segmenting eggs were obtained by this procedure. The disadvantages of this method are two : first, a large quantity of fluid must be examined, and, second, the presence of cellular debris in the washings makes it difficult to locate the single egg.

In rodents the cornua may be removed from the body and separated into their right and left halves. Each cornu is then flushed with physiologic saline by inserting a fine hypodermic needle into the oviductal end. During the flushing, the cornu should be gently stretched so as to release ova that may be trapped within the endometrial folds.


In the cow relatively large quantities of physiologic solutions are used to flush out tlie cornu on the side on which the corpus luteum has been detected by rectal palpation (Rowson and Bowling, 1949). The recovered fluid is poured into a series of French separatory funnels and allowed to stand for 20 minutes. Ordinarily, this interval is long enough for the ovum to gravitate to the bottom. A few milliliters of fluid are removed from each funnel and the egg searched for. By this method, Dracy and Petersen reported the recovery of 10 fertilized ova from a single cow which had been superovulated.

3. Recovery of Attached Embryos

The techniques devised by Dr. Chester Heuser, thus far unsurpassed in the degree of their perfection, provide the safest method of obtaining blastocysts or early implanting embryos. Uteri of man or other primates which have been removed by hysterectomy are completely immersed in Locke's solution. The uterus is cut coronally into dorsal and ventral halves. The surface of the mucosa can then be examined under a binocular dissecting microscope in order to locate the site of the implanting embryo (Heuser and Streeter, 1941 ; Hertig and Rock, 1951 ).

A somewhat similar procedure can be followed in observing and recovering implanting embryos of the guinea pig, rat, and rabbit. The cornu is cut longitudinally along the mesometrial border with iridectomy scissors and the entire cornu laid open as a book. The mucosa of the antimesometrial area is examined under a binocular dissecting microscope in order to find the implanting embryos and, when they are found, fixatives can be added directly and only a small segment of the uterus removed for sectioning (Blandau, 1949b; Boving, personal communication).

B. Egg Culture and Preservation In Vitro

Studies of the effects of various environmental conditions on mammalian eggs and zygotes are of more than academic interest. The possibility of applying such knowledge to artificial insemination and intergeneric and reciprocal transplantation of eggs is of economic importance, especially in animal husbandry. Consequently, for years special attention has been given to the problem of finding satisfactory media for the successful culture and transplantation of eggs.

Gates and Runner (1952) compared Ortho-bovine semen-diluter containing egg yolk with regular Locke's solution as a medium for transplanting mouse ova and concluded that the semen diluter was the more satisfactory medium. Many other media have proved successful. These include, to list only a few, Ringer-Locke solution with an equal volume of homologous blood serum (Pincus, 1936), Krebs' solution (Black, Otto and Casida, 1951), phosphate-buffered Ringer-Dale solution mixed with an equal volume of homologous plasma (Chang, 1952b), and Krebs-Ringer bicarbonate containing 1 mg. per ml. glucose and 1 mg. per ml. crystalline bovine plasma albumin (Armour) (McLaren and Biggers, 1958).

Rabbit eggs have been used most often as test objects in the evaluation of media. The eggs of this animal are particularly hardy during manipulation and storage in vitro, a condition which may be related to the presence of the mucous coat. Aqueous humor from sheep's eyes has been used successfully for the transfer of eggs from sheep to sheep (Warwick and Berry, 1949) . Willett, Buckner and Larson (1953) obtained pregnancies in cows from eggs suspended in homologous blood serum during transfer.

Except when the rabbit was used, attempts at growing fertilized eggs in vitro in the same media used for their transfer have not been successful. The pioneering work on the cultivation of mammalian eggs under conditions of tissue culture must be attributed to Brachet (1913), Long (1912), Lewis and Gregory (1929), Pincus (1930), and Nicholas and Hall (1942). Lewis and Gregory recorded their notable success in culturing fertilized rabbit ova in homologous blood scrum in vitro by means of cinemicrophotography. Fertilized rabbit ova will cleave regularly in vitro up to and beyond the initial stages of blastocyst expansion (Pincus and Werthessen, 1938). Lewis and Hartman (1933) succeeded in culturing the fertilized eggs of Macacus rhesus for a number of divisions. Eggs of guinea pigs, cultured in vitro, rarely divide beyond the first few blastomeres (Squier, 1932). Guinea pig blastocysts, however, grow quite well in a culture medium consisting of equal parts Locke's solution (pH 7.5), serum from guinea pigs pregnant from 20 to 24 days, and embryo extract prepared from 19- to 20day-old guinea pig embryos (Blandau and Rumery, 1957). As yet, no success has been obtained with the very early fertilized eggs of the hamster and rat (Wrba, 1956) .


Hammond (1949.) cultured fertilized mouse ova in dilute suspensions of whole hen's egg in saline to which had been added Ca, K, Mg, and glucose. No 2-cell ova developed beyond the 4-cell stage; 8-cell ova ordinarily developed into blastocysts. Whitten (1956) found that 8-cell mouse eggs developed into blastulae in an egg white-saline mixture or in Krebs-Ringer bicarbonate solution to which 0.003 m glycine had been added. There seems to be some physiologic difference between the 2- and 8-celled ova in this animal because the 2-celled mouse eggs are refractory to in vitro cultivation unless calcium lactate replaces the calcium chloride in the culture medium (Whitten, 1957).


Considerable success has attended the in vitro culture of embryos which are beyond the blastocyst stage at the time of transfer to tissue culture (Brachet, 1913; Waddington and Waterman, 1933; Jolly and Lieure, 1938; Nicholas, 1947; Moog and LutwakMann, 1958). Nicholas (1933) obtained better growth in vitro when the embryos were cultured in a circulating medium.


Several investigators have studied the effects of cooling mammalian eggs in vitro. Chang (1948a, b) found that rapid lowering of the temperature of 2-celled rabbit ova that had been suspended in a mixture of equal parts of buffered Ringer's solution and rabbit serum was harmful to subsequent development. However, the important factor was not the rate of cooling but whether the process was continued until +10°C. was reached. Apparently, that is the optimal temperature for the storage of fertilized rabbit eggs. At this temperature eggs can be kept in vitro up to 168 hours without loss of viability. At +22°C. to +24°C. ova lived for only 24 to 48 hours. Attempts to maintain glycerol-treated rabbit ova at temperatures ranging from -79° to -190°C. have so far been unsuccessful (Smith, 1953).


C. Intraspecific Egg Transfer

The technique for the transfer of unfertilized and fertilized eggs between the members of the same species was first described by Heape (1890). He used this method in rabbits to demonstrate that the genetical characteristics of mammals are fixed at the time of fertilization and are not influenced by the intra-uterine environment of the foster mother. Biedl, Peters and Hofstatter (1922) and Pincus (1930) used Heape's technique during investigations on fertility and demonstrated that it is possible to transplant fertilized rabbit eggs to pseudopregnant does.


In animal husbandry artificial insemination has been an important method for the widespread distribution of desirable genes by way of the spermatozoa. Similar genetical improvement through the egg has been greatly limited in domestic farm animals by the small number of offspring. A single cow, for example, will produce 1 calf per year and seldom more than 5 in a lifetime. If transplantation of eggs could be perfected, the number of genetical experiments could be increased at least 2-fold. That the prospect is favorable, is indicated by the fact that transfers which have resulted in pregnancies have been reported for mice (Bittner and Little, 1937; Fekete and Little, 1942; Fekete, 1947; Runner, 1951; Gates and Runner, 1952; Runner and Palm, 1953; McLaren and Michie, 1956; Tarkowski, 1959; McLaren and Riggers, 1958); rats (Nicholas, 1933; Noyes, 1952); rabbits (Heape, 1890; Bicdl, Peters and Hofstatter, 1922; Pincus, 1936, 1939; Chang, 1947, 1948a, b, 1949a, 1952b; Chang, Hunt and Romanoff, 1958; Venge, 1953; Avis and Sawin, 1951; Black, Otto and Casida, 1951; Adams, 1953); sheep and goats (Warwick and Berry, 1949; Averill and Rowson, 1958) ; swine (Kvasnickii, 1951) ; and cows (Willett, Buckner and Larson, 1953).

The majority of successful egg transfers have been accomplished by exposing the oviducts and cornua surgically and placing the eggs within them (Fig. 14.2). Introducing fertilized eggs into the cornua by way of the vagina and cervix has usually failed to result in pregnancy (Dowling, 1949; Umbaugh, 1949; Rowson, 1951). Two exceptions have so far been reported. Kvasnickii (1951) obtained one pregnancy in the sow from eggs placed in the uterus per vaginam and Beatty (1951) obtained 5 young from 55 mice morulae and blastulae introduced into the cornua by the same approach. Since the normal development of ova in artificial pregnancy is wholly dependent upon the environment into which they have been placed, day-old rabbit ova would develop into normal young only when transferred to oviducts of animals in which ovulation had been induced at approximately the same time. Similarly, blastocysts would develop into young only when transplanted into 2day or 5-day cornua (Chang, 1950c). Again in transferring fertilized tubal ova to the cornua of rats, Nicholas (1933) reported that when the host animal ovulated later than the donors, implantations were greatly reduced as compared to those instances in which the cycles were more closely synchronized. Dickmann and Noyes (1960) transferred ova that were one day younger than the cornua to host females and found that they developed at a normal rate until the fifth day, when they degenerated and failed to implant. On the other hand, ova that were one day older than the host's cornua delayed their development until the endometrium had "caught up" and was ready for implantation. This implies that there is a very critical egg-uterine interrelationship that is established on the fifth day of pregnancy in the rat. Transplantation of rat ova beneath the kidney capsule (Nicholas, 1942) and of mouse ova into the abdominal cavity and anterior chamber of the eye (Fawcett, Wislocki and Waldo, 1947; Runner, 1947) have resulted in only partial embryonic development.

D. The Production of Eggs by Superovulation

Many studies have been directed to methods for superovulating various animals, then fertilizing the eggs in vivo, recovering and transferring them to recipient females (Clewe, Yamate and Noyes, 1958; Noyes, 1952; and Chang, 1955a).

Sucli possibilities have been realized especially by Chang (1948a), who obtained 53 2-celled rabbit ova from a single doe. These ova were transplanted to 4 other females and yielded 45 normal young. Using somewhat similar techniques of superovulation and in vivo fertilization in rabbits, Avis and Sawin (1951) obtained 81 per cent successful impregnations and Dowling (1949) 78 per cent pregnancies.


Fk;. 14.2. Result of autotransfer of a 4-cell goat egg, B. Tlio mother was operated upon on the second day after breeding, the oviduct was removed and the 4-cell egg (A) was washed out. The egg was then injected into the opposite horn of its mother (Warwick and Berry, 1949).


Subsequently, Marden and Chang (1952) performed the novel experiment of shipping superovulated, fertilized rabbit ova by way of aerial transport from Shrewsbury, Massachusetts, to Cambridge, England, for successful transplantation into recipient does. While in transport, the eggs were stored in a flask containing whole rabbit serum kept at temperatures from 12 to 16°C. In domestic animals, the economic importance of such transfer of eggs from genetically superior animals is receiving considerable attention (see Proceedings of the First National Egg Transfer Breeding Conference, 1951). Unfortunately, superovulation in cattle which has been achieved by the administration of gonadotrophic hormones (Casida, Meyer, McShan and Wesnicky, 1943; Umbaugh, 1949; Hammond, 1950a, b) has met with little success as a means of inducing pregnancy (Willett, Black, Casida, Stone and Buckner, 1951 ».

III. Biology of the Mammalian Egg

A. Oogenesis

The literature is now revealing a more clear cut opinion as to whether or not the primordial germ cells from the yolk sac of the embryo are set aside at the beginning of ontogenesis, or whether they arise de novo from the somatic cells of the gonadal peritoneum in the embryo and particularly the sexually mature female. Knowledge in this field has been significantly advanced by employing the techniques of experimental embryology, organ and tissue culture, histochemistry, x-rays, ultraviolet irradiation, genetics and statistics. The Gomori alkaline phosphatase procedure lias been used by a number of investigators to distinguish selectively the primordial germ cells in the human (McKay, Hertig, Adams and Danzigcr, 1953), the mouse (Chiquoinc, 1954; Mintz, 1959), and the rat (McAlpine, 19551. Using the same technique, Bennett (1956) reported the absence of germ cells in strains of mice known to be sterile. It has been suggested that the high alkaline phosphatase activity in the germ cells may be related to their active movement through tissues. This speculation has merit when it is noted that alkaline phosphatase activity is greatly reduced in amblystoma, in which the germ cells do not actively migrate, and in the chick where these cells are apparently transported by way of the blood stream (Chiquoine and Rothenberg, 1957, Simon, 1957a, b). It should be noted that the primordial germ cells may be identified by other techniques. For example, in the rat and man the use of the periodic acid-Schiff (PAS) reaction and a hematoxylin counter stain gives such excellent cytologic differentiation of the germ cells that they can be counted and their migratory course followed (RoosenRunge, personal communication).

It is beyond the scope of our discussion to present the details of the controversy of germ cell origin, migration, localization, and proliferation. Excellent reviews of the betterknown theories are contained in the papers and monographs of Heys (1931), Cheng (1932), Swezy (1933), Pincus (1936 », Bounoure (1939), Everett (1945), Nieuwkoop (1949), Zuckerman (1951), Brambell (1956), and Nieuwkoop and Suminski ( 1959) . Evidence for the extragonadal origin of the primordial germ cells has been significantly enhanced by the more recent investigations in amphibia, birds, and various mammals such as the armadillo, mouse, rat, cat, rabbit, and man. In an excellent paper dealing with the migration of the germ cells in the human, Witschi (1948) points out that in embryos of less than 16 somites all of the primitive germinal elements are located in the endoderm of the yolk sac splanchnol)leure near the site of evagination of the allantois (Fig. 14.3). From this location the individual germ cells appear to migrate to the genital folds by various routes. Witschi concludes from studies of sectioned human embryos that the migration of the germ cells is accomplished by active autonomous movements and cites evidence of proteolysis of the cells and tissues in the immediate vicinity of the forward moving cells. He suggests that the specific orientation of the cell is directed by some chemical substance released by the peritoneum of the gonadal regions.


A very important contribution to the solution of the problem of seeding the primitive gonads by germ cells from extragonadal origin is described in the contributions of IVIintz (1957, 1959) and Mintz and Russell (1957). These authors noted that the gonads of mice of the WW, WW and WW^ genotyi^es are almost devoid of germ cells at birth. The application of the alkaline phosphatase technique revealed that the cells are present in their usual numbers in the yolk sac splanchnopleure by the 8th day of development. The mutant genes apparently do not impair the initial formation of the primordial germ cells. By the 9th day of development, however, many of the germ cells had already degenerated at their site of origin. Some of them escape destruction and migrate toward the genital ridge. The migratory cells fail to divide so that the total number reaching the gonads is small. These findings were in strong contrast to the behavior of germ cells of the normal mouse.


Fig. 14.3. Drawings of graphic reconstructions of a 16- and 32-somite human embryo. A. The bhick dots within the circle represent the location of the germ cells in the yolk sac and ventral wall of the hind-gut in the 16-somite embryo. B. Position of indi\-idual germ cells (black dots) in the 32-somite embryo. Larger dots indicate an endodermal position. Few germ cells remain in the ventral mesenchyme. (After E. Witschi, Contr. Embryol., Carnegie Inst. Wasliington, 32, 67-80, 1948.)


By use of a genetical marker, further experimental proof of extragonadal origin of germ cells was obtained. From theoretic expectations, experimental matings using heterozygotes should yield 25 per cent defective offspring. The actual frequency of embryos with gonads containing few germ cells was 28 to 29 per cent. The observations of Mintz and Russell give significant verification of the initial extragonadal origin of primordial germ cells in the mouse. Their work demonstrates further that mice of different strains lose oocytes at different rates depending on their genetical characteristics.

In some of the mutant mice, there is a complete absence of ovocytes in the ovaries of the adults. Russell and Fekete (1958) have shown that when chimeric organ cultures were made in vitro, combining onehalf of a fetal ovary from the mutant strain with one-half of an ovary from a normal animal, no germ cell differentiation occurred despite active proliferation of the germinal epithelium.

The sterility pattern described for the female has been observed also in the male mouse. Primordial germ cells are very poorly represented in the testes of WW, WW" and W^'W"' embryos and newborn. The mature males of these strains are invariably sterile. Veneroni and Bianchi (1957) reported some success in treating such sterile males with follicle stimulating hormone and testosterone propionate. They conclude that the problem of sterility is related not only to the reduction in the number of primordial germ cells but also to an endocrinologic deficiency.

Willier (1950) studied the developmental history of the primordial germ cells in the chick by preparing chorio-allantoic grafts of the blastoderm at certain critical stages, namely, (1) at the time the germ cells were still near the site of their origin, (2) during their migration, and (3) when they had arrived in the prospective gonadal areas. He found that under these experimental conditions the ovarian cortex never forms; he attributed this deficiency, at least in part, to a failure of the development of a mechanism in the graft for transporting the primordial germ cells to the areas of the developing gonad. Swift (1914), Dantschakoff, Dantschakoff and Bereskina (1931 ) , Willier (1950), and Weiss and Andres (1952), suggested that the primary germ cells are carried to the primitive sex glands of the chick embryo by way of the blood stream. Thus the cells are originally distributed at random, but they accumulate and persist only in the gonadal primordium.


Recently, Simon (1957a, b) confirmed the vascular transport of the germ cells in the chick by the application of several ingenious experimental embryologic techniques of transplantation and parabiosis. In the developing chick of less than 10 somites the primitive germ cells are localized in the germinal crescent zone in the anterior part of the yolk sac. The caudal part of the embryo containing the future genital ridge was severed and moved some distance from the original embryo. Vascularity of both parts was interfered with as little as possible. Stained sections of embryos examined on the 4th day of development revealed that the gonads had been populated by germ cells which could have reached them only by way of the vascular stream. In other experiments the caudal areas of 10 somite embryos, where gonads were not seeded by germ cells, were transplanted to the area vasculosa of other 10 somite embryos. The developing gonads in the transplants were colonized by germ cells. In still another experiment chick embryos were placed in parabiosis. In one of the transplanted embryos the anterior crescent containing the primordial germ cells was cut away. In cases of successful parabiosis the gonads of both embryos were seeded by germ cells.


Even though it is recognized that in many mammals and the chick the germ cells of the primitive sex glands are derived from migratory primordial germ elements, a more difficult problem remains of a possible second source of germ cells arising from somatic cells in the gonad of embryos, fetuses, and mature animals. It has been proposed that the original germ cells degenerate after having reached the gonads and having effected their inductive roles, and that newcells arise secondarily by proliferation of cells in the germinal epithelium (Allen, 1911; Firket, 1914; Kingery, 1917). On the otlier hand, Essenberg (1923), Butcher (1927), Brambell (1927, 1928), and Swezy and Evans (1930) postulated a dual origin for the germ cells, i.e., they may arise both from the primordial germ cells, and directly from somatic cells.


The ingrowth of new cells from the germinal epithelium, resulting in the production of new oocytes, was thought to have been demonstrated for both the eutherian mammals (Pincus, 1936; Duke, 1941; Slater and Dornfeld, 1945), and birds (Bullough and Oibbs, 1941). However, various opinions flourished as to whether these oocytes were produced continuously throughout the reproductive life of the female (Robinson, 1918; Papanicolaou, 1924; Hargitt, 1930j, or whether they arose from a cyclically stimulated germinal epithelium. On the basis of Allen's (1923) investigations on the mouse, and Evans' and Swezy 's (1931) work on a variety of mammalian species, it was widely accepted that a large number of oocytes make their appearance from the germinal epithelium about the time of estrus. According to these investigations the oocytic population reaches its peak during the period of heat and ovulation. On the other hand. Green and Zuckerman (1951a, b, 1954) analyzed the difference in the number of oocytes during the menstrual cycle in 12 pairs of ovaries of Maccica mulatta by both quantitative and statistical methods. Their results did not support the accepted view that the total number of oocytes in the ovaries of the monkey varies during the cycle and reaches a maximum near the time of ovulation. They concluded that there is no significant difference between the average total number of oocytes present at the beginning, middle, and end of the cycle. From the results of the experiments of Papanicolaou (1924), Moore and Wang (1947), Mandl and Zuckerman (1951), Mandl and Shelton (1959), Enders (1960), and others, one would assume that the germinal epithelium is not essential for oogenesis in the adult mammal. If oogenesis is to continue after puberty in the absence of a germinal epithelium, are there alternative sources for the new oocytes? It has been proposed that either the concentration of primordial germinal cells in the region of the hilum of the ovary, redescribed by Vincent and Dornfeld (1948), may be a source, or that specialized cells, histologically indistinguishable from other stromal cells, may be transformed into germ cells. In support of the latter, Dawson (1951) suggested that in polyovular follicles in which there is a great disproportion in the size of the ova, the accessory egg may have arisen by delayed oocytic differentiation of a cell temporarily incorporated in the follicular epithelium.


Of the numerous experimental approaches to the problem of the origin of the germ cells in the sexually mature animal, the action of various hormones on the germinal epithelium has received particular attention. Bullough (1946) claimed that at the time of ovulation the estrogen-rich follicular fluid which bathes the ovary induces mitotic activity of the germinal epithelium. Stein and Allen (1942) demonstrated a stimulating effect of estrogen on the proliferation of the germinal epithelium of the mouse when this hormone was injected directly into the periovarial sac. On the other hand, thyroxine similarly applied retarded mitoses of the germinal epithelium (Stein, Quimby and Moeller, 1947). More recently Simpson and van Wagenen (1953) reported an enhancement of all the processes concerned with the development of oocytes and follicles in prepubertal monkeys (Macaca mulatta) that had been injected subcutaneously with either highly purified folliclestimulating hormone (FSH) extracted from the sheep pituitary or extracts from homologous pituitaries ( also see van Wagenen and Simpson, 1957, and Simpson and Van Wagenen, 1958). The germinal epithelium was stimulated to such an extent that there was an active ingrowth of germinal cords which closely simulated the development of Pfliiger's tubes. Small oocytes appeared to be developing within the germinal cords and there were evidences which one could interpret as reactivated oogenesis. An attempt was made to carefully quantify the response of the ovaries by counting the number of oogonia and growing follicles. In general the follicular counts remained unchanged, but primary follicles with a single granulosa cell laver were fewer in the stimulated ovaries than in the controls, indicating that more of them had been started on the course of fm^ther development. From the evidence presented in the monkey and from a variety of other observations one must conclude that, once reproductive life has begun, there is no neonatal growth of germinal epithelium.


One of the major difficulties is the problem of distinguishing germinal epithelial cells from adjacent oogonia. A similar difficulty is encountered when attempts are made to remove only the germinal epithelial cells by surgical or chemical means (]Moore and Wang, 1947; Mandl and Zuckerman, 1951). This problem is further emphasized by Everett (1945) when he states, "It seems probable that the cells of the epithelium, which form functional sex elements, are not and never were a part of the mesothelial covering, but are cells which were segregated early and are merely stored in the epithelium."


From some of the earlier work, it was felt that much would be gained if some technique were devised whereby individual cells could be marked and their subsequent fate determined. Latta and Pederson (1944) initiated such experimentation when they injected India ink into the periovarian space and examined the ovaries at varying intervals thereafter. Ova and follicular cells with carbon particle inclusions were seen in various stages of growth and maturation and these observations were interpreted as demonstrations of the origin of ova and follicular cells from "vitally stained" germinal epithelium. It is suggested, however, in light of recent evidence that many cells are capable of moving such particles across the cells and transferring them to others (Odor, 1956; Hampton, 1958), that the validity of using colloidal particles for labeling epithelial cells should be re-evaluated.


Theoretically, the study of tissue culture preparations of fetal and adult ovaries by phase contrast and time-lapse cinematography might be a better approach to the problem of the neoformation of oocytes in mammals and a few experiments of this type have been performed. Long (1940) reported oocytes developing from newborn and adult mice ovaries growing in vitro. These findings were not confirmed by similar studies of Ingram (1956) in which he found no signs of oogenesis in tissue culture preparations of either mouse or rat ovaries. Gaillard (1950) suggested that the germinal epithelium was essential for survival of explants of human embryonic ovaries in that explants without germinal epithelium invariably died. On the other hand, Martinovitch (1939) cultured fetal mouse ovaries for as long as 3V2 months. Although the ovarian epithelium disappeared after one week in vitro, the ovocytes continued to grow.


The covering epithelium of the ovary is capable of proliferation, and mitotic figures are frequently demonstrable. As the size of the ovary changes during the normal cycle or upon stimulation with exogenous hormones, the covering epithelium must keep pace with the changing surface contour. As mentioned above, the primordial germ cells in the embryo are strongly phosphatasepositive. Careful evaluation of the cells arising from the germinal epithelium have so far shown negative enzymatic reactions.


Furthermore it is a consistent finding that when mice are x-rayed in late fetal life or at birth with sufficient dosages to eliminate the ovogonia, no new ovocytes form from the cells of the germinal epithelium (Brambell, Parkes and Fielding, 1927; Mintz, 1958) .


It is an obvious conclusion that any attempt to ascertain the origin of germ cells cannot be considered adequate without thoroughly investigating the entire germ-cell cycle from tlie very earliest stages to the formation of the definitive sex elements in the fetal and postnatal periods. This must include also the origin of the functional germinal cells in the sexually mature animal. There is an urgent need for a comprehensive comparative study of the cytology, distribution, and migration of these cells. Inasmuch as the germ cells often contain nuclear and cytoplasmic features which are highly characteristic, they offer unusual advantages for various experimental analyses using some of the moi'e modern techniques of experimental embryology, tissue culture, and microscopy.


Even though we have confined our remarks here to the chick and mammal, we recognize the importance of the considerable body of descriptive and experimental information that has been recorded for the amphibia and invertebrates (Tyler, 1955). Heteroplastic transplantations and other experimental procedures which can be performed more easily in these animals may lead to explanations of the fundamental patterns of germ cell-inducing influences by the surrounding cells and to other problems bearing on the question of the origin of second generation germ cells in the genital ridge.

B. Growth, Composition, and Size of the Mammalian Egg

The rate of growth of the oocyte in relation to the stage of development of the ovarian follicle has been investigated in a numl)er of placental mammals (Brambell, 1928, mouse; Parkes, 1931, rat, ferret, rabl)it, pig; Zuckerman and Parkes, 1932, baboon; Green and Zuckerman, 1951a, 1954, Macaca mulatta and man). The available information indicates that size relationship of ovum and follicle has the same c^uantitative aspect in all animals studied. It is interesting that the regression line relating to the size of egg and follicle is steep in the first phase and almost horizontal in the second (Fig. 14.4). It is generally believed that the ovum attains its mature size about the time antrum formation begins in the follicle. Further, it is also believed that follicular response to pituitary hormones is confined primarily to those follicles in which the ova have attained their full dimensions (Pincus, 1936). It is well known that not all ova grow to mature size. Factors determining which of the ovarian eggs are destined to begin their growth or to complete their growth during a reproductive cycle are unknown and present very challenging problems. Growth of the follicle beyond the antrum stage may be quite independent of the presence of an ovum. This has been demonstrated in a variety of ways, but particularly by the observation that in senile rats large anovular follicles are of common occurrence (Hargitt, 1930). The converse has been reported; ova may grow to full size within the stroma of an ovary without being invested by follicular cells.


Of particular interest, also, are the questions raised by Gaillard (1950) and Dawson (1951) of the histogenetic relationship between the oocyte and follicular cells and the oocytic potentiality of the follicular cells themselves. In tissue culture explants from human fetal ovarian cortex, Gaillard described the development of cord-like groups of cells from the germinal epithelium. A second group of cord-like outgrowths developed from the follicular cells of the primordial follicles in which the oocytes had degenerated. New oocytes developed within these follicular cords and the surrounding cuboidal epithelial cells arranged themselves in a single layer to form the corona radiata. The observations of Gaillard emphasize the potential histogenetic interrelationships between the egg and the first layer of follicular cells. The possible inductive relationships of the ovarian egg and the various components of the follicle need to be clarified and offer excellent opportunities for more detailed investigation.


Fig. 14.4. Regression lines relating size of ovum and follicle in human ovaries (Green and Zuckerman, 1951b).


Studies of the various microscopically visible components of the ooplasm of mammalian eggs have not advanced as rapidly and significantly as have studies dealing with similar elements in the eggs of the lower vertebrates and invertebrates (Claude, 1941; Holtfreter, 1946a, b; Schrader and Leuchtenberger, 1952; Rebhun, 1956; Yamada, Muta, Motomura and Koga, 1957; Nath, 1960).


Relatively little information is availal)lo on the historv, biocliemical significance, and function of the cytoplasmic inclusions during the period of growth, maturation, or fertilization of the mammalian oocyte. In the dog, cat, and rabbit Golgi material of the young oocyte is first localized in the region of the nucleus, but it is later distributed throughout the ooplasm and finally aggregates near the cell periphery. The submicroscopic details of these shifts in the organelles of the oocyte have now been described for the rat and mouse. In oocytes with a single layer of granulosa cells the large Golgi complex lies at one pole of the nucleus (Fig. 14.5). This position of the Golgi complex is characteristic of primary follicles before zona pellucida formation. Large mitochondria with relatively few cristae are present also and at this stage are rather evenly distributed throughout the egg.



Fk;. 14.,5. Electron micrograpli of a portion of a imilammar or prniiary follirle obtained fronn a rat 2 days postpartum. The large mitochondria have much matrix and few cristae. The large Golgi complex is located at one pole of the nucleus. Note close apposition of granulosa cell membranes to oolemma! membrane. (Courtesy of Dr. L. Odor.)



Fig. 14.6. An electron micrograph of a small segment of a multilaminar follicle from a 15day-old rat. The peripheral location of the Golgi elements, its parallel stacked double membranes and associated vesicles are well shown. The relations between the microvilli and the granulosa cell profiles in contact with the oolemma may be observed. (Courtesy of Dr. L. Odor.)


As the egg continues to develop the follicle becomes multilayered and the Golgi complex now appears as a number of smaller units with a complex of stacked, parallel, double membranes lying relatively near the surface of the egg (Fig. 14.6). The mitochondria and other organelles also assume a more peripheral position. The behavior of the Golgi complex varies greatly from animal to animal (Zlotnik, 1948), and there are diverse opinions concerning its role in yolk production. Some investigators suggest that the Golgi material is concerned with the production of protein yolk, whereas others, working on different animals, maintain that it is always associated with the fatty yolk (Gresson, 1948 ».


During the early stages in the development of the follicle, the Golgi material in those cells arranged to form the corona radiata lies nearest the zona pellucida. Small granules from the vicinity of the Golgi material have been described, in fixed and stained cells, as migrating toward the egg (Gresson, 1933; Moricard, 1933; Aykroyd, 1938; Beams and King, 1938; Zlotnik, 1948) . How the yolk material is transferred from the cells of the corona radiata into the egg itself has not been miequivocably demonstrated. A reversal of the polarity of the Golgi complex in the follicular cells of the more mature follicles suggested to Henneguy (1926), Gresson (1933), and Aykroyd (1938.) that it may be responsible, at least in part, for the elaboration of the follicular fluid.


The appearance and distribution of the mitochondria in the mammalian egg also vary greatly from animal to animal. Rodlike or granular mitochondria have been described as being concentrated around the Golgi material in the fixed and stained eggs of the dog (Zlotnik, 1948) and in the cortical zones of the eggs of the bat, cat, and dog (Van der Stricht, 1923). In the mature unfertilized eggs of the rabbit, mouse, and hamster the mitochondria are concentrated in the peripheral zones. At the time of fertilization they migrate to the region of the developing pronuclei and tend to aggregate around them (Lams, 1913; Gresson, 1940).


Observations of the living eggs of the rat and guinea pig by time-lapse cinematography at the time of fertilization do not reveal a significant displacement of the cytoplasmic inclusions such as have been described in fixed and stained preparations.


The ultracentrifuge has been used in an investigation of the cytoplasmic components of the eggs of the mouse and human (Gresson, 1940; Aykroyd, 1941). In the human ovarian egg coagulated cytoplasm occupies more than one-half of the cell, whereas the nucleus, mitochondria, and Golgi material are confined in the remaining half. During ultracentrifugation the mouse egg is stratified into four distinct layers: (1) a centripetal layer, which stains very lightly and which may contain a few small Golgi aggregations, (2j a thin layer of yolk, (3) a relatively wide band containing the major portion of the Golgi material and the nucleus, and (4) a wider band containing principally the mitochondria (Gresson, 1940).

The distribution of nucleic acids in the developing and the mature rat and rabbit egg has been studied histochemically by Vincent and Dornfeld (1948),Dalcq (1956), Dalcq and Jones-Seaton (1949), Austin (1952b), Van de Kerckhove (1959); and Sirlin and Edwards (1959). As the oocyte grows, the desoxyribonucleic acid content of the nucleus is reduced and a perinuclear band of ribonucleic acid makes its appearance in the cytoplasm. Vincent and Dornfeld attributed the organization of the primary follicle to the evocating action of the ribonucleic acid elaborated by the oocyte. Alicrophotometric determinations of desoxyribonucleic acid (DNx\) have been reported on Feulgen-stained nuclei of mouse oocytes and of cleaving eggs (Alfert, 1950). The data indicate that the amount of DNA {present in a primary oocyte nucleus is constant, but that as the nucleus grows the DNA is progressively diluted. On the other hand, just before the first cleavage in fertilized eggs the amount of DNA in the pronuclei is doubled. The nuclei of each of the succeeding cleavage stages contain twice the amount of DNA present in the early pronuclei. In addition, studies were carried out on the protein concentration in oocytes and cleavage nuclei using the Millon reaction. The ripe egg contains a reserve of proteins which is divided among the cells and nuclei of the cleavage stages.


Attention should be directed to the raj)idly expanding literature dealing with the cytology and biochemistry of the eggs of amphibia and the chick. Clues for experimental methodology on the eggs of mammals may be found within these rejiorts (Bieber, Spence and Hitchings, 1957; Flickinger and Schjeide, 1957; Rosenbaum, 1957, 1958; Wischnitzer, 1957, 1958; Bellairs, 1958; Tandler, 1958; also see Tyler, 1955, and Brown and Ris, 1959).


The use of compounds labeled with radioisotopes is an important tool for the study of the transport and utilization of various substances by eggs (^Moricard and Gothie. 1955, 1957, Lin, 1956; Friz, 1959). Most of the tracer experiments have been done in the chick and amphibia in which it is clear that such egg storage materials as lecithin, cephalin, and vitellin are formed in organs outside the ovary and transported by way of the plasma to the egg. Greenwald and Everett (1959) injected pregnant mice with S^*" methionine and subsequently studied the eggs by radioautographic techniques. Ovarian ova and the blastocysts recovered from the cornua showed active protein synthesis. Similar synthesis was noted in the early fertilization stages. However, eggs in the 2-cell through the morula stages contained no demonstrable S^^ methionine. From these observations one would conclude that there is a basic difference in the metabolism of tubal and cornual ova, and again raises the question of the importance of the environmental fluids in providing materials necessary for the growth and development of the eggs.


Earlier investigators directed attention to the fact that in many mammalian eggs the deutoplasm is arranged in such a way as to exhibit an obvious polarity. Such polarity was described particularly for the eggs of the guinea pig by Lams (1913) and is conspicuous in a newly ovulated egg found in section by Myers, Young and Dempsey (1936). Such a polarity has been observed also in eggs of the cat (Van der Stricht, 1911), bat (Van Beneden, 1911), dog (Van der Stricht, 1923), and ferret (Hamilton, 1934).


Attention has recently been redirected to the fact that the mammalian egg may have a specific cytologic organization which is important in establishing its symmetry and polarity. This pattern of symmetry is based on the crescentic distribution of a primary basophilia and the localization of the mitochondria. The significance of the cytoplasmic organization in relation to the morphogenetic pattern in the mammalian egg must await the elaboration of new techniques of experimental embryology which can be applied to mammalian material ( Jones-Seaton, 1949; Dalcq, 1951, 1955; Austin and Bishop, 1959).


There are striking species differences in the amount and distribution of yolk material within the cytoplasm of living mammalian eggs. In the eggs of the horse, cow, dog, and mink the cytoplasm is so filled with fatty and highly refractile droplets that the vitellus under phase microscopy appears as a dark mass obscuring the nucleus (Squier, 1932; Enders, 1938; Hamilton and Day, 1945; Hamilton and Laing, 1946). In living eggs of the monkey, rat, mouse, rabbit, hamster, and goat the yolk granules are finely divided and vmiformly distributed; thus the various nuclear changes occurring during meiosis and fertilization are more readily visible (Long, 1912; Lewis and Gregory, 1929; Lewis and Hartman, 1941; Amoroso, Griffiths and Hamilton, 1942; Samuel and Hamilton, 1942; Austin and Smiles, 1948; Blandau and Odor, 1952). The ooplasm of human and guinea pig eggs is of intermediate density when compared to the two groujis mentioned above (Squier, 1932; Hamilton, 1944).


The mature mammalian egg is a cell of extraordinary size, and even the smallest (field vole, 60 //,) is large when compared with any of the somatic cells within its environment. It is remarkable that throughout the eutheria there should be so little relationship between the size of the adult animal and the volume of the egg (Hartman, 1929). Data on the apparent sizes of the vitelli of living eggs of various animals are summarized in Table 14.1. The need for more accurate measurements on the diameters and volumes of the living eggs of mammals still exists.


C. Egg Membranes

1. The Zona Pellucida

The zona pellucida is usually classified as a secondary egg membrane. It is believed to be a product of the primary layer of follicular cells which surround the oocytes in the ovary (Corner, 1928a). Under the light microscope the fresh zona pellucida appears as a more or less homogeneous membrane with a somewhat irregular surface, the amount of irregularity depending upon the species. As mentioned earlier the immature mammalian oocyte is surrounded by a single layer of cuboidal "follicle cells" whose plasma membranes are in intimate contact with the vitelline membrane. This relationship is partially altered in the growing egg by the gradual deposition of a mucopolysaccharide membrane which when fully formed constitutes the zona pellucida. At first the zona pellucida appears in irregular patches and in the form of an homogeneous secretion (Fig. 14.7). Slender microvilli which extend from the surface of the vitelline membrane are embedded in the zona. Short, blunt cellular processes also arise from the granulosa cell surfaces facing the zona and as the cells recede clue to the thickening of the zona they maintain contact with the vitelline memIjrane (Fig. 14.6). Several investigators have called attention to an agranular layer of cytoplasm of the granulosa cells in contact with the developing zona (Trujillo-Cenoz and Sotelo, 1959; Odor, 1960). This layer may indicate the elaboration of secretory material for the building of the zona pellucida. The agranular layer is certainly suggestive but not conclusive evidence for the follicular cell origin of the zona, for a similar layer of dense substance has been described just below the oolemmal membrane (Fig. 14.8). Some interpret the granular layer below the plasma membrane of the egg as indicative of the transfer of material of large molecular weight from the granulosa cells into the egg.


TABLE 14.1 Estimates of the diameter of the viteUus of various

mammalian ova (Modified from C. G. Hartman, Quart. Rev. Biol.,


4, 373-388, 1929)


Animal


Most Probable Size of Egg



M


Monotremata



Platypus

Echidna


2.5 mm. 3.0 mm.


Marsupialia

Dasyurus


240


Didelphys


140-1 GO


Edentata



Armadillo


80


Cetacea



Whale


140


Insectivora



Mole (Talpa)

Hedgehog (Erinaceus)

Rodentia


125 100


Mouse


75-87.8


Rat


70-75


Guinea pig


75-85


Hamster


72.2


Field vole


60


Lagomorpha

Hal)hit


120-130


("arnivora



Mink


107


Dog

Cat


135-145 120-130


Ferret


153


Ungulata Cow


138-143


Horse


105-141


Sheep


147


Goat


145


Pig


120-140


Chiroptera

Bat


95 105


Lemurs



Tarsius


90


Primates



Gibbon


110-120


M. mulatta


125-143


Gorilla


130-140


Man


130-140



As the zona pellucida increases in thickness the number of microvilli also greatly increase and extend into the zona for api:)roximately one-third of its width (Figs. 14.6 and 14.8). In eggs with fully developed zonae pellucidae, membrane profiles of the granulosa cell processes traversing this membrane have been observed in intimate contact with the oolemma (Fig. 14.8) (Yamada, Muta, Motomura and Koga, 1957; Odor, 1959; Sotelo and Porter, 1959; Anderson and Beams, 1960).

If the living tubal ova of mammals are examined with the phase microscope, the protoplasmic extensions of the corona radiata cells also may be seen penetrating the zona pellucida in an obliciue or irregular direction. These canaliculi are the radial striations of the zona pellucida described by Heape (1886) andNagel (1888).

It is well known that after ovulation and sperm penetration the egg shrinks and the ])eri vitelline space makes its appearance. At this time the surface of the vitellus appears quite smooth with the microvilli no longer demonstrable.

As mentioned above, the jn'otoplasmic extensions of the corona radiata are in intimate contact with the surface of the egg membrane. A number of investigators have described the passage of Golgi material from the follicle cells into the eggs in fixed preparations in fishes, reptiles, bii'ds, the sciuirrel, rabbit, and rat (Brambell, 1925; Bhattacharya. Das and Dutta, 1929; Bhattacharya, 1931). Zlotnik (1948) described the migration of small .sudanophilic granules from the vicinity of the follicular Golgi material into the oocytes of the dog, cat, and the rabbit. There is great need for clarification of the role of the cells of the corona radiata in the transport of various materials into the ooplasm and in the formation of yolk in the mammalian egg (Gatenby and Woodger, 1920; Kirkman and Severinghaiis, 1938).


Fig. 11.7. I'uiiKju ui uiiiLiiuiiiai- luilirli Hum :ai b-Jay-old rat. Tlic zona prlluri.la '././',' is just forming, and is deposited in irregular patches. The Golgi complex, not shown in this micrograph has begun to break up into smaller units. The mitochondria still have a random distribution. (Courtesy of Dr. L. Odor.)


Also awaiting clarification is the problem as to whether the retraction of the corona radiata cell processes alters the morphology and/or physical characteristics of the zona IK'llucida. The zona apparently is able to function as a differential membrane. It has been observed in the rat that accessory spermatozoa within the perivitelline space remain intact even until the time of implantation whereas those suspended in the fluids of the oviduct and not incorporated in j^hagocytic cells undergo complete disintegration within 12 to 24 hours after insemination. Furthermore, if the zona pellucida of a rat ovum is removed mechanically, the ooplasm then lying free in Ringer-Locke's solution will undergo visible plasmolysis within a few minutes.



Fig. 14.8. Small M-cUuii noiii all egg williin a laultilainiiiai l\)lln h in ui,i. w .< .-iu,.ll ,iii,.iiin was present. Continuity and extent of ovular microvilli are well shown. Note dense substance just inside the oolemma. (Courtesy of Dr. L. Odor.)



The physical properties of the zona pellucida vary according to the animal species and the experimental conditions under which the membrane is examined. Ordinarily the zona pellucida of a newly ovulated ovum is glassy, resilient, and tough. It is moderately elastic and may be considerably indented with fine needles without rupturing. Chemically the zona is composed chiefly of neutral or weakly acidic mucoproteins (Leach, 1947; Wislocki, Bunting and Dcmpsey, 1947; Barter, 1948; Leblond, 1950; Konecny, 1959; Da Silva Sasso, 1959). It is exceedingly sensitive to changes in hydrogen ion concentration: for example, the rat zona pellucida softens in buffers more acid than pH 5 and passes into solution in pH 4.5, but the rabbit zona rec}uires buffers of pH 3 or lower to accomplish the same effect (Hall, 1935; Braden, 1952).


The dissolution of the zona may also be effected by hydrogen peroxide and certain other oxidizing and reducing agents. Furthermore, the zona pellucida in fresh rat eggs may be dissolved readily by trypsin, chymotrypsin, and mold protease (Braden, 1952). In the rabbit the zona is removed by trypsin but is not affected by chymotrypsin or mold protease (Braden, 1952) . These data indicate that in both rat and rabbit ova the zona contains protein, but that the type of protein is not the same in the two species (Chang and Hunt, 1956). In rat eggs which are undergoing cleavage and which are examined immediately after being flushed from the oviduct the external surface of the zona is suflEiciently smooth so that the eggs may roll down the incline of a concave dish containing them. But after a short interval in the new environment, the zonae may become sticky and chng to the glass surface of the dish or to the pipettes and needles used in transporting them. Nonmotile spermatozoa caught within or on the zona pellucida have been pictured many times in the eggs of the human (Shettles, 1953), the rhesus monkey (Lewis and Hartman, 1941), the guinea pig (Squier, 1932), and the rabbit (Pincus, 1930). The same phenomenon has been observed only on rare occasions in rat eggs, again emphasizing differences in the physical characteristics of the zona from animal to animal.


There is very little information as to the permeability of the various membranes enclosing the mammalian egg. Recently the eggs of the rabbit, rat, and hamster were exposed to dyes such as toluidine blue and alcian blue and to a 1 per cent solution of heparin and digitonin in order to test the selectivity of the membranes (Austin and Lovelock, 1958) . It was found that the zonae pellucidae of all three animals were permeable to the dyes and digitonin but not to heparin.


There is too little known of the changes which occur in the zona pellucida and other egg membranes under varying environmental conditions to draw conclusions as to the nature of its selectivity. Techniques whereby invertebrate egg membranes are impaled with microelectrodes have yielded new information as to membrane potentials and resistance at varying stages of fertilization (Tyler, Monroy, Kao and Grundfest, 1956). Similar investigations on mammalian eggs would be valuable in solving the problems of selectivity of the egg membranes and in evaluating the response of eggs to various environmental fluids.


The question should also be raised as to whether or not the zona pellucida and/or the mucin coating may present barriers to the diffusion of gases and thus constitute a limiting factor to the rate of development. Fridhandler, Hafez and Pincus (1957) found no differences in the 0^ uptake when comparing normal rabbit eggs and eggs in which the mucin coat and zona pellucida had been punctured. Other properties of the zona pellucida will be considered later when the problem of the means by which spermatozoa penetrate it is discussed.


2. The Mucous Or "Albuminous" Layer

Unlike the zona pellucida, which is formed in the ovary, the "albumin" or mucous layer is deposited on the zona by secretions of the glandular cells in the oviducts or uterus and is therefore classified as a tertiary membrane.


In the monotremes (Hill, 1933) and many marsupials (Hartman, 1916; McCrady, 1938) an abundant albuminous coat is deposited on the zona pellucida as the egg moves through the oviduct. A similar deposit, but composed principally of mucopolysaccharides has been described for the eggs of various animals forming the order Lagomorpha (Cruikshank, 1797; Gregory, 1930; Pincus, 1936). A thinner but cheniically identical coat has been described in the ova of the horse and dog (Lenhossek, 1911; Hamilton and Day, 1945). It is only in the rabbit that the mucous coat has been charged with limiting the period during which the ovum can be penetrated by spermatozoa. A very thin layer of mucus has been observed on rabbit eggs removed from 5 to 8 hours after ovulation (Pincus, 1930; Braden, 1952). Furthermore, it has been shown that the rabbit egg must be penetrated by a spermatozoon before the 6th hour after ovulation if normal development is to ensue (Hammond, 1934). That the mucous membrane inhibits sperm penetration is confirmed by the fact that unfertilized rabbit ova may be stored in vitro for 48 to 72 hours without, in many instances, losing their fertilizing capacity after being transferred into the oviducts of properly timed recipients (Chang, 1953). It has been clearly demonstrated that the mucin is stored in the secretory cells of the oviduct and that estrogens are necessary for the synthesis of the mucin granules (Greenwald, 1958a). Discharge of the mucin granules is apparently controlled by progesterone. The thickness of the mucin coat on rabbit eggs, or glass beads placed in the oviduct, can be either significantly increased by injecting progesterone in properly conditioned females, or greatly reduced by injecting estrogens immediately after ovulation.


Apparently the ovum plays only a passive role in the process of mucin deposition. The remarkably even distribution of mucin on living eggs or glass beads implies that the oviduct has a specific pattern of muscular contraction so as to rotate the eggs as they move forward.

If the mucous coat is vitally stained with toluidine blue, one observes a concentric stratification which may indicate an appositional growth as the egg proceeds through the oviduct. Chemically the mucous coat is composed chiefly of strongly acid mucopolysaccharides. It is readily dissolved by trypsin, chymotrypsin, and pepsin. It is not affected by hydrochloric acid solutions as strong as 0.1 M but it may be slowly removed by solutions more alkaline than pH 9. A peculiar and important proi)erty of the albuminous coat is that at pH 9 or 10 it becomes exceedingly sticky. As will be noted later, this may be of importance for the adherence of the egg to uterine tissue at the time of implantation. The possible role of the mucous coat in the development of the egg was not realized until the investigations of Boving (1952c) in which certain details of rabbit blastocyst implantation were observed directly. A plastic chamber was developed for examining the interior of the pregnant rabbit uterus. It was noted that the mucous coat participates actively in the initial adhesive attachment of the blastocyst to the uterus. Such localized attachment precedes by several days the cellular adhesion and invasion of the uterus by the blastocyst. Boving observed further that the adhesion to the uterus is localized in the abembiyonic hemisphere of the blastocyst, probably because it is in this region that an alkaline reaction, produced by secretions of the embryo, enhances the stickiness of the mucous coat. The polar localization of the adhesive attachment of the mucous coat not only provides a mechanism for the initial blastocyst attachment, but also is important in establishing the orientation of the blastocyst within the uterus (see section on "Spacing and orientation of ova in utero").

Boving (1954) observed that still another membrane is deposited on the rabbit egg by secretions of the uterus. The membrane forms a sticky covering that stains metachromatically in toluidine blue and functions as an adhesive attachment during positioning and orientation of the blastocyst in utero. He proposed that the noncellular, adhesive layer be called the "gloiolemma."

D. The First Maturation Division

Meiotic division is not a phenomenon which is confined entirely to the ova in the preovulatory follicles. It may be encountered in egg cells in the latter part of embryonic development, in immature follicles undergoing atresia, and in ovaries stimulated excessively by the animal's own pituitary hormones, or by pituitary hormone preparations which have been injected (Evans and Swezy, 1931; Guthrie and Jeffers, 1938; Dempsey, 1939; Witschi, 1948).


Fairly complete descriptions of the various stages in the formation of the first polar body and second maturation spindles are available for a number of mammals (Hartman and Corner, 1941, the macaque; Hoadlcy and Simons, 1928, Hamilton, 1944, and Rock and Hertig, 1944, the human; Kirkham and Burr, 1913, Blandau, 1945, Odor, 1955, the rat; Long and Mark, 1911, the mouse; Moore, 1908, the guinea pig; Langley, 1911, the cat; Van Beneden, 1875, Pincus and Enzmann, 1935, the rabbit; Robinson, 1918, the ferret).


Specific data on the temporal relationship between ovulation and the first maturation division are available primarily for the rabbit (Pincus and Enzmann, 1935-1937), guinea pig (Myers, Young and Demi-)sey, 1936), cat (Dawson and Friedgood, 1940), rat (Odor, 1955), and mouse (Edwards and Gates, 1959).

The rabbit is an animal particularly suited for studies of maturation phenomena because it ovulates regularly between 9 and 10 hours after copulation. The first evidence of change in the nucleus of a ripe ovum may be seen 2 hours after copulation. At this time the nuclear membrane is intact but tetrad formation is in evidence. Four hours after copulation the nuclear membrane has disappeared and the first polar spindle, with tetrads located on the metaphase plate, occupies a paratangential position near the periphery of the ooplasm. Abstriction of the first polar body is completed about 8 hours after copulation. Shortly thereafter, the second metaphase spindle is formed and remains in position just below the surface of the primary egg membrane. It remains in this condition until the fertilizing spermatozoon penetrates the egg.


Similar observations on successive phases of the first maturation division have now l)een completed for the rat (Odor, 1955). In over 1500 living and fixed eggs examined at specific times before and after the onset of heat it was observed that by the onset of heat, the germinal vesicle has lost its memiM'ane in most animals, and has been transformed into a a dense chromatic mass which then quickly moves towards the periphery of the ooplasm. Between the 3rd and 4th hours the chromosomes have arranged themselves in the metaphase plate. Abstriction of the first polar body is usually completed between the 6th and 7th hour, and positioning of the second metaphase spindle by the 8th hour. It is interesting that, even though there was considerable variation in the stages of maturation found in animals killed at the same time after the onset of heat, 83 per cent of all the ova were in the same stage of maturation or in a very closely related phase.

In all mammals studied, except the dog and fox (Van der Stricht, 1923; Pearson and Enders, 1943), the first maturation division is completed within the ovarian follicle several hours before it ruptures.


There is evidence that a specific correlation exists between the gonadotrophins and the maturation phenomena within the oocytes (Bellerby, 1929; Friedman, 1929; Friedgood and Pincus, 1935). Apparently the threshold of response of oocytes for maturation is lower than is the threshold for ovulation (Hinsey and Markee, 1933). Moricard and Gothie (1953) injected small quantities of chorionic gonadotrophin directly into the ovarian follicles of unmated ral)bits and observed the formation of the first metaphase spindles and the abstriction of the first polar bodies. This was interpreted as showing the direct effect of pituitary hormones in inducing meiosis. On the basis of a study on oocytes recovered from ral)bit ovaries Chang (1955b) concluded that once the oocytes have attained the vesicular stage maturation can be readily induced by a variety of experimental procedures the most effective of which is the subnormal temperature treatment of unfertilized ova. According to his investigations first polar body formation is not immediately dependent on gonadotrophic stimulation.


A number of investigators who have examined mammalian ova have commented on the rapid disappearance of the first polar body. Sobotta and Burckhard (1910) saw the first polar body in only 2 of 100 recently ovulated mouse ova. The infrequent presence of the first polar body in postovulatory ova in which the second maturation spindle was completed suggested that possibly the first polar body was not always formed (Sobotta, 1895) . Yet from a variety of studies on meiosis in fixed and living eggs, it may be concluded that the abstriction of the first polar body invariably occurs. In addition it may not disappear as rapidly as some of the older investigators believed. The first and second polar bodies are visible in a 4-celled guinea ])ig embryo photographed by Squier (1932). There has been considerable speculation as to the method whereby the first polar body disappears. Kirkham ( 1907) suggested that the first polar body in the mouse either was forced through the zona pellucida or escaped from the perivitelline space by its own ameboid movement. Similar theories have been held by Moricard and Gothie ( 1953) for the rat, in which they maintain that the polar body passes directly through the zona pellucida. From the observations of Lams and Doorme (1908) in the mouse. Mainland (1930) in the ferret, and Odor (1955) in the rat, it is almost certain that the first polar body undergoes rapid fragmentation and cytolysis within the perivitelline space so that only some finely granular material remains. Ameboid movement of the first body has never been documented in the thousands of living mammalian eggs examined.


E. The Ovulated Egg

The appearance of tubal ova from a single animal varies considerably depending on the lapse of time between ovulation and examination and the environmental fluids in which they are kept. When the eggs are shed from the follicles they are ordinarily surrounded by a variable number of layers of granulosa cells and a matrix of more or less viscid follicular fluid. The vitellus does not completely fill the zona pellucida, and the first polar body, if it has not already disintegrated, may be pressed between the zona and the ooplasmic membrane. An exception to this may be found in the Canidae in which formation of the first polar body is apparently delayed for some time after ovulation. The length of time that the coronal cells persist varies greatly in the eggs of different species.


A well developed corona radiata is regularly found in newly ovulated ova of the mouse (Lewis and Wright, 1935), the hamster (Ward, 1946), the rat (Gilchrist and Pincus, 1932), the rabbit (Gregory, 1930), the cat (Hill and Tribe, 1924), the dog (Evans and Cole, 1931) , the monkey (Lewis and Hartman, 1941), and man (Hamilton, 1944; Shettles, 1953). The rapid dispersal or even absence of the cells forming the corona radiata has been reported for the sheep (McKenzie and Terrill, 1937), the cow (Evans and Miller, 1935; Hamilton and Laing, 1946; Chang, 1949b), the pig (Corner and Amsbaugh, 1917; Heuser and Streeter, 1929), the horse (Hamilton and Day, 1945), and the deer (Bischoff, 1854), and would seem to be a characteristic of the newly ovulated guinea pig ovum (Myers, Young and Dempsey, 1936).


The eggs of unmated females gradually lose their investment of granulosa cells as they pass through the oviducts. The cells become rounded and drop away from the cumulus, a process that occurs first in the more peripheral cells. The cells of the corona radiata which are adjacent to the zona pellucida are the last to fall away and when they are brushed from the surface of the zona in living eggs in vitro their long and irregularly shaped protoplasmic processes extending into the zonal canaliculi can be seen (Squier, 1932; Duryee, 1954; Shettles, 1958). The mechanism which effects the dispersal and final dissolution of the cumulus oophorus and corona radiata in unmated females is not known. It has been suggested that an enzyme, elaborated by the tubal mucosa, is responsible for the dispersal of the cells (Shettles, 1958).


When an observer follows the cytologic changes in the cells forming the cumulus oophorus as ovulation approaches and notes their behavior in tissue culture preparations in vitro he is impressed with the suggestion that separation of the cells involves a gradual depolymerization of the intercellular cement substance and a change in the activity of the cell surface.


If time-lapse photographs are made of the coronal cells surrounding ovulated eggs, a very active bubbling and "blister" formation of the surface membranes is apparent. "Bubbling" activity of the cell surfaces is frecjuently seen in cells which are losing their vitality (Zollinger, 1948). These surface changes occur at the time when the cells are undergoing most active separation and accounts for the withdrawal of the cytoplasmic processes from the zona pellucida. Further evidence that the behavior of the cell is related to loss of vitality is shown by their very poor growth in tissue culture.


F. Respiratory Activity of Mammalian Eggs

There have been only limited investigations on the energy-yielding mechanisms and energy-requiring processes of the developing eggs of mammals (rat, Boell and Nicholas, 1948; rabbit, Smith and Kleiber, 1950, Fridhandler, Hafez and Pincus, 1957; and cow, Dragoiu, Benetato and Oprean, 1937).


The fertilized ovum during its various stages of cleavage and differentiation is an ideal experimental object for such studies and has been used extensively in the invertebrates, amphibia, and birds where large numbers of eggs are readily available (Boell, 1955). Refinements in the Cartesian diver technique have made possible the measurement of gas exchange of less than 1 mfxi.; thus the number of mammalian eggs required to obtain significant data need not be large (Sytina, 1956). Furthermore, the effectiveness of gonadotrophins in inducing ovulation in the sexually immature female rodents and their willingness to mate after such treatment provides a ready source of eggs independent of ovulation at specific phases of the sexual cycle.


The type of information which can be obtained by measuring the Oo uptake of fertilized rabbit ova placed in the Cartesian diver and subjected to a variety of metabolites and inhibitors can be seen in Tables 14.2 and 14.3 (Fridhandler, Hafez and Pincus, 1957). In the rabbit egg, as in other cells, cyanide has a markedly inhibiting effect on respiration. This inhibition is reversible and presumably cyanide acts through the cytochrome oxidase system. Of significance is the finding that glucose is not an obligatory substrate for respiratory activity of the fertilized rabbit egg.


If glucose is added to the medium containing 2- to 8-cell eggs, there is little capacity to carry out glycolysis. However, such capacity develops during the late morula and blastocyst stages. This change may indicate either an alteration in the membrane characteristics of the egg, or the develojmient of a new enzyme system as the egg develops.


The electrical characteristics of eggs and their changes during activation and fertilization have been studied in frogs, echinoderms, and fish (Maeno, 1959; Ito and Maeno, 1960). The electrical properties and membrane characteristics of mammalian eggs are entirely unknown. The use of the ultramicro-electrode which has been so helpful in nerve and muscle electrophysiology offers an unusual research tool for examining the primary process of activation of


G. Transport Of Tubal Ova

The mammalian oviducts must perform a variety of functions in the transport and development of the gametes (also see "Sperm transport in the female genital tract") . They must provide some means for transporting the ovulated ova from the ovary or periovarial space into the infundibulum. Secretions must be elaborated within the infundibulum in order to provide an environment favorable for sperm penetration. In some animals, such as the rabbit, opossum, horse, and dog, specialized cells secrete materials which form tertiary membranes for the eggs. Still other cells secrete nutritional and possibly other substances which may be essential for the normal growth and development of the fertilized eggs. Furthermore, the peristaltic and antiperistaltic activities of the oviducts must be regulated in such a way that the ova are propelled forward at a definite rate and in proper rotational sequence so as to be evenly coated with the tertiary membranes. The oviducts are indeed highly specialized organs whose anatomic differences in the various regions have been described by many investigators but whose specific physiologic functions still present many unsolved problems.

As evidence accumulates, a happier middle ground of opinion is forming as to the roles of the musculature and ciliary activity in the downward propulsion of the eggs and in the ascent of the spermatozoa. Comprehensive summaries of observations and theories dealing with these particular problems may be found in the papers and monographs of Westman (1926), Parker (1931), Hartman (1939), Alden (1942b), Kneer and Cless (1951).

The more extensive investigations of the oviducts during the estrous cycle include: (1) The observations of Snyder (1923, 1924), Andersen (1927a, b), Anopolsky (1928), Westman (1932), and Stange (1952), on the lymphatics, the size of muscle fibers, and the cyclic changes in the epithelium of the Fallopian tubes of the rabbit, sow, and man. (2) The alterations of rhythmic contractions in the oviducts of the rat (Alden, 1942b; Odor, 1948), the sow (Seckinger, 1923, 1924), the rabbit (Westman, 1926), the rhesus monkey (Seckinger and Corner, 1923; Westman, 1929), and man (Seckinger and Snyder, 1924, 1926; Westman, 1952).

The specific method whereby the newly ovulated egg is moved from the site of rupture of the ovarian follicle to the infundibulum is poorly understood. There is considerable species variation in the relationship of the fimbriated end of the oviduct to the ovary proper. In the Muridae and Mustelidae the ovaries are almost enclosed by the thin, membranous periovarial sac (Alden, 1942a; Wimsatt and Waldo, 1945). The medusa-like infundibulum is enclosed within the sac but occupies a relatively small area of the periovarial space. It is believed thai in those animals in which fluids accumulate within the ovarian bursa at the time of ovulation the ova are directed to the ostium by movement of these fluids into the oviduct (Fischel, 1914). However, observations on normal fluid flow within the periovarial sac are very limited. It has been demonstrated that if dyes such as Janus green or particulate material are introduced into the periovarial space in the immediate vicinity of the ostium, the material quickly passes into the first loop of the oviduct (Alden, 1942b). Transport is effected primarily by the ciliary activity of the fimbriated end of the ostium (Clewe and Mastroianni, 1958) . Furthermore, if newly ovulated eggs are placed on the surfaces of the fimbriae in the rat, mouse, or hamster the cilia will sweep them into the infundibulum within 8 seconds (Blandau, unpublished observations). How those ova located at some distance from the oviduct reach the fimbria has not been observed.


TABLE 14.2 Effect of pre -incubation on O2 uptake of fertilized ova Incubating medium: Ca++-free Krebs-Ringer phosphate, pH 7.4. Gas phase: air. (After L. Fridhandler, E. S. E. Hafez, and G. Pincus, E.xper. Cell Res., 13, 132-139, 1957.)


Developmental Stage


Pre-incubation of Ova


Metabolites Added to RP in Diver


Average O2 Uptake


Morphology


Hr postcoitum


r'c


Time





min.



m fil . / oTu»! / hr .




23 23 23


120 120 120


None

0.1% ghicose

10~^ M pyruvate


0.45 0.49 0.47


2-4 cell


20-28


29 29 29


90 90 90


None

0.1% glucose

10"" M pyruvate


0.45 0.42 0.41




29 29 29


180 180 180


None

0.1%, glucose

10~" M pyruvate


0.39 0.59 0.53


Blastocyst


108


37 37


150 150


None

10^2 M pyruvate


1.84 2.42


TABLE 14.3 O2 uptake of fertilized ova in different media Gas phase: air. (After L. Fridhandler, E. S. E. Hafez, and G. Pincus, Exper. Cell Res., 13, 132-139, 1957.)


Developmental Stage



Medium in the Divers


Average O2 Uptake




Morphology


Hr. Postcoitum


Basic medium


Added substances (m)


mill./ ovum /hr.


2-8 cells


24-30


RPG


None

lO" M NaCN (appr.)

10^" M phlorizin


0.41 0.02 0.41




RPG


None

2 X 10-3 M Na fluoride


0.56 0.47


Morulae


68


RP


None

10-2 M malonate 10-2 M malonate plus 10-3 M fumarate


0.48 0.42

0.47


Blastocysts


78


RP


None

10 2 M malonate 10-2 M malonate plus 10-3 M fumarate


1.71 1.69

1.74


Blastocysts


88


RP


None

10-2 M malonate 10-2 M malonate plus 10-3 M fumarate


2.92 2.36

2.70


Blastocysts


115


RPG


None

10-3 M NaCN (appr.)


78.00 0.00


BIOLOGY OF EGGS AND IMPLANTATION


821



Under normal physiologic conditions the ovary moves backwards and forwards within the periovarial sac. These movements are accentuated at the time of ovulation and are effected by the abundant smooth muscle in the mesovarium. Such activity keeps the fluids of the periovarial sac in motion. Those eggs ovulated at the opposite side of the ovary away from the infundibulum are passively moved into its vicinity where ciliary currents then aid in completing transport.

A potentially wide communication between the ostium of the oviduct and the peritoneal cavity exists in a variety of animals such as the guinea pig, rabbit, monkey, and man (Sobotta, 1917; Westman, 1952). The extent of the communication varies with the stage of the menstrual or estrous cycle. Ordinarily in a rabbit not in heat, the fiml)riae do not cover the ovary. As the time of ovulation approaches, there is a great increase in motility and turgidity of the fimbriae so that they almost enclose the ovary (Westman, 1926, 1952). Recently attempts have been made to observe the activities of the human fimbriae by means of abdominal l)eritoneoscopy or exploratory culdotomy. Elert (1947) has seen the elongated fimbria grasp the lower pole of an ovary for as long as 2 minutes. Doyle (.1951, 1954), how^ever, failed to observe either a sweeping or grasping motion of the fimbriae before or during the rupture of the follicle. He suggests that in the human female the initial transport of the ovum is by a process in which it floats into the cul-de-sac and from there is siphoned into the ampulla by simple peristaltic contractions which originate at the region of the fimbriae. Doyle's (1956) recent observations are more in line with those described by Elert above.

It has been suggested that the activity of the abundant smooth musculature of the adnexa and the fimbriae produces a powerful suction effect on the ovary, thus drawing the ovulated eggs into the tube (Sobotta, 1917; Westman, 1952). It is a fact, however, that no one has made measurements of this presumed negative pressure, nor, as pointed out earlier, has anyone observed a newly ovulated mammalian ovum transported from the surface of the ovary into the oviduct in animals in which the ovaries are not enclosed in periovarial sacs. During laparotomy there are very real problems in maintaining the normal anatomic position and physiologic condition of the oviducts so that their actual function in vivo can be assessed accurately. In general the muscular activity of the fimbriae has received more enthusiastic support than the cilia as being the agent for the transport of eggs from ovary to oviduct. However, in the few instances in which eggs were placed close to the fimbriae and egg transport observed directly, the ciliary activity of the fimbriae appeared to be primarily responsible.

The rate of the ciliary beat in the rabbit Fallopian tubes has been studied by Borell, Nilsson and W^estman (1957) ; during estrus the cilia beat at a rate of 1500 beats per minute.

The rate increases about 20 per cent on the 2nd and 3rd day after copulation and at the time of implantation. By the 14th day of pregnancy the rate of beat had returned to normal. There was no significant difference in the rate of beat in cilia removed from various segments of the oviduct. Many more direct and continuous observations on the intact oviducts of different animals are needed before definite conclusions may be reached as to the mechanics of egg transport from the ovary to the infundibulum.

In the rat, mouse, and hamster, one of the most striking changes in the oviduct is the dilation of the ampulla during the heat period (Sobotta, 1895; Alden, 1942b; Burdick, Whitney and Emerson, 1942). In the rat several of the loops of the ampulla begin to dilate between the 3rd and 4th hours after the onset of heat, maximal dilation being about the time of ovulation (Odor, 1948) . A constriction at the distal end of the dilated loop is frequently visible as a distinct blanched segment a few millimeters in length and in which the mucosal folds fit snugly against each other. This valve-like constriction is responsible for the retention of the oviducal fluids and eggs for at least 18 to 20 hours. Nothing is known of the nervous or hormonal mechanisms effecting the constriction, nor how spermatozoa cope with the stenosis as they proceed through the oviducts to reach the ampullae where sperm penetration occurs. The eggs of the mouse, rat, and hamster are fertilized in the dilated ampullae and I'emain there for approximately 20 to 30 hours after ovulation (Burdick, Whitney and Emerson, 1942; Odor and Blandau, 1951 ; Strauss, 1956 1 . In the rabbit the freshly ovulated eggs pass through the upper half of the oviduct within 2 hours after ovulation and come to lie at the junction of the ampulla and isthmus. They remain here for the next day and a half (Greenwald, 1959).

Normally sperm penetration into the eggs of mammals takes place in the ampullae. There are, however, several interesting exceptions. In ferrets, tenrecs, and shrews spermatozoa somehow enter the ovarian follicles containing the ripe eggs and penetrate them before ovulation.

Both ciliary activity and peristalsis are involved in moving the eggs into the dilated ampullae. Burdick, Whitney and Emerson (1942) showed that ciliary action in the second loop of the oviduct in the mouse is sufficiently strong to rotate a whole cluster of eggs. Vigorous, localized peristaltic waves, spaced 12 to 16 seconds apart, seemed to be more important than the cilia in moving the eggs towards the entrance of the isthmus. Almost identical observations have been reported for the transport of eggs in the ampulla of the rat (Alden, 1942b; Odor, 1948).


As the time of ovulation approaches in the rat, the contractions of the dilated loops of the ampulla increase in amplitude more than in rate. The force of the aduterine contraction waves, measured by the rate of movement of particulate matter in the lumen, greatly exceeds that of the antiperistaltic activity. The contraction waves do not extend beyond the constriction at the uterine end of the dilated ampulla. Clumps of ovulated eggs, stained lycopodium spores, or ascaris eggs were moved vigorously backwards and forwards within the lumen of the tube and then forced gradually into the distal, most dilated loop. This vigorous activity subsided rapidly after ovulation and would have been missed completely if continuous observations had not been made. It would be important to determine more accurately the temporal relationship between ovulation, the dilation of the ampulla, and the changes in the pattern of muscular contractions of this area as compared with the remaining coils of the oviduct.

The passage of ova through the isthmus and intramural regions proceeds at a remarkably constant rate in various animals. The principal forces invoked are muscular or ciliary or both. Whatever the mechanism for propulsion may be, it is not necessarily similar for all species nor for any particular segment of the oviduct within a single animal (Sobotta, 1914; von ]\Iikulicz-Radecki, 1925; von ]\Iikulicz-Radecki and Nahmmacher, 1925, 1926; Kok, 1926; Alden, 1942b; Burdick, Whitney and Emerson, 1942; Odor, 1948).

On the basis of their studies on the behavior of the rabbit oviduct in vitro, Black and Asdell (1958) suggested that the movement of the luminal contents imparted by the circular muscles is ample to account for the transport of sperm and eggs through all of the oviduct except the isthmus. When the ova reach the isthmus they w^ait until sufficient fluid "surges down the tube to sweep them through the tubo-uterine junction" (Black and Asdell, 1959).

When the in vivo movements of oviducts are studied by short interval time-lapse cinematography one is impressed wdth the variety of contraction patterns exhibited at different times in the cycle. These observations re-emphasize the importance of applying a host of techniques to chirify the physiology of the oviducts.

The normal functional state of the oviducts is dependent on the maintenance of a delicate balance between estrogen and progesterone. In the mated mouse and rabbit, injections of estrogen result in tube-locking the ova for as long as 7 days after copulation, at which time the eggs degenerate (Burdick and Pincus, 1935; Burdick, Whitney and Pincus, 1937). By contrast, the injection of progesterone (Alden, 1942c) and induced superovulation (Wislocki and Snyder, 1933) accelerate the passage of eggs. Fertilized ova introduced into the oviducts of pseudopregnant rabbits will continue to develop normally but they are not transported into the uterus. Similarly the eggs of donor rabbits will not be transported if they are introduced into the oviducts of estrous females in which there is no luteal growth (Austin, 1949b). Alden (1942c) carefully removed the ovaries from the periovarial sacs in mated rats and observed the position and development of ova. Ovariectomy after ovulation did not prevent the normal development or transport of the eggs through the oviduct and, in fact, hastened their transport. Noyes, Adams and Walton (1959) ovariectomized rabbits and found that when freshly ovulated eggs from donor females were transplanted into the ampulla of the oviduct, the eggs were transported into the uterus in 14 hours.

There is very little pertinent information concerning the role of the cilia in moving the ova through the isthmus and intramural portions of the oviduct. Because of the thickness of the muscular wall in these areas it is difficult to observe the activity of the cilia in living specimens even by transillumination (Alden, 1942b). Also the number, size, and arrangement of the ciliated cells in the oviduct varies greatly from species to species. In addition, individual variations within a given species have been described throughout the reproductive cycle (Sobotta, 1914; Novak and Everett, 1928; Hartman, 1939; Burdick, Whitney and Emerson, 1942; Odor, 1948).

The earlier observations of Parker (1928, 1931) on the ciliary currents in the opened oviduct of the turtle. Chryseunis picta, have recently been repeated and extended by Yamada (1952) to the tortoise, Clemmys japonicus, and the frog, Rana nigromaculata. Yamada described a reverse ciliary movement beating toward the ovarian end of the oviduct in both animals. The rate of the descending current was about two times faster than that of the ascending current. In the frog the activities of the cilia cause the eggs to rotate as they descend. This may be an important mechanism for coating the eggs evenly with egg jelly. Crowell (1932) also described a tract of cilia beating toward the infundibulum in the oviducts of several species of lizards. It is generally assumed that during the period in which eggs are being transported the oviducts of most mammals undergo a secretory phase, but it is not known what proportion of the fluid within the lumen is contributed by the secretions of the oviduct, the lining of the periovarial sac when present, the follicular fluid, and the peritoneal fluid. Even less is known concerning the chemistry of these fluids. The rabbit, hare, opossum, and possibly the dog and horse present peculiar problems because of the specialized mucous secretions which coat the eggs and form the tertiary membranes.


The cytology and secretory activity of the epithelial lining of the oviduct have been the subjects of many studies in mammals, but there is little unanimity of opinion regarding (1) the changes in cellular morphology during the cycle, (2) the types of secretions elaborated, and (3) the cyclic variations of the particular secretory products which have been identified. In the oviducts of the pig and man both secretory and ciliated cells are present in the same proportions in all phases of the cycle. The height of the ciliated cells varies periodically, reaching a maximum during the time the eggs are passing through the tubes (Snyder, 1923, 1924; Novak and Everett, 1928; Bracher, 1957). Allen (1922), among others, expressed the view that there are no ciliated cells in the isthmus of the oviduct of the mouse or rat. This interpretation must be modified at least for the rat, in view of the findings of Alden (1942b), Kellog (1945), and Deane (1952) that both ciliated and secretory cells are present in the isthmus of this animal. Alden (1942b) and Deane (1952) were unable to observe cyclic variations in the histologic or histochemical picture of the oviducts of the rat. In the mouse the primary cyclic alteration of the epithelium is restricted to a slight but significant variation in the height of the ciliated cells ('Espinasse, 1935). In the sheep the majority of the secretory cells are confined to the ampulla, few being found in the isthmus (Hadek, 1953). Hadek describes a significant increase of secretory products in the lumen of the oviduct during estrus and early in the metestrum.


Studies of electron micrographs of ultrathin sections of oviducts of the mouse, man (Fawcett and Porter, 1954), rabbit (Borell, Nilsson, Wersall and Westman, 1956; Nilsson, 1957), and rat (Odor, 1953; Nilsson, 1957, 1959) have demonstrated the similarity of the ciliary apparatus of epithelial cells in the various species. Of special interest was the presence of tiny, filiform projections on certain of the cells interspersed among the ciliated cells (Fig. 14.9). Similar projections are also found on the luminal surface of what are probably the secretory cells. These processes do not have the longitudinal fibrils nor basal corpuscles that are essential components of cilia. A comparative study of the fine structure of the mammalian oviducts at carefully timed intervals and under different hormonal influences may lead to important observations of cyclic variations in both the ciliated and secretory cells (Borell, Nilsson and Westman, 1957).


The histochemical characteristics of the epithelium of the oviduct have been studied particularly by Deane (1952) and Milio (1960) in the rat, Hadek (1955) in the sheep, Fredricsson (1959b) in the rabbit, Fawcett and Wislocki (1950) and Fredricsson (1959a) in man. In the rat alkaline phosphatases occur on the ciliated borders of the cells of the isthmus, which suggests that this material has a role in the transfer of phosphorylated compounds. The rat differs from many other species in that glycogen could not be demonstrated in the epithelium of the oviduct at any time of the cycle. Quantities of esterase were present in the cells of all regions but only the cells of the fimbriated end contained lipid droplets. It is interesting as noted earlier that in the rat no histochemical changes could be demonstrated during the various phases of the estrous cycle. In the sheep an acid mucopolysaccharide is secreted by the oviduct most profusely at the time of ovulation (Hadek, 1955). Amylase is present in the .secretions of the oviducts of man, cow, rabbit, and sheep in concentrations above that found in homologous sera. The significance of the relatively high concentrations of this enzyme in relation to the reproductive process is not clear (McGeachin, Hargan, Potter and Daus, 1958).


Fig. 14.9. Electron microgiaph of a thin section of the oviduct of the rat. Note nonciliated cell with microvilli wedged between ciliated cells. NN, nucleus of nonciliated cell ; NC, nucleus of ciliated cell; BB, basal bodies; C, cilia; MV, microvilli. (Courtesy of Dr. L. Odor.)




In man glycogen occurs not only in the ciliated cells but also in the nonciliated epithelia. Even though it is impossible to draw a firm conclusion regarding the correlation of glycogen in tubal epithelium with the menstrual cycle, it is generally believed that the maximal amount is present during the follicular phase (Fawcett and Wislocki, 1950).

It is generally assumed that the luminal fluids of the oviducts and cornua undergo cyclic changes, not only in amounts secreted, but also in their chemical composition. Such assumptions are based on very tenuous evidence; actually these fluids have received very little attention primarily because of the problems in obtaining adequate samples and in correlating the chemical and physical characteristics of the tract fluids with the endocrinologic and histochemical activity of the cells forming the stroma. With the development of a method for the volumetric collection of tubal fluid <Clewe and Mastroianni, 1960; Mastroianni. Beer, Shah and Clewe, 1960), accurate information with respect to the cjuantity and nature of the secretion may now be obtained. In the meantime we are dependent on the reports by Bishop (1956a, c) and Olds and Van Demark (1957a, b) who have recently summarized and extended the information available on the composition and endocrine control of luminal fluids in the female genital tracts of the rabbit and cow.

Observations on hydrogen ion concentrations of the fluids within the periovarial sac, the dilated ampullae, and uterine cornua in the rat have revealed that the fluids of the periovarial sac and oviduct have a pH of approximately 8.05 ± 0.18, whereas the mean pH of the cornual fluid is 7.74 ± 0.12 (Blandau, Jensen and Rumery, 1958). The fluids from the reproductive tract were significantly more basic than the peritoneal fluids. The results from biochemical studies on tubal fluid and ligation experiments reveal clearly that tubal fluid is the product of the oviduct itself and that contributions from the peritoneal cavity or uterus are either minimal or absent. Much more information is necessary to learn the nature of the molecular species present in the fluids of the reproductive tract. Free electrophoretic patterns of the cornual fluids of rats in heat demonstrate the presence of four major components in low concentrations. The leading major component has mobility values somewhat faster than albumen and the remaining components have mobilities within the range of normal serum proteins. Studies of cornual fluids by paper electrophoresis, however, suggest that the distribution of the proteins is not the same as in rat serum (Junge and Blandau, 1958). Previous observations on the washings of a sheep's oviduct examined 45 to 60 minutes after death showed a |)H of 6 to 6.4 during the diestrum and 6.8 to 7.0 during estrus and the metestrum (Hadek, 1953). The pH of the uterine fluids in cattle has been reported as ranging from 5.8 to 7.0 with very minor changes during the cycle (Sergin, Kuznecov, Kozlova and Nesmejanova, 1940).

Respiratory differences between the epithelium of the ampulla and the infundibulum of the human oviduct have been studied by Kneer, Burger and Simmer (1952) and Mastroianni, Winternitz and Lowi (1958). They found an increase in the respiratory rate of botii segments during the follicular phase, but not during the secretory phase. During all phases of the cycle the oxygen consumption of the epithelium of the amindla was consistently higher than that of the epithelium of the infundibulum. Bishop (1956b) measured oxygen tension in the lumen of the rabbit oviduct by electrochemical techniques and found that the luminal environment is aerobic and that the oxygen tension is in equilibrium with that of the arterioles within the endometrium.



TABLE 14.4

Volume of fluid secreted by the doubly ligated rabbit

oviduct during a three-day interval

(After D. W. Bishop, Am. J. Physiol.,

187, 347-352, 1956a.)


Condition of Animal


Number of Tracts


Volume of Tubal Secretion



Ligated


Range


Average


Estrogen-dominated. .

Progestational

Castrate (9-day)


18 11


ml.

1.3-4.5 0.3-2.3 0.0-2.1


ml.

2.62 1.10 0.80



Finally, it has been established that secretions of the oviduct undergo changes in response to hormonal variations (Table 14.4). Bishop (1956a) studied the rates of fluid production and the secretion pressures in rabbit oviducts under a variety of experimental conditions. Ligatures were secured around the uterotubal junctions. Polyethylene tubes were then inserted into the fimbriated ampullae and securely tied, and manometric changes in fluid pressures were recorded continuously for periods up to 52 hours. The mean rates of oviduct secretion are recorded in Table 14.4 and the maximal secretory pressures graphically in Fig. 14.10. The data indicate that the oviducts of rabbits exhibit an active process of secretion against a gradient. The variations in secretory pressures are related to changes in hormonal activity in the normal female or to hormonally induced responses in the castrate animal. Corner (1928b) showed that if the ovaries of rabbits are removed 4 to 8 hours after ovulation, all of the eggs are transported to the uterus, but that the blastocysts die soon after entering the uterine cavity. He concluded that the presence of actively secreting corpora lutea is essential for the continued nutrition of the free blastocyst. Westman (1930) also removed the ovaries of rabbits 12 hours after mating. All the ova recovered from the oviducts 72 hours later showed some signs of degeneration. Subsecjuently Westman, Jorpes and Widstrom (1931) cauterized the corpora lutea of mated rabbits and recorded a degeneration of the tubal ova similar to that observed after ovariectomy. Injections of corjius luteum extract into the operated animals prevented degeneration of the ova.



Fig. 14.10. Tubular secretion pressure of right and left oviducts of rabbit under Dial anesthesia. Vertical bars indicate pulsations due to visceral movements at the time of reading. (After D. W. Bishop, Am. J. Physiol., 187, 347-352, 1956.)



Current investigations on fluids of the rabbit oviduct have shown that the secretions of the upper, fimbriated third are necessary for normal enlargement of the blastocyst (Bishop, unpublished data). The oviducts of pregnant females and castrates who have received progesterone secrete copious quantities of fluids. If these fluids are prevented from entering the uterus about the 5th day, by double ligation, the blastocysts remain small and do not reach their normal size by the 8th day or the time of implantation.

If fertilized ova of the Muridae are preA-ented from entering the uterus, either by ligation of the oviduct or by the administration of hormones which inhibit the normal jiropulsive mechanism of the tube, the eggs develop to the blastocyst stage before degeneration begins (Burdick, Whitney and Pincus, 1937; Burdick, Emerson and Whitney, 1940; Alden, 1942d). The occurrence of tubal pregnancies, especially in the human female, indicates that under some circumstances development may continue within the oviduct beyond the stage of normal implantation.

IV. Fertilization and Implantation

Fertilization involves the penetration of a fully developed egg by a motile, mature spermatozoon, and the subsequent formation, growth, and karyogamy of the sperm and egg nuclei. An integral part of this process is the physical act of penetration of the spermatozoon into the "karyocytoplasm" which results in the "activation" of the egg. The classical experiments of Loeb (1913) in the invertebrates and Rugh (1939) in amphibia have shown that "activation" does not depend on a specific property of the spermatozoon, but may be effected by chemical, mechanical, or physical stimuli (see also Wilson, 1925). Unfertilized mammalian eggs may likewise be activated by a variety of stimuli, but ordinarily do not proceed far in embryonic development (Pincus and Enzmann, 1936, Chang, 1954, 1957. in the rabbit; Thibault, 1949, Austin, 1951a. in the rabbit, rat, and sheep).


Although Barry (1843) was the first investigator to observe a spermatozoon within the mammalian egg, no detailed description of the process of fertilization appeared until Van Beneden published his observations on the rabbit in 1875. Since then, numerous investigations on the cytology and physiology of fertilization in the mammal have formed a large volume of literature (Van der Stricht, 1910, the bat; Sobotta, 1895, Lams and Doorme, 1908, Gresson, 1948, the mouse; Rubaschkin, 1905, Lams, 1913, the guinea pig; Gregory, 1930, Pincus and Enzmann, 1932, the rabbit; Tafani, 1889, Sobotta and Burckhard, 1910, Kirkham and Burr, 1913, Huber, 1915, Kremer, 1924, Gilchrist and Pincus, 1932, MacDonald and Long, 1934, Austin, 1951a, b, Blandau and Odor, 1952, Austin and Bishop, 1957, the rat; Van der Stricht, 1910, Hill and Tribe, 1924, the cat; Mainland, 1930, the ferret; Van der Stricht, 1923, the dog; Pearson and Enders, 1943, the fox; Wright, 1948, the weasel; Hamilton and Laing, 1946, Piykianen, 1958, the cow; Amoroso, Griffiths and Hamilton, 1942, the goat; and others). The specific point of emphasis and the degree of completeness of these studies vary widely and in a number of instances only discontinuous and isolated stages were observed and reported.

Certain of the many changes occurring during the process of sperm penetration and fertilization can be studied best in fixed material properly sectioned and stained. Many features, however, can be observed most clearly only in the living egg. Obviously one way of studying fertilization phenomena is to look at them. But microscopic observations on the living egg even with the newer phase-contrast objectives and other techniques have been disappointing to many because of the problems in establishing and maintaining an environment in which the processes can take place. There is such an array of observations of sperm penetration and fertilization in the invertebrates that there has been a tendency to translate these observations directly to the mammalian egg. It is becoming increasingly clear that there is not necessarily a common denominator for these vital processes and that they vary widely. The interesting differences in the shape of the heads of spermatozoa from species to species alone may indicate the existence of a variety of mechanisms for penetrating the various barriers encountered before the vitellus can be entered.




Fig. 14.11. A living rat ovum with cumulus oophorus intact and a fertilizing spermatozoon in the ooplasm (A). B. Living rat ovum with cumulus intact and showing the earW development of the male and female pronuclei. X 450.



Quantitative data on the temporal relationship between ovulation, penetration of sperm, and syngamy are lacking for most mammals. Before this information can be had for any animal, the time of ovulation must be easily and accurately determinable, the rate of ascent of spermatozoa to the site of fertilization must be known, and the rate


of sperm passage through the cumulus oophorus, zona pellucida, and vitelline membrane must be established. Information of this sort is now available for several species, particularly that obtained by the use of phase-contrast microscopy and time-lapse cinemicrophotography in the study of living eggs. These methods have supplemented the earlier observations and made possible a more complete account of the process of fertilization (Austin and Smiles, 1948; Odor and Blandau, 1951; Austin, 1951b, 1952a).

A. The Cumulus Oophorus and Sperm Penetration

The number of layers of cells and the compactness of the cumulus oophorus of newly ovulated eggs varies greatly in different animals. Cumulus cells and the mucopolysaccharide matrix enclosing them have been reported as sparse or absent in the tubal eggs of the sheep (Assheton, 1898; McKenzie and Allen, 1933; Clark, 1934), the roe deer (Bischoff, 1854), the cow (Hartman, Lewis, Miller and Swett, 1931 1, the pig (Corner and Amsbaugh, 1917), the horse (Hamilton and Day, 1945), and the opossum (Hartman, 1928). In other species such as the rat, mouse, hamster, mink, rabbit, monkey, and man (Boyd and Hamilton, 1952), many layers of granulosa cells form the cumulus oophorus. Furthermore, in certain rodents the ovulated eggs clump together within the dilated ampullae of the oviducts, greatly increasing the number of cell layers and viscous gels the spermatozoa must penetrate in order to reach the more centrally lying eggs. If attempts are made to remove the cells forming the cumulus of newly ovulated eggs by pulling them away with fine needles, the tenaciousness of this investment is impressive and one wonders how a spermatozoon ever reaches the vitellus (Fig. 14.11).

In the preovulatory follicle the cells of tiie cumulus oophorus become loosened from the follicular wall and somewhat separated one from another. This is seen most spectacularly in the guinea pig and cat (Myers, Young, and Dempsey, 1936; Dawson and Friedgood, 1940). The ovum and enveloping cumulus cells have frequently been observed to lie free within the antrum before the follicle ruptures. Although only limited observations have been made, some reports indicate that the cumulus oophorus in the preovulatory follicles cannot be dispersed as readily by the methods that are effective in ovulated eggs (Farris, 1947; Shettles, 1953). It is important to determine what chemical or physical alterations occur in the intercellular cement substances of the cumulus during the time the follicle is ripening and to learn why this should differ in the cells surrounding the egg from other similar cells lining the walls of the follicle.


The existence of a "cumulus-dispersing" factor in mammals was brought to light by the experiments of Gilchrist and Pincus (1932), Yamane (1935), Pincus (1936), and Pincus and Enzmann (1936). These investigators demonstrated that either living sperm suspensions or sperm extracts of the rabbit, rat, and mouse rapidly disperse the cells of the cumulus oophorus of tubal ova. Yamane (1930) inferred that the presence of a proteolytic enzyme in the spermatozoa was responsible for both follicle-cell dispersion and "activation" of the egg to produce the second polar body.

In a series of carefully controlled experiments Pincus (1936) showed that a heatlabile substance was present in sperm extracts which caused follicle-cell dispersion, but that this substance would not effect second polar body formation. Pincus demonstrated further that the rate of cell dispersion in vitro was roughly proportional to the number of spermatozoa in the suspension. It was discovered later that the "cumulus-cell-dispersing substance" was the enzyme hyaluronidase (Duran-Reynolds, 1929). The enzyme depolymerizes and liydrolyzes the hyaluronic acid cement substance binding the granulosa cells together. This discovery at first seemed to provide a happy solution to the problem of how spermatozoa penetrate the cumulus oophorus (McClean and Rowlands, 1942; Fekete and Duran-Reynolds, 1943; Leonard and Kurzrok, 1945). Numerous observations cpickly demonstrated that the testes and spermatozoa of mammals are the richest sources of animal hyaluronidase. The enzyme first appears in the testes when spermatogenesis begins in the pubertal animal and before fully developed spermatozoa are present in the tubules (Riisfeldt, 1949).


It became clear that there is a proportional relationship in vitro between sperm count and the hyaluronidase concentration ; further, that the enzyme is associated with the spermatozoa and not with the seminal plasma (Werthessen, Berman, Greenberg and Gargill, 1945; Kurzrok, Leonard and Conrad, 1946; Swyer, 1947a; Michelson, Haman and Koets, 1949). Hyaluronidase concentration per sperm is highest in the bull and rabbit, somewhat less in the boar and man, still lower in the dog, and very low in birds and reptiles (Swyer, 1947a, b; Mann, 1954). Observations on the in vitro dispersal of granulosa cells by hyaluronidase suggested that large numbers of spermatozoa are necessary in the semen in order to provide a sufficient concentration of the enzyme.


The in vitro observations of Pincus and Enzmann (1936) strengthened this assumption when they demonstrated that a minimum number of 20,000 spermatozoa per cubic millimeter of rabbit semen is necessary if the cumulus cells surrounding one ovulated egg are to be dispersed. Such observations seemed to explain the necessity of the "sperm swarms" described in the oviducts of mated rabbits. The swarms created and maintained a sufficiently high concentration of the enzyme to permit the denudation of the eggs so that certain of the spermatozoa could approach and penetrate the zona pellucida.


Attempts were then made to increase the fertilizing capacity of a subnormal number of spermatozoa by adding hyaluronidase extracts to semen suspensions used for artificial inseminations. In 1944, Rowlands proposed that such a procedure had increased the fertilizing capacity of rabbit spermatozoa. This could not be confirmed by Chang (1950b) ; indeed, it was observed that seminal plasma in which the hyaluronidase had been inactivated by heat was as effective as untreated plasma. Kurzrok, Leonard and Conrad (1946) outlined a method for adding bull hyalurodinase to oligospermic specimens of human semen which was to be used for artificial insemination. This method was employed in the treatment of sterility and reported to have been notably successful.


Many further attempts to demonstrate the therapeutic value of hyaluronidase in mammalian infertility have met with failure (see Siegler, 1947; Tafel, Titus and Wightman, 1948; Johnston and Mixner, 1950). The generally poor results obtained by the addition of hyaluronidase to semen introduced into the vagina or uterus by artificial insemination may be explained by the later experiments of Leonard, Perlman and Kurzrok (1947), which conclusively demonstrated that hyaluronidase inserted into the lower reproductive tract is not transported to the oviducts. The systematic studies of Austin (1949b) and Chang (1947, 1951a) revealed that in the rabbit only 100 to 1000 spermatozoa reach the site of fertilization. Even though in one experiment 600,000,000 spermatozoa were artificially introduced into the female reproductive tract, only approximately 2000 of them were found in the tubes. An even smaller number (10 to 50) have been shown to reach the ampulla of the rat oviduct at the time of sperm penetration (Blandau and Odor, 1949; Moricard and Bossu, 1951).


It is probably correct to assume that any hyaluronidase which reaches the cumulus at the time of semination is transported by relatively few spermatozoa. Although the enzyme has not been localized in the sperm itself, it is assumed that it is an integral part of the cell and is liberated in a relatively localized region as the spermatozoon makes its way through the cement substance. The spermatozoon is remarkably permeable in that such large molecules as cytochrome c or hyaluronidase can detach themselves from the sperm cell and pass into the extracellular en^'ironment by the so-called "leakage" phenomenon (Mann, 1954).


In vitro tests have shown that the enzyme hyaluronidase diffuses into the suspending fluid at a definite rate depending on the type of medium and the temperature. New formation of the enzyme by spermatozoa does not seem to occur (Meyer and Rapport, 1952). The possibility exists that the enzyme may be able to exert its action while still bound to the sperm cell.

A recent development in the study of hyaluronidase action and its possible role in fertilization has been the attempt to utilize certain inhibitors of the enzyme as systemic contraceptives. Among the naturally occurring and extraneous inhibitors may be listed heavy metals, heparin, quinones, "rehibin" or trigentisic acid, and antihyaluronidase antibodies, as well as a nonspecific, electrophoretically identifiable serum factor (Leonard and Kurzrok, 1945; Beiler and Martin, 1947; Glick and Moore, 1948; Meyer and Rapport, 1952; Hahn and Frank, 1953; Parkes, 1953). Many of these substances are highly active inhibitors of hyaluronidase and may reduce or prevent fertilization when added to semen in vitro before artificial insemination. Attempts to inhibit fertilization by giving these substances orally or by injection have not been repeatedly successful, but several derivatives of hyaluronic acid obtained by acetylation or nitration and added to rabbit semen in vitro seemed to have inhibited dispersion of follicle cells and to have impaired fertility (Pincus, Pirie and Chang, 1948).


It has now been demonstrated repeatedly that ova in the ampulla of the oviduct may have been penetrated by spermatozoa without evident dispersal of the granulosa cells (Lewis and Wright, 1935; Leonard, Perlman and Kurzrok, 1947; Austin, 1948b; Bowman, 1951; Odor and Blandau, 1951, in the rat; Chang, 1950b, in the rabbit; Amoroso, personal communication, in the cat). Again, dog spermatozoa do not contain hyaluronidase yet they are capable of penetrating the many layers of granulosa cells comprising the cumulus. Inasmuch as a generalized dispersal of the cells of the cumulus does not occur at the time of sperm penetration, the pendulum has swung to the present view that the individual spermatozoon carries sufficient enzyme to make a path for itself through the cumulus layer and the gel matrix. If rat spermatozoa are added to slides containing cumulus masses from freshly ovulated eggs and their movement through the cumulus matrix observed with phase objectives, one is led to conclude that an intact cumulus is essential if sperm penetration is to be successful, i.e., the cumulus may act as a base against which the sperm flagellum can push as it moves forward towards the zona pelliicida. The spermatozoa may move through the cumulus with broad sweeps of their flagella and at a rate of forward i^rogression which makes it difficult to conceive of the del)olymerization of the matrix to form a tunnel for the sperm. It must be concluded therefore that the role of hyaluronidase in sperm penetration is unknown and that much more critical evaluation needs to be directed into this area.

Even though the outer layers of the cumulus oophorus of ovulated eggs iti vitro may be removed readily by hyaluronidase, the corona radiata may not be dispersed with the same rapidity, especially in eggs treated immediately after ovulation. The basis for this difference lies in the fact that the cells forming the corona radiata send liolar, cytoplasmic extensions into the zona liellucida, thereby anchoring them firmly, although temporarily. In the newly ovulated eggs of the rat, hamster, and mouse the corona cells cannot be removed mechanically without breaking the zona pellucida. It is only after the eggs have been in contact with spermatozoa or have resided in the oviducts for a number of hours that the corona cells may be either brushed off the zona pellucida or drop away spontaneously.

Swyer (1947b) and Chang (1951b) suggested that the coronal cells are removed mechanically by being more or less brushed off by the ciliary and muscular activity of the oviduct. This may be true for human and rabbit eggs, but in the rat, mouse, and hamster in which the eggs lie in the dilated ampulla, and thus at a distance from the wall of the oviduct, it would seem appropriate to assume that factors other than mechanical are involved in dispersing the corona. If rat eggs are examined approximately 24 hours after ovulation, one can observe that their zonae are completely free of the coronal cells, but that they may be still enclosed in an abundant viscous matrix. It appears that the corona cells gradually retract their cytoplasmic extensions from the zonal canaliculi.

Interesting observations can be made by growing freshly ovulated eggs and their attached corona cells in tissue culture. Timelapse cinematography reveals that the cells forming the cumulus and corona, although alive, have lost much of their vitality. The surfaces of the cells undergo peculiar bubbling movements. This "bubbling" is similar to that described in cells in the late stages of cell division or in cells which are about to die. Changes in the fluidity of the cell surface apparently account for the bubbling which continues for hours in favorable preparations. This i)henomenon accounts for the retraction of the cell processes from the zona and the gradual dispersal of the cells. That the cumulus and coronal cells lose their vitality rather quickly after ovulation is shown further by their very poor growth in tissue culture compared with that of similar cells removed from young follicles.

The rate of the dispersal of cumulus cells after ovulation varies in different animals. In mated ral)bits the eggs are completely denuded of cumulus and corona cells 4 to 6 hours after ovulation. After sterile matings, however, the cumulus and corona are not dispersed until 7 to 8 hours after ovulation (Pincus, 1930; Chang, 1951b; Braden, 1952). In the rat there is relatively little change, either in the cumulus mass or in the corona cells for many hours after ovulation and fertilization (Blandau, 1952). Shettles (1953) suggested that in addition to hyaluronidase there may be a tubal factor which is important in the removal of the cumulus oophorus in the human egg. He found that hyaluronidase had little effect in removing the cumulus cells in ovarian eggs, but, if bits of homologous tubal mucosa were added, the cumulus oophorus was dispersed readily.

In spite of the formidable barriers interposed by the cumulus and corona, they do not prevent the entrance of sperm into the egg ; in fact, as suggested earlier, their presence seems to be important in some animals if penetration is to be effected (Fig. 14.11). Chang (1952a) demonstrated that, in the rabbit at least, there is a relationship between the loss of the granulosa cells and fertilizability. He counted the spermatozoa in eggs fixed at different intervals after ovulation and found that the greatest number entered the eggs between the 2nd and 4th hours. Once the denudation of the eggs is completed (approximately 6 hours after ovulation), penetration of spermatozoa no longer occurs, despite the presence of adequate numbers in the environs. It is important to remember that the deposition of the mucous coat in the rabbit ovum may limit its fertilizable life (Pincus, 1930; Hammond, 1934). The actual time after ovulation that mucous deposition begins has been variously reported as 5, 6, 8, and 14 hours (Pincus, 1930; Hammond, 1934; Chang, 1951b; Braden, 1952). It remains to be determined whether failure of sperm penetration into the rabbit egg after 6 hours' sojourn in the ampulla is related to the loss of the cumulus, the deposition of the mucous coat, or to a specific change in the physical characteristics of the zona pellucida itself.

B. The Zona Pellucida And Sperm Penetration

The general appearance and properties of the zona pellucida were described earlier. The manner whereby spermatozoa penetrate the zona pellucida and the conditions influencing this process are poorly understood. Despite the numerous attempts to fertilize mammalian ova in vitro, only a few investigators have described isolated stages in the process of sperm penetration through the zona pellucida or into the vitellus. Shettles (1953) described in some detail the behavior of a human spermatozoon passing through the zona pellucida of an isolated follicular ovum. As the spermatozoon became attached to the zona it rotated on its longitudinal axis. As the head was observed in focus in the equatorial plane, the rate of rotation decreased until, by the time the tip of the head was midway in the zona, the front and side views of the head could be seen to alternate. The progression of the head through the zona pellucida was intermittent until only the tail lay within it. The head and body then underwent several intermittent side-to-side, jerky movements and finally slipped into the peri vitelline space. It required 18 minutes for a spermatozoon to traverse the zona pellucida. Duryee (1954) described the consistency of the zona pellucida of the human follicular egg as jelly-like, much less tough and resilient than the tubal egg. It would be interesting to know whether these differences in the physical properties of the zonae of ovarian and tubal eggs in the human affect the manner of spermatozoon penetration.


On two occasions Pincus (1930) found rabbit ova with the heads of spermatozoa partially embedded within the zonae, and described the slow yet perceptible forward progress until the heads penetrated the vitelli. Pincus believed that the flagellae did not enter the ooplasm but were left behind in the zonae pellucidae.

There is no sound evidence of a predetermined pathway or "micropyle" in the zona pellucida of mammals. In the few instances where attention has been paid to this matter, spermatozoa seem to be able to penetrate the zona at any point on its surface. A small elliptical slit with the sperm tail partially projecting through it has been noted in the zona pellucida of living fertilized eggs of the rat, guinea pig, and Libyan jird (Austin, 1951b; Austin and Bishop, 1958). The slits in the zona are not seen in eggs which do not contain spermatozoa. It is usually possible to discern as many slits as there are sperm within the perivitelline space. The general appearance of the slit and the manner in which the perforatorium of the sperm head attacks the zona pellucida in vitro creates the impression that the zona may be fractured by the spermatozoon. Similar slits can be made by fracturing rat zonae with tungsten needles sharpened electrolytically to several micra in thickness.

Recently Austin and Bishop (1958) have presented observations suggesting that the acrosome is lost as the sperm passes through the female reproductive tract and postulate that the perforatorium elaborates an enzyme which depolymerizes the zona pellucida in a very restricted zone as the sperm moves through it.

Discussions on the mechani.sms involved in sperm penetration of the zona have implicated a variety of conditions and substances as being of importance in changing the physical characteristics of the zona in the localized area of contact. As mentioned earlier, the zona pellucida can be softened or disintegrated in rat and rabbit eggs by buffers with pH values from 3 to 5 (Hall, 1935; Harter, 1948; Braden, 1952). Various reducing agents such as glutathione and cysteine in Tyrode's solution cause rapid dissolution of the zona. Oxidizing agents such as the hydrogen peroxide which is produced by sperm (Tosic and Walton, 1946) are particularly efficacious in removing this membrane. Several investigators favor the possibility that a specific mucolytic enzyme, "zona lysin" (Austin and Bishop, 1958) may be secreted by the sperm as it makes contact with the zona pellucida (Leblond, 1950; Austin, 1951b). It seems likely that the passage of the spermatozoon through the zona pellucida may occur in a variety of ways in different animals. Too few observations have been made to significantly implicate any of the physical, chemical, or mechanical mechanisms suggested for sperm penetration of the zona pellucida in the mammalian egg.


It has been suggested that the physical jiroperties of the zona pellucida in the dog, hamster, and sheep are altered after the first sperm passes through it and enters the vitellus. It is postulated that a substance is secreted by the vitellus which "tans" the zona so that additional sperm cannot penetrate it (Braden, Austin and David, 1954). Smithberg (1953) reported that the zonae l)ellucidae of the unfertilized mouse eggs are more readily removed by proteolytic enzymes than those of fertilized eggs.


Chang and Hunt (1956) tested the effects of a variety of proteolytic enzymes on the zonae pellucidae of fertilized and unfertilized eggs of rabbits, rats, and hamsters. Even though none of the fertilized hamster eggs contained more than one sperm, there was no evidence that the zonae pellucidae of the fertilized eggs were more resistant to digestion than those of unfertilized eggs. In contrast Austin (1956c) reported that the zonae pellucidae of fertilized hamster eggs were dissolved more quickly by trypsin than those of unfertilized eggs. Blockage of the zona pellucida in the rat and rabbit egg is not as definite, yet there are indications that fertilized and unfertilized eggs react differently to proteolytic enzymes.


In many animals the sequence of the reproductive processes are arranged in such a manner that spermatozoa must wait at the site of fertilization for several hours before ovulation occurs and the eggs have arrived in the ampullae. If freshly ejaculated spermatozoa of rats or rabbits are transferred directly to oviducts containing newly ovulated eggs, relatively few if any of the eggs will be fertilized. If, however, spermatozoa are introduced into the genital tract several hours l)efore the expected time of ovulation, they undergo some kind of change by which they gain the capacity to fertilize eggs on contact. Chang ( 1951 ) was the first to report this j^henomenon in the rabbit and termed it "development." In the same year, Austin (1951) working in Australia independently described the phenomenon and called it "capacitation." Chang (1959a) further api)i-oached this question by artificially inseminating rabbits that acted as "incubator" hosts. He subsequently withdrew sperm samples at stated intervals and injected them into the oviducts of rabbits that had just ovulated. Chang concluded that 6 hours of such "host incubation" was necessary before rabbit sperm could fertilize the majority of ova ovulated. Similar observations by Austin (1951), Noyes (1953), and Noyes, Walton and Adams (1958) on rats indicated that approximately 3 hours is the time required for capacitation in this animal. There has been some success in the intrajieritoneal insemination of the rabbit doe 8 hours before ovulation with sperm which had been washed several times in a sodium citrate buffer solution (Hadek, 1958). Attempts to induce capacitation in vitro by exposing rabbit spermatozoa for varying lengths of time to a variety of physiologic solutions and solutions containing endometrial tissue have been largely unsuccessful (Chang, 1955b). Partial capacitation has been reported when rabbit spermatozoa are incubated in diverticula of the bladder and colon which had been created surgically (Noyes, Walton and Adams, 1958). Capacitation was also effected when spermatozoa were stored in the seminal vesicles and anterior chamber of the eye. There is no evidence as yet which favors the need for capacitation in the mouse and guinea pig during normal mating. According to Austin and Bishop (1958) there are changes in the optical properties of the acrosomes of rabbit, rat, and hamster spermatozoa as they traverse the female reproductive tract. When a sperm reaches the egg in the ampulla, the acrosome is detached, exposing the perforatorium. Austin and Bishop propose that the acrosome is the carrier of the enzyme hyaluronidase which allows the sperm to depolymerize the hyaluronic acid jelly of the cumulus oophorus. The exposed perforatorium, then, may be a carrier of a lysin which may alter the physical characteristics of the zona pellucida so that the sperm may pass through it. There has been much speculation on the importance of capacitation in fertilization, but there is little significant evidence to support the various theories proposed (Chang, 1955a, b, and 1959b; Strauss, 1956).


C. Sperm-Egg Interacting Substances

The phenomenon of agglutination by "egg water" has been observed and described many times for the spermatozoa of echinoderms, annelids, molluscs, ascidians, cyclostomes, fish, and amphibia (Rothschild, 1956; Tyler, 1957) . The compound in the egg water responsible for tlie effect is derived from a jelly-like membrane which is secreted on the egg by the follicular cells. On ovulation the jelly gradually dissolves in sea water and composes the fertilizin first described by Lillie (1919). Experiments with invertebrate eggs have demonstrated that fertilizin is responsible for the specific sperm-agglutinating power and for the initial specific adherence of the sperm to the egg. One of the interesting chapters in biology has been the attempt to characterize the biologic and chemical properties of these interacting substances.


Whether sperm-egg interacting substances are present in the fluids forming the environment of ovulated mammalian eggs has been very little investigated. Recently Bishop and Tyler (1956) and Thibault and Dauzier (1960) have reported the presence of fertilizin in the eggs of rabbits, mice, and cows. The reaction was found to be primarily species specific and its source is believed to be the zona pellucida.

Much more experimental testing must be done to amplify knowledge in the field of interacting substances of mammalian eggs and spermatozoa.


D. Sperm Penetration of the Vitelline Membrane

The penetration of a spermatozoon into the ooplasm in vitro has been observed on so few occasions in mammals that it is not yet possible to give an accurate account of this phenomenon. Pincus (1930) records a slight bulging of the ooplasm in rabbit eggs at the point where the head of the sperm made contact with the vitelline membrane. Because of the opacity of the egg cytoplasm, no further i^rogress of the head could be observed. Studying rat, mice, and hamster eggs, Austin (195ibj and Austin and Braden (1956) described a more or less passive penetration of the ooplasm by the fertilizing spermatozoon, as if the ooplasm "pulled" the entire sperm into its substance or "phagocytized" it. Austin (1951b) and Austin and Bishop (1957) ascribed some peculiar property to the head of the sperm which results in its being "absorbed" into the vitellus. The investigations of Dan (1950) on the changes in the acrosome of the sea urchin at the time of sperm penetration of the egg have an interesting bearing on this problem. She believed that as the spermatozoon swims actively through the jelly layer of the egg, the acrosome responds to the chemical stimulation of the egg jelly by a localized breakdown of its membrane. By the time the spermatozoon reaches the vitelline membrane a few seconds later, it carries at its tip a labile mass of lysin with which it effects penetration of the ooplasm.


The observations of Austin are at variance with those made by others also in the rat and in which it appeared that ooplasmic penetration was accomplished primarily by the activities of the flagellum of the fertilizing sperm (Blandau and Odor, 1952). Although discontinuous, the forward progression of the spermatozoon into the ooplasm seemed to depend on a propulsive type of undulating movement of the tail which forced the head forward a distance of 10 to 20 /x at a time. While that portion of the flagellum within the ooplasm was retarded in its amplitude of motion by the viscosity of the egg cytoplasm, that which was still in the pehvitelline space lashed about vigorously. These observations are similar to those described by Shettles (1960) in the human. As mentioned earlier, the technical problems in observing in vitro fertilization will no doubt be solved when the molecular species of the fluids forming the normal egg environment is known.


There is no specific information with respect to the nature of the vitelline membrane of the mammalian egg or to the changes it may undergo on sperm entry. It would be desirable to know whether the vitelline membrane undergoes modification after penetration by the fertilizing spermatozoon. An interesting procedure for measuring the solidification of the egg membranes of salmonid eggs has been described recently by Zotin (1958). Even though there is no clear evidence of a comparable phenomenon in mammalian eggs, some factor appears to control the number of spermatozoa which enter the vitellus. Cortical granules have been described in the unfertilized hamster egg which disappear on fertilization, but apparently they are not associated with the block of l)olyspermy (Austin, 1956a). Quantitative data are necessary to clarify the relationship between the number of spermatozoa which may enter the periovarial space, the rate of the "tanning" reaction of the zona, if such a ])henomenon exists, and the reaction of the perivitelline membrane which blocks the entry of further spermatozoa.


Shrinkage of the vitellus after sperm penetration has been described in the rabbit and rat (Gilchrist and Pincus, 1932; Pincus and Enzmann, 1932), but a comparable shrinkage can be noted in unfertilized ova recovered from the oviduct several hours after ovulation, and thus shrinkage per se cannot be used as a criterion for sperm penetration. The shrinkage of the vitellus is related in some way to changes in the vitelline membrane because the numerous microvilli present in the young ovarian egg have disappeared and the total surface of the egg has been greatly reduced.


E. Fertilizatiox In Vitro

During the past century one of the most challenging and frustrating problems was the attempt to fertilize mammalian ova in vitro and to follow their cleavage. Even though several successes were recorded, it could not be maintained unequivocally until the recent work of Chang ( 1959a) that sperm penetration has been accomplished and that the divisions of the eggs noted were the result of fertilization rather than of an "activation" of the egg instituted by some other factor in the environment, or just plain fragmentation.


Relatively little has been added to our understanding of the mechanism of sperm penetration into the ooplasm since the extensive experiments of Long (1912) in which he attempted to fertilize rat and mice eggs in vitro. He described penetration of the follicle cells and observed the sinuous movements of the sperm as they advanced within the cunmlus. The role of the spermatozoa in the dispersal of the granulosa cells was noted and this was interpreted as being due to the lashing activities of the sperm fiagellum. Long also described the formation of the second polar body in eggs which had been placed in sperm suspensions. Polar body formation began within 2 hours and abstriction was completed within 4 hours of the time of immersion. Unfortunately, his description leaves one uncertain as to whether penetration by the sperm was actually observed or merely confirmed by sectioned material.

Some success with fertilization in vitro was also achieved by Pincus (1930, 1939), Pincus and Enzmann (1934, 1935), Venge (1953), and Thibault and Dauzier (1960) in their extensive experiments with both ovarian and tubal eggs of rabbits. These investigators described the abstriction of the second polar body, the shrinkage of the vitellus, the penetration of the zona by spermatozoa partially embedded within it, and the presence of spermatozoa in the perivitelline space in fixed and stained preparations. Transplantation of living eggs into the oviducts of pseudopregnant rabbits, following the addition of sperm to the eggs, resulted in the birth of live young possessing the genetic characteristics of coat color which had been used as markers. It is suggested in a later report (Chang and Pincus, 1951) that the results "may have been due to adherent sperm effecting fertilization in the fallopian tubes."


The mammalian egg may be "activated" to various degrees according to the balance of thermal, osmotic, and chemical factors in its environment. Thus eggs "activated" by being placed in a cold environment may form double nuclei which closely resemble normal pronuclei (Thibault, 1947a, b, 1948). The eggs of the opossum, rat, mouse, hamster, mink, and ferret also will show varying degrees of "activation" and may be difficult to differentiate from normally cleaving ova (Smith, 1925; Chang, 1950a; Austin, 1951a, 1956c; Blandau, 1952). Attempts to fertilize the timed human ovarian ova recovered by Corner, Farris and Corner (1950), were unsuccessful. Rock and Menkin (1944) and Menkin and Rock (1948) also attempted to achieve fertilization of human ovarian eggs in vitro and reported several successes. The first egg recovered from a large follicle was cultured in the patient's serum for 27 hours. It was then placed in a washed suspension of sperm for 1 hour and observed continuously. Penetration of the ovum by sperm was not observed. When the same egg was inspected 40.5 hours later, it consisted of two blastomeres each measuring 86 /a in diameter. A second egg treated in much the same manner also was found to contain two blastomeres 45 hours after exposure to spermatozoa. The stage of maturation of these ovarian eggs could not be determined and it is assumed that the meiotic divisions occurred in vitro. Since the fertilizable life of the human ovum is unknown, and there is no specific information on sperm penetration, the role of the flagellum in semination, pronuclei formation, karyogamy, and the rate of cleavage, it is clear that the true identification of a fertilized human ovum has not been achieved. In the rat, for example, one can find unfertilized cleaved ova which on first inspection closely resemble fertilized eggs even containing modified nuclei or nuclear fragments. When examined in detail the fragmenting eggs do not contain the flagellae of spermatozoa, a positive indication that penetration has not been accomplished (compare 2 and 3 in Figure 14.15).

Various criteria have been accepted as an indication of fertilization in vitro such as polar body formation, shrinkage of the vitellus, presence of one or more pronuclei, and cleavage of the ooplasm. As emphasized earlier, all of these phenomena have been observed many times in eggs which have not been penetrated by a spermatozoon and which are in varying stages of degeneration and fragmentation. Too little is known concerning the processes of semination and fertilization in mammals, with the possible exceptions of the rat and rabbit, to judge uneciuivocally whether normal sperm penetration and fertilization have been accomplished in vitro.


The freshly ovulated eggs of most mammals are notoriously sensitive to changes in environment and one is concerned lest the eggs cultured in vitro may simulate the events occurring in vivo without activation by a spermatozoon. If sperm penetration and the various fertilization i)henomena cannot be followed continuously by direct visualization, it is generally agreed that, unless viable embryos are obtained by transplanting the supposedly fertilized eggs to recipient animals, the success of fertilization is not sufficiently proven. Recently Chang (1959a) was successful in fertilizing the rabbit egg in vitro and obtaining living young by transplanting them to host animals. Thus for the first time a repcatable procedure for fertilizing mammalian ova in vitro has been perfected. Chang obtained unfertilized rabbit eggs by intravenous injection of sheep pituitary extract into estrous rabbits. Sperm were obtained 12 hours after mating females with fertile bucks by washing the uterus with a Krebs-Ringer bicarbonate solution. Unfertilized ova were obtained by flushing the oviducts of the animals which had received the gonadotrophins. Both sperm and eggs were placed in a small Carrel flask and kept at 38°C. Three to 4 hours later the ova were transferred to a second Carrel flask containing rabbit serum and cultured for another 18 hours. At this time the eggs were recovered and examined, and those that appeared to be cleaving were transferred to recipient rabbits. Approximately 42 per cent of the transferred ova that appeared to be fertilized were delivered at term as viable young.

F. Fate of the Unfertilized Egg

Evidence that ovulation without fertilization is followed by rapid degeneration and fragmentation of the vitellus has been obtained for many different species (Hartman, 1924, Smith, 1925, in the opossum; Sobotta, 1895, Kirkliam, 1907, Long, 1912, Charlton, 1917, in the mouse; Chang and FernandezCano, 1958, in the hamster; Long and Evans, 1922, Mann, 1924, Blandau, 1943, 1952, in the rat; Squier, 1932, Blandau and Young, 1939, in the guinea pig; Chang, 1950a, in the ferret; Heape, 1905, Pincus, 1936, in the rabbit; Dziuk, 1960, in the gilt; Hartman, Lewis, Miller and Swett, 1931, in the cow; and Allen, Pratt, Newell and Bland, 1930, in man ) .

With the possible exception of the rat, the jiroblem of the ultimate fate of the degenerating ova has not been satisfactorily resolved for any mammal. It is generally accepted that as the unfertilized eggs undergo coml)lete fragmentation and dissolution they are absorbed either in the oviducts or uterus (Corner, 1928a; Pincus, 1936). Charlton { 1917) suggested that final disintegration of unfertilized ova in the mouse is effected by means of phagocytic leukocytes. It is assumed further that the unfertilized ova disappear from the female reproductive tract before the succeeding ovulation. However, Hensen (1869) described the retention of approximately 100 rabbit ova in a blocked oviduct in which presumably the eggs had accumulated from a number of ovulations.

The unfertilized ova in the rat do not undergo complete dissolution during the normal 4- to 5-day estrous cycle. The vitellus fragments ordinarily into a number of units of varying sizes and the eggs, with their zonae intact, are eliminated near the end of the succeeding heat period by being washed out through the vagina (Blandau, 1943). Attention has been directed to the freciuent occurrence of abortive "cleavages" in the unfertilized tubal eggs of the ferret and rat (Austin, 1949a; Chang, 1950a). This phenomenon is more common in the prepubertal rat treated with gonadotrophins than in the adult animal. In the "cleaved" unfertilized ova, the blastomeres and their nuclear configurations may appear identical with those of fertilized ova and can, indeed, be differentiated only by the absence of the flagellum of the fertilizing sperm. Most unfertilized ova. however, fragment into a number of units of unequal size, each containing one or more abortive nuclei.


G. Formation of the Second Polar Body

The penetration of the vitellus by a spermatozoon is not the only stimulus which will induce the formation of the second polar body. Yamane (1930) observed that if rabbit eggs are placed in solutions containing rat or horse spermatozoa, or immersed in pancreatic solutions, cytoplasmic masses similar to the second polar body will be abstricted. Similar "false polar bodies" or extrusions of clear, chromatin-free masses were produced when rabbit eggs were immersed in various concentrations of trypsin ( Pincus and Enzmann, 1936). Both the abstriction of the second polar body and shrinkage of the ooplasm may be induced in rabbit, rat, and mouse eggs by a variety of other nonspecific stimuli such as ether, Nembutal, nitrous oxide anesthesia, and "cold shock" (Pincus and Enzmann, 1936; Thibault, 1949; Austin and Braden, 1954b; Braden and Austin, 1954). By contrast, colchicine or "hot shock" inhibits the emission of the second polar body (Austin and Braden, 1954b). Austin (1951b) described the formation of the second polar body in rat eggs in which spermatozoa were in the perivitelline space but had not yet penetrated the vitelline membrane. It is uncertain whether "activation" is caused by a substance released into the perivitelline space by the spermatozoa, or by the mechanical impact of the spermatozoa on the vitelline membrane. There are relatively few data on the temporal relationship between penetration of the vitellus by the sperm and the abtrusion of the second })olar body. Pincus and Enzmann (1932) reported that in rabbit ova 45 minutes or more elapse between the time the sperm enters the vitellus and the formation of the second polar body is completed. Formation of the second polar body in vitro has also been observed in mouse eggs that had been penetrated by spermatozoa. The time required for the complete process was over 2 hours (Lewis and Wright, 1935). Long (1912) pointed out that second polar body formation in the rat began within 5 minutes to 2 or more hours after the spermatozoa were added to tlie eggs in in vitro preparations. Abstrictions of the polar bodies were completed 45 minutes later.


The interesting observations of Austin (1951c) on the sequence of events during formation of the second polar body in the living rat ova deserve special mention. In the unfertilized egg the chromosomes are arranged on the metaphase plate with the spindle lying paratangentially to the surface, usually in close association with the abstricted first polar body. Within a few minutes after the sperm head has penetrated the vitellus, and before it shows any detectable change, the chromosomes on the second maturation spindle pass to anaphase. The telophase stage is reached about 75 minutes after the initial penetration by the sperm. Then, there is a 20-minute period during which no further change is noted. Subsequently, the spindle slowly moves away from the surface and begins to rotate in such a way that its final position is at right angles to its original location. Rotation is completed in about 50 minutes. The spindle then elongates and becomes narrower, the process terminating in abstriction of a clear vesicle containing the clumped chromosomes. Since it was necessary to flatten the egg considerably in order to be able to observe the spindle under the phase microscope, complete abstriction of the polar body did not occur.


Similar observations on the formation of second polar bodies in rat ova were re]iorted by Odor and Blandau (1951). Approximately 2000 eggs were removed at varying intervals after ovulation and sperm penetration. The eggs were examined either in the fresh condition or after histologic preparation. In the majority of ova, the second polar body had been abstricted completely by the end of the 4th hour after semination.


H. Pronuclei Formatiox, Syngamy, and First Segmentation Division

As mentioned earlier, the general concept of the mechanism of fertilization in mammals has been based almost entirely on the examination of fixed and stained material. Even so, it is remarkable that a story of continuing development should have evolved by the piecing together of evidence from killed eggs, the age of which could not be determined within narrow limits. The more recent advances involving an evaluation of the temporal relationship between ovulation and the various phenomena of fertilization may be said to be due largely to the application of phase contrast microscopy to the studies of living rat ova (Austin and Smiles, 1948; Odor and Blandau, 1951; Austin, 1951a, b, 1952a; Blandau and Odor, 1952; Austin and Braden, 1954a, b).


Employing this method, Austin and Smiles observed fertilized eggs that were obtained by inducing ovulation in immature rats by means of gonadotrophins and subsequently allowing the females to mate. The recovered zygotes were kept at body temperature and development was followed continuously with the phase microscope. The details of the fertilization process described by Odor and Blandau were the result of examining several thousand living and fixed fertilized eggs recovered from sexually mature females at specific time intervals after ovulation and fertilization.


In the rat the complete process of fertilization, from the penetration of the ooplasm by sperm until the first segmentation division, requires approximately 24 hours. In general, the first 8 hours after sperm penetration is the period of the formation of the second polar body and the initial development of the male and female pronuclei (Fig. 14.12).


Changes in the morphology of the living sperm head can be noted as early as 10 minutes after penetration of the ooplasm and involve a loss of sharpness of outline and contrast, first in the posterior and caudal regions of the head. The decrease in contrast continues until finally the whole nuclear part is almost invisible in the living specimen, even under the phase-contrast objectives (Fig. 14.12, Jf). Concomitantly the head increases greatly in size and fluidity. During the initial period of swelling of the nuclear portion, the bifid perforatorium becomes detached (Fig. 14.12, 3). Approximately 2 hours after the sperm has entered, the j^rimary nucleoli make their appearance within the enlarged sperm nucleus. Time-lapse cinemicrophotography has shown that the nucleoli enlarge by the fusion of minute nucleolar aggregations. The larger nucleoli then fuse one with another until only a single large nucleolus is present (Fig. 14.13, 1 and 2). Throughout this period of transformation, the fiagellum may remain attached to the head and may undergo a very fine, intermittent, vibratory motion, especially in the region of the middle piece. The formation of the definitive female pronucleus begins soon after the second polar body has been coml)letely abstricted. The chromosomes remaining within the ooplasm after extrusion of the polar body are clumped together in the form of a small, compact mass (Fig. 14.13). The first indication of transformation of this chromosomal mass into the female pronucleus is the appearance of several minute nucleoli within a homogeneous nucleoplasm. As the nucleoli increase in size and number, certain of them coalesce, eventually producing one or two large nucleoli. As the pronucleus grows, the optical density of its nucleoplasm decreases to such an extent that it becomes clear and translucent.



Fig. 11.12. \'nri()U.< ,-l;i-iv.s in the traii.-loiiuat ion ol I lie Ina.l nl iIm I. i i ili/iii- -|m i m leading to the formation of the male pronucleus. Note loss of contrast of the liead as it enlarges. The changes in the head from 1 through 6 require 2 to 3 hours. Observations were made on the living egg, in vitro, and examined with phase contrast objectives. P, perforatorium; N, sperm nucleus; SF, sperm flagellum (Austin, 1951c).





Fig. 14.13. Further transformation of the sperm head into the male pronucleus, 1 and 2. Note the large nucleolus, formed by fusion of smaller nucleoli. NC, nucleolus. This stage has been reached approximately 5 hours after that in part 1, Figure 14.12, 1 and 2 (Austin, C. R., 1952). Developing male ( i ) and female ( $ ) pronuclei, in situ, as observed in living rat eggs, 3, 4 and 5. The entire sperm flagellum enters the ooplasm at the time of sperm penetration, 3 (Odor and Blandau, 1951).




Although there may be considerable variation in the development of the pronuclei between the 9th and 19th hours after sperm penetration, this is the time of active growth of the pronuclei and of increase in the numher of their nucleoH (Fig. 14.13, 5). During the early hours of this period, the male pronucleus grows at a more rapid rate than that of the female, and this differential is maintained even until karyogamy. At the stage of greatest development, the number of nucleoli in the male pronucleus may have increased to approximately 30 and that within the female nucleus to 10. Near the end of this interval, the pronuclei gradually approach one another. For some time after actual contact, the pronuclei retain their identity and the female pronucleus may considerably indent the larger male pronucleus (Fig. 14.14, 2). Approximately one-half hour before karyogamy begins, the nucleoli in both in'onuclei disappear from view and there is some shrinkage in the size of the pronuclei (Fig. 14.14, 3). Even after the complete disappearance of the nucleoli, the nuclear membranes may still be intact. Soon, however, they become irregular in outline and disappear. Shortly before the first segmentation division, an aggregation of the pi'ophase chromosomes may be observed. Within a brief period, the chromosomes are arranged on the metaphase plate. After an interval of 30 to 40 minutes, the chromosomes begin to divide and pass through the anaphase and telophase stages (Fig. 14.14, 4 and 5). The first segmentation spindle is observed most commonly between the 21st and 23rd hours after the entrance of the sperm. Even though Austin ( 1951c) followed the formation of the segmentation spindle, cleavage of the rat zygote did not occur in vitro.


It is often difficult to differentiate between the male and female pronuclei in sectioned material. Hence, their identification has not been clearly established for most mammals. The male pronucleus has been reported to be larger in the cat (Hill and Tribe, 1924) , vole ( Austin, 1957 1 , guinea pig ( Lams, 1913 ) , and rat (Odor and Blandau, 1951 ; Austin, 1951c; Austin and Braden, 1953) , and of approximately equal size in the mouse, guinea pig (Lams and Doorme, 1908), bat (Van der Stricht, 1910), cat (Van der Stricht, 1911), and hamster (Boyd and Hamilton, 1952; Austin, 1956b).


Edwards and Sirlin (1956a, b, 1959) have demonstrated that the male pronucleus within the fertilized mouse egg could be identified by injecting adult males with C14-labeIed adenine approximately 1 month before mating. The male pronuclei showed autoradiographs which could be related to the labeled sperm particularly in di- and trispermic eggs. Lin ( 1956) labeled unfertilized mouse eggs with DL-methionine while they were still within the follicles. Ovulation was induced by gonadotrophins and the unfertilized eggs were transplanted to mated females where they were fertilized and subsequently delivered as normal young.


The acridine orange-staining tcchni(iue has been applied recently to living rat eggs and the localization of the stain determined by fluorescence microscopy (Austin and Bishop, personal communication). The distribution of DNA may be determined by this technique and the ]H-eliminary data give support to the earlier rejjorts of Dalcq and Pastcels (1955) that duplication of DNA occurs within the pronuclei.


Information regarding the temiwral relationship between the formation of the first segmentation spindle and karyogamy is also very meager. In the guinea pig (Rubaschkin, 1905; Lams, 1913), bat (Van der Stricht, 1910), and rat (Odor and Blandau, 1951), the pronuclei have not completely fused by the time the spindle is formed. Isolated phases of this stage have been described also for the mouse (Lams and Doorme, 1908), rabbit (Gregory, 1930), and goat (Amoroso, Griffiths and Hamilton, 1942).

I. Fate of the Cytoplasmic Components of the Fertilizing Sperm Flagellum

Observations on the extent to which the flagellum of the fertilizing spermatozoon is carried into the ooplasm of the mammalian egg are contradictory and incomplete. The majority of the reports deal with sectioned material in which the identification of the whole flagellum may be very difficult. Yet, knowledge of the fate of the cytoplasmic components of the sperm is essential to an understanding of the role of the male gamete and must be pursued further.


In the mammals, the entire tail has been reported to be lodged within the ooplasm in the bat (Van der Stricht, 1923 ) ; mouse (Van der Stricht, 1923; Gresson, 1948) ; guinea pig (Lams, 1913) ; rat (Gilchrist and Pincus, 1932; Austin, 1951b; Blandau and Odor, 1952) ; and ferret (Mainland, 1930). Pineus (1930) and Nihoul (1926) were not convinced that in rabbits the flagellum enters the ooplasm. In the vole the flagellum enters the vitellus in only 55 per cent of fertilized eggs (Austin, 1957).



Fig. 14.14. Migration of the male and female prouuclei towards the center of the egg, 1 and 2. The male pronucleus is frequently indented by the female pronucleus, 2. At 3, note the disappearance of the nucleoli in the pronuclei immediately before the appearance of recognizable chromosomes as seen in 4. Telophase stage during first segmentation division in 5. PB, polar body (Odor and Blandau, 1951).



Except for the investigations by Gresson in the mouse, and Blandau and Odor in the rat, there are no detailed accounts of the fate of the flagellum after it enters the fertilized egg. In the mouse the mitochondria and Golgi material of the sperm become dispersed throughout the egg cytoplasm and the axial filament of the flagellum disappears before the first cleavage. But in the rat the flagellum is of such length and rigidity that it assumes an eccentric position within the periphery of the cell. Probably this explains why the male pronucleus ordinarily begins its development in the outer zones of the egg. Between the 15th and 19th hour after penetration, the external sheaths of the middle- and main-pieces begin to lose their smooth contours and they gradually disappear (Fig. 14.15). When this has been accomplished, the spiral mitochondrial sheath of the middle piece and the axial filament of the main piece can be clearly visualized. Immediately before the first cleavage, the continuous helical mitochondrial thread begins to swell. During the 2-cell stage, the mitochondrial thread is broken vip into globules that are dispersed throughout the egg cytoplasm. The remains of the axial filament have been observed in the 2-cell stage of the bat (Van der Stricht, 1902), guinea pig (Lams, 1913), and vole (Austin, 1957) and as late as the blastocyst stage of the rat (Blandau and Odor, 1952). Van der Stricht (1902) and Lams (1913) believed that, in the 2-cell stage of the bat and guinea pig, the sperm tail is present in only one of the blastomeres. This was partially substantiated for the rat by Blandau and Odor, who noted that in 58 per cent of 329 2-celled ova a greater portion of the axial filament was located within one blastomere and that in 12 per cent it lay entirely within a single blastomere. In the remaining 30 per cent, the axial filament was equally divided between the two. The significance of the various positions of the axial filament in the cleaving egg is not clear. It may represent merely the mechanical difficulty of moving an inert body. Of greater significance is the meaning of the cytoplasmic contribution of the sperm midpiece to the developing embryo in those animals in which its component parts are despersed within the vitellus.



Fig. 14.15. Chromosomes from the metaphase of the first segmentation division removed from a living, fertilized rat egg, 1. The sperm flagellum from the same egg lies just below the chromosomes. Note that the spiral mitochondrial sheath (SMP) is still present. At 2, twocell rat egg with the remains of the sperm flagellum at arrow. At 3, unfertilized rat egg in which the fragments appear similar to the blastomeres of a normally fertilized egg but there is no sperm flagellum present (Blandau and Odor, 1952).



Fig. 14.16. Somewhat flattened, living rat ovum with 13 accessory spermatozoa in the perivitelline space and a single fertilizing spermatozoon in the ooplasm. X 450.



J. Supernumerary Spermatozoa and Polyspermy in Mammalian Ova

The terms "supernumerary sperm," "accessory sperm," and "polyspermy" have been used to mean either the penetration of more than one spermatozoon into the ooplasm with the subseciuent development of multiple sperm nuclei, or the location of one or more spermatozoa in the perivitelline space. Inasmuch as polyspermy is used widely in the literature of invertebrates to designate the penetration of the ooplasm by multiple spermatozoa, it is suggested that this meaning should be retained for mammals and that the terms supernumerary or accessory spermatozoa should be utilized just to indicate the presence of nonfertilizing spermatozoa in the perivitelline space.


Intact spermatozoa have been observed many times within the perivitelline spaces of ova of various mammals (Sobotta and Burckhard, 1910, Gilchrist and Pincus, 1932, Odor and Blandau, 1949, Austin, 1951b, in the rat; Lams and Doorme, 1908, Lewis and Wright, 1935, in the mouse; Van der Stricht, 1910, in the bat; Hcnsen, 1876, Lams, 1913, in the guinea pig; Hill and Tribe, 1924, in the cat; Heajie, 1886, in the mole; Harvey, 1958, in the i)ika; Pincus and Enzmann, 1932, Chang, 1951c, in the rabbit; Hancock, 1959, in the pig). Quantitative data on the presence of supernumerary spermatozoa are available for the rat and several strains of mice. Austin (1953) and Odor and Blandau (1951) found that approximately 23 per cent of seminated rat ova contained supernumerary spermatozoa. The number of sperm per egg ranged from 1 to 23 (Fig. 14.16). After mating various strains of mice, Braden (1958a, b); and Piko, 1958) reported that the percentage of ova containing more than one sperm was more significantly related to the strain of the male than to the female. Matings with C57 males resulted in a consistently higher number of eggs with more than one sperm, irespective of the strain of the females used.

Apparently supernumerary spermatozoa have no effect on the rate of development of the ovum. In the rat, at least, the fluids of the perivitelline space offer an environment which is considerably more favorable for these spermatozoa than that of the oviduct. Except for a separation of the head from the neck-piece, the accessory spermatozoa in the rat, at least, show no evidence of cytolysis in any of the developmental stages including the late blastocyst. As mentioned earlier, spermatozoa from the same insemination that are lying free in the oviduct will have undergone extensive cytolysis in less than 24 hours. Finally, with the disappearance of the zona pellucida at the time of implantation, the accessory spermatozoa are cast forth into the uterine lumen. Austin (1957) suggests that the flagellum within the perivitelline space of the vole egg may undergo dissolution in situ.

The penetration of more than one sperm into the ooplasm is a common phenomenon in birds, rei:»tiles, urodeles, selachians, and insects (Fankhauser and Moore, 1941). Ordinarily, the additional sperm nuclei do not interfere with the development of the egg.

Until recently, polyspermy in cutherian eggs was considered to be relatively rare (Austin and Braden, 1953). Nevertheless, trinucleate eggs have been described in the rat (Tafani, 1889; Kremer, 1924; Pesonen, 1949; Austin and Braden, 1953); cat (Van der Stricht, 1911; Hill and Tribe, 1924); ferret (Mainland, 1930) ; and rabbit (Amoroso and Parkes, 1947; Austin and Braden, 1953).

According to Austin and Braden, the incidence of polyspermy in the normally mated rat is approximately 1.2 per cent; in the rabbit 1.4 per cent. If mating is delayed until after ovulation or if rats are subjected to hyperthermia, the figure rises to as much as 8.8 per cent. The incidence of polyspermy is no doubt influenced by a variety of conditions including hereditary variations within various strains (Odor and Blandau, 1956; Braden, 1958a, b). Austin and Braden (1953) concluded from their work that polyspermy in rats gives rise to triploidy in the embryo and that the polyspermic male pronuclei and the female pronucleus contribute to the formation of the first cleavage spindle. To the present, the polyspermic rat embryos have been found to develop to at least the 8-cell stage without showing abnormality. Fischberg and Beatty (1952a, b) have observed a normal-appearing triploid mouse embryo at 9^2 days. It is not known wdiether the triploid embryos can survive to birth. More recently. Gates and Beatty (1954) have stated that delay of fertilization by 5^/2 hours or more in the mouse did not result in an increased number of triploid embryos.


K. Stages of Development and Location of Eggs

The zygotes of the eutherian mammals are remarkably similar in their appearance and rate of development through the various stages of cleavage and formation of the blastocyst. Cleavage consists of a succession of mitotic divisions of the zygote at specific time intervals after karyogamy. The partitioning of the zygote occurs with little or no increase in the total amount of cytoplasm. Salient features of the mechanism of cleavage in different vertebrate types have been reviewed bv Bovd and Hamilton (1952).

Data on the rate of cleavage and transport of fertilized ova through the oviduct in different animals have accumulated much more slowly than one would expect from the availability of material. The most complete information has been obtained for some of the ungulates and laboratory rodents and is presented in tabular form (Table 14.5) from the summary of Hamilton and Laing (1946). The rate of cleavage is an inherent property of the zygote. Thus the cleavage rates of different species of amblystoma reared at the same temperature are significantly different. Similarly, in the rabbit the cleavage rate is consistently more rapid in strains of larger-sized animals than in the smaller-sized races (Castle and Gregory, 1929; Gregory and Castle, 1931). It is interesting that, although the zygotes of the larger-sized race divided more rapidly and contained more cells, embryonic differentiation occurred at the same rate in both races.

Altering the environment of zygotes may also effect the rate of cleavage. Thus the early fertilized eggs of the rat, mouse, hamster, and guinea jiig cleave only irregularly if at all under tissue-culture conditions. If various thio-amino acids are added to the medium in which rabbit zygotes are being cultured, cell division will proceed normally and may even be accelerated (Pincus, 1937; Pincus and Werthessen, 1938; Miller and Reimann, 1940).


Again cleavage may be either partially or completely inhibited by the addition of colchicine to a culture medium containing the fertilized eggs of frogs (Samartino and Rugh, 1946) or rabbits (Pincus and Waddington, 1939). A similar effect is observed when this drug is injected into mated mice (Waldo and Wimsatt, 1945). The rate of cleavage also depends on the amount of stored yolk. This is particularly true in the macrolecithal eggs of frogs and birds.

The peculiar phenomenon of deutoplasmolysis, or extrusion of yolk from fertilized and cleaving eggs, has been described in the bat (Van der Stricht, 1909) , various marsupials (Hartman, 1928), the horse (Hamilton and Day, 1945), the guinea pig (Lams, 1913), the cat (Hill and Tribe, 1924), the pig (Heuser and Streeter, 1929), and the ferret (Hamilton, 1934). In the cleaving eggs of the horse, a large amount of the yolk is extruded into the perivitelline space. The significance of this process is unknown, but it has been suggested that elimination of yolk may be necessary in order to establish a normal nucleocytoplasmic ratio (Levi, 1915).

Except for the monotremes, all mammals have meiolecithal eggs which undergo a complete or holoblastic type of cleavage. A discrepancy in the size of the first two blastomeres has been reported for a number of mammals and seems to be the usual condition (see Amoroso, Griffiths and Hamilton, 1942, for review of this subject).

The second cleavage division occurs in two planes at right angles to each other. Division of the two blastomeres is not necessarily synchronous and accounts for the frequent observation of a 3-cell stage. In an ovum containing two blastomeres of unequal size, the larger cell apparently has some priority in the next two divisions and this probably explains the origin of eggs containing an unequal number of cells. In the 4-cell stage, the blastomeres are arranged in the form of a tetrahedron, due to the preceding orientation of the two mitotic spindles at right angles to each other. Differences in the size of the blastomeres have been recorded in almost every species.

By the end of the 16-cell stage, several of the blastomeres have been moved centrally thus forming the morula. In subsequent cleavages, the smaller, peripheral cells divide more rapidly and an asynchrony, already present, is accentuated. Then fluids begin to accumulate between peripheral and central cells, giving rise to the cavity of the blastocyst.

During cleavage there is a significant diminution in the volume of the total ooplasm. In the first cleavage division of the monkey {Macacus rhesus), Lewis and Hartman (1933) recorded a shrinkage of 44 per cent. During the 1-cell stage of the mouse, Lewis and Wright (1935) noted shrinkage of as much as 25 per cent with a further decrease in volume as cleavage continued. The hamster egg is even more remarkable for the very large volume of its perivitelline space (Austin, 1957).

As mammalian ova of various species are studied, attention is being directed to the dift'erences in the size of blastomeres and rate of cleavage in the hope of finding evidence for the sorting and localization of specific determining substances in the zygote. It has been suggested that in the eggs of the monkey (Lewis and Hartman, 1941), pig (Heuser and Streeter, 1929), goat (Amoroso, Griffiths and Hamilton, 1942) , rabbit (Van Beneden, 1875), and mouse (Sobotta, 1924), the more rapidly dividing blastomeres are the precursors of the trophoblast and the more slowly cleaving cells the precursors of the inner cell mass or the embryo proper.

Even though discrepancies in the size of the first two blastomeres have been described in many mammals, there is at yet little evidence of a qualitative difference between them. Heuser and Streeter (1929) could not find a demonstrable cytologic difference between the first two blastomeres of the pig. Hamilton (1934) suggested that, at least in the ferret, size differences of the blastomeres can be explained by the chance division of the cytoplasm in the first cleavage. Despite the difference in size of the first two blastomeres in the mouse, Gresson

(1941) has shown that the mitochondria are equally divided between them. Furthermore, the observations of Nicholas and Hall

(1942) do not support the theory of absolute determination of the early blastomeres.



These investigators obtained normal young after separating the first two blastomeres of a rat embryo and transplanting them into pregnant host females. From the results of these experiments, they concluded that "the rat egg possesses the capacity to satisfy two of the criteria for equipotentiality: (1) each of the first two parts of the egg may form a whole embryo which develops further than the cleavage stages, and (2) the fusion of two eggs produces one single individual of large size." More recently, Tarkowski (1959) destroyed a single blastomere in 2- and 4-celled mouse eggs by piercing them with a micropipette directly through the zona pellucida. The eggs were then transferred to properly timed recipients. Of 175 half-blastomeres transplanted, 30 per cent had implanted and appeared to be normal except that they were significantly smaller than the controls. Three females gave birth to a total of 6 young which had developed from the experimental eggs. All of the animals were fertile and subsequently gave birth to several litters. Much more experimental work is needed in this area, and perhaps the techniques of transplanting individual blastomeres to the anterior chamber of the eye may open possibilities for further investigations (Fawcett, Wislocki and Waldo, 1947; Runner, 1947).

L. THE AGE OF THE EGG AT THE TIME OF FERTILIZATION

In 1913, Jacques Loeb in his book "Artificial Parthenogenesis and Fertilization" wrote as follows: "The unfertilized mature egg dies in a comparatively short time, which may vary from a few hours to a few weeks according to the species or the conditions under which the egg lives. The death of the unfertilized egg is possibly the only clear case of natural death of a cell, i.e., of death which is not caused by external injuries, and the act of fertilization is thus far the only known means by which the natural death of a cell can be prevented." As studies on the physiology of mammalian gametes are pursued, it is evident that these cells must indeed be listed among those having the shortest life span in the body.

The reproductive processes in most mammals are so timed that spermatozoa reach the site of fertilization and are ready to penetrate the eggs almost immediately after their extrusion from the follicles.

Developmental defects which result from the over-ripening of gametes before fertilization have been studied in greatest detail in invertebrates and lower vertebrates (Gemmill, 1900; Bataillon, 1901; Grave and Oliphant, 1930). Because of their ready availability, the eggs of the fish and amphibia have been found particularly useful in experimental investigations of this type (Mrsic, 1923, 1930; Witschi, 1952).

Witschi descril)ed a method whereby frogs were induced to retain their mature eggs within the uteri for varying intervals by separating the females from the males and keeping them in dry containers at room temperature. By subsequent removal of the eggs, by either laparotomy or strijiping, the aging gametes could be fertilized by artificial insemination and their development followed. He found that fertilization and development remained relatively normal in eggs which had been retained for 3 to 4 days. However, the sex-determining mechanism was affected in that almost 90 per cent males were produced. Similar alterations in the sex ratio have been reported by Mrsic in the aging eggs of the rainbow trout. If the frog eggs were retained for more than 4 days without fertilization, they gradually became over-ripe and either failed to be fertilized or, if penetrated by a spermatozoon, developed abnormally. After approximately 1 week, all of the eggs retained in the genital ducts became unfertilizable. In amphibia, as in other species, the aging eggs gradually lose their vitality. Witschi's observations are particularly significant in that he followed the development of the over-ripe eggs beyond the stage of metamorphosis and described a number of teratogenic effects of widely divergent nature. Some of the developmental abnormalities encountered were polymyelia, Polydactyly, axial duplications (especially in the region of the head), anencephaly, microcephaly, and failure of normal differentiation of various tissues and organs.

In evaluating the bases for the widely divergent nature of these abnormalities, Witschi suggested that they are expressions of an interference with either the normal processes of producing or liberating evocators, or the capacity of the embryonic tissues to respond to induction. Of special interest is the finding that the older fertilized eggs frequently gave rise to teratomatous proliferations in the endoderm. When these tumor-like masses were transplanted to older larvae they grew rajjidly and metastasized. Needham (1950) proposed that, because the primary evocator, the principal sex-hormone, and various carcinogens belong to the steroid compounds, the effect of over-ripeness may be related to a disturbance of embryonic sterol metabolism.



TABLE 14.6 The fertilizable life of the mammalian ovum


Anim.il


Length of Fertilizable Life


Investigator



Morphologic signs of degeneration appear within 24 hours after ovulation.


Hartman (1924a)



Mouse


(a) 12 hours. Matings 13 hours after ovulation results in reduced fertility

(b) 6 hours, estimation

(c) 8 hours, experimental


Long (1912)

Lewis and Wright (1935) Runner and Palm (1953)


Hamster


5 hours, experimental


Chang and Fernandez-Cano (1958)


Rat


>12 hours, experimental


Blandau and Jordan (1941)


Guinea pig


>20 hours, experimental


Blandau and Young (1939); Row lands (1957)


Ferret


>30 hours, experimental


Hammond and Walton (1934)


Rabbit


6 hours, experimental 8 hours, experimental


Hammond (1934) Chang (1953) Braden (1952)


Sheep


24 hours, estimation


Green and Winters (1935)


Cow


18-20 hours, experimental


Barrett (1948)


Mare


Short


Day (1940)


Monkey


23 hours, estimation


Lewis and Hartman (1941)


Man


6-24 hours, estimation


Hartman (1936)



The fertilizable life of the mammalian ovum has been experimentally determined in only a few rodents, carnivores, and ungulates (Table 14.6). In the ferret, for example, Hammond and Walton (1934) found that the ovum remains capable of fertilization for not more than 30 hours after ovulation. In the rabbit, delay in fertilization


results in lowered fertility and smaller size of litters (Hammond, 1934). In the hamster 50 per cent of ova are incapable of fertilization 4 to 5 hours after ovvdation (Chang and Fernandez-Cano, 19581.

In rats the spermatozoa may penetrate eggs which have been aged 12 hours before fertilization or to a point of devitalization but not of death. In such eggs they may even undergo transformation into the male pronuclei and form segmentation spindles, but the female nucleus in the same egg either fails to develoj) or fragments into a number of nuclei of varying sizes. Even though 70 per cent of the greatly over-ripe rat eggs may be penetrated by spermatozoa, various abnormalities of development result which are not compatible with continued growtii and development. Thus, at the time of implantation only 4 per cent of the experimental rats are impregnated. Furthermore, the ova which do implant successfully are retarded in their development, and the


«50


SPERM, OVA, AND PREGNANCY


majority die before the fetal period is reached (Blandau, 1952; also see Braden, 1959).

A strikingly similar picture is presented by delayed fertilization in the guinea pig. The fertilizable life of the egg in this species is approximately twice (20 hours) that of the rat (Blandau and Young, 1939; Rowlands, 1957). The first effects of over-ripeness are seen in embryos from females inseminated approximately 8 hours after ovulation. No normal development followed inseminations more than 20 liours after ovulation. As far as could be determined, the principal effects of aging were either the early death of the zygote in the pre-implantation period or retardation in the rate of growth in embryos which were capable


of implanting. A moderate delay in fertilization has been shown to lead to polyspermy particularly in rats and rabbits (Austin and Braden, 1953; Odor and Blandau, 1956).

M. Implantation

The blastula of the placental mammal is called the blastocyst. In the fully developed stage it is still enclosed in the zona pellucida and shows the inner cell-mass attached to the embryonic pole of the trophoblast. During the early period of its existence the blastocyst is spherical to somewhat oval in shai)e and except for size appears remarkably similar from animal to animal (Fig. 14.i7).


In most mammals, the blastocyst does not come into firm contact with the maternal endometrium for a number of days after reaching the uterus. In the mouse, mole, shrew, and guinea pig, the free uterine period is from 3 to SVk days; in the rabbit, 5 to 6 days; in the rhesus monkey and possibly the human, 4 to 6 days ; in the cat, 8 to 9 days; in the dog, 9 to 10 days; and in the ungulates probably somewhat longer.



Fig. 14.17. The similarity of the free uterine bhistoc-y.^t.s of various mammals: 1, 5!/2-day human blastocyst (photograph courtesy, Hertig, A., and Rock, J.); 2, 6-day guinea pig blastocyst; 3, 9-day monkey blastocyst (Heuser and Streeter, 1941); and 4, 9-day sheep blastocyst (Boyd and Hamilton, 1952).



Under the conditions of "developmental diapause" or delayed implantation, the free uterine period of the blastocysts may be significantly prolonged. Delayed implantation occurs naturally in a variety of species such as the pine marten, 6 months; American badger, 2 months; European badger, 3 to 10 months; European roe deer, 4 months; armadillo, 14 weeks; fishers, 9 months; and bears, 6 months. Delayed implantation has also been recorded in the stoat, weasel, sable, and fur seal. In the rat, mouse, and certain insectivores, implantation may be delayed several days to 2 wrecks if there is concurrent lactation (Lataste, 1887; Daniel, 1910; King, 1913; Hamlett, 1935; Brambell, 1937; AVeichert, 1940, 1942). In the mouse and rat the delay varies roughly with the number of young suckled, and this, in turn, prolongs the period of gestation. According to Lataste, the duration of gestation is normal in mice suckling only 1 or 2 young but prolonged in those suckHng 3 or more. If certain hormonal conditions are satisfied, implantation will occur in normal females suckling large litters (Kirkham, 1916; Weichert, 1940, 1942, 1943; Krehbicl, 1941). Delay of implantation is very likely due to an inhibitory effect by some uterine or nutritional factor acting on the blastocysts (Whitten, 1958). Various experimental methods may successfully delay implantation without destroying the ova. Ovariectomy the second day after mating in the rat, followed by subliminal doses of progesterone (0.5 mg. per day ) , will keep the eggs alive for 6 to 45 days, but the decidual cell response and implantation do not take place. If more progesterone than 0.5 mg. per day is injected into these animals, implantation may occur. A combination of injections in which a small dose of estradiol benzoate is added to the subliminal dose of progesterone is very effective in consummating iml^lantation. In contrast, if the ovaries are removed from pregnant rats on the fourth


day when the blastocysts have reached the cornua, progesterone, even in dosages of 10 mg., cannot effect implantation. If estrogen and progesterone are injected simultaneously, the blastocysts will resume their growth and will implant (Canivenc and Laffargue, 1957; Cochrane and Meyer, 1957; Mayer, 1959).


The blastocysts of pregnant rats spayed the 4th day may remain alive for as long as or longer than 21 days. Rat blastocysts apparently do not reciuire adrenocortical hormones to remain viable. Mayer (1959) and his co-workers have demonstrated that the blastocysts in the cornua of rats which have been ovariectomized and adrenalectomized on the 4th day after mating can implant on the 10th day, provided estrogen and progesterone are both injected simultaneously.

The experiments of Cochrane and Meyer, and others that have been mentioned, suggest that the optimal conditions for embryo attachment and implantation depend on a delicately balanced, synergistic action of estrogen and progesterone on the endometrium. But nothing is known as to what is happening within the egg during its dormant state and what factors control the dormancy, nor do we understand what changes occur within the uterine lumen which may eventually satisfy the conditions of the embryo to continue its growth, make attachment to the uterine epithelium, and implant. Our point of view will no doubt be broadened as experimental approaches to the problem are varied and more species are studied.


Runner (1947), Fawcett (1950), and Kirby (1960) found tliat, irrespective of the state of the host's gonads, implantation occurred when mouse ova were transplanted either to the kidney capsule or to the anterior chamber of the eye. Whitten (1958) transplanted 8-celled mouse eggs to the surface of the kidneys of normal and hypophysectomized mice. Ten days later successful grafts were found in 10 of 15 normal and in 13 of 18 hypophysectomized animals. Successful implantation of mouse eggs onto the kidney apparently does not depend on the secretion of the pituitary.


Buchanan, Enders and Talmage (1956) reported that implantation occurs in ovariectomized armadillos that are not receiving hormonal replacement. In the European badger ovulation occurs during delayed implantation. The new set of corpora lutea does not hasten implantation because delay in implantation may continue for 2 months after the last ovulation (Harrison and Neal, 1959).


The phenomenon of delayed implantation offers an excellent experimental approach to the general problem of embryo-endometrial interrelationships and the specific factors that control embryo attachment and implantation.


N. Spacing and Orientation of Ova In-Utero

The specific sites of implantation in mammals having multiple young, as related both to the longitudinal axis and to the surface of the endometrium, are remarkably constant (Mossman, 1937). Even in animals having only a single young and a simplex uterus, such as man, monkey, sloths, and others, the location of the implantation site and the orientation of the blastocyst to the endometrium are quite definitely regulated (Mossman, 1937; Heuser and Streeter, 1941).


Various explanations have been proposed to account for the intra-uterine spacing of blastocysts in polytocous mammals. Mossman suggested that the implanting blastocyst may interact in some manner with the surrounding endometrium so as to create a local refractory zone in which no other embryos can implant. The results obtained by Fawcett, Wislocki and Waldo (1947) after transplanting several mouse ova into the same anterior chamber of the eye are of interest in this connection. They found that fertilized eggs continue to develop in close proximity to one another only until one of them begins to implant. Thereafter, the remaining embryos degenerate. The onset of the degenerative changes in the surrounding blastocysts is coincident with the extravasation of blood into the tissues in the immediate vicinity of the attaching embryo. They suggest that possibly a cytolytic ferment of the trophoblast may cause edema or hemorrhage into the maternal tissues which so alters the local environment that it is untenable for the remaining blastocysts.


According to Mossman's theory, the blastocyst that enters the uterine cavity first establishes a refractory zone near the uterotubal junction and begins the process of attachment. The remaining blastocysts establish similar zones in the fashion of a gradient toward the cervix until all become evenly spaced. It has been frequently observed in pregnant animals with bicornuate uteri that the embryos which are implanted nearest the oviducts are slightly more advanced in development than those nearest the cervix. It has also been observed that the embryos which are implanted nearest the cervix show a higher incidence of resorption than those implanted at other sites.


Recently McLaren and Michie (1959) have taken issue with Mossman's theory that implantation is serial and that refractory zones are established. These investigators induced ovulation and mating in mice by hormone treatment. At 18V^ days after mating, the cornua were divided into 6 equal segments and the embryos weighed. They found that the embryos in the middle of the cornua actually weighed less, on the average, than those at either end. The embryo lying nearest the oviduct was usually significantly lighter than its neighbor.


It may be questioned whether the differences in weight of mice fetuses at ISV^ days post coitum have any relationship to differences in size and differentiation of the embryos during the first 5 to 10 days of development or during the period of orientation in utero or of attachment and implantation.


Investigators who have observed blastocysts and implanting embryos have frequently commented on the variations in the early stages of development in the same animal and the variation from animal to animal when they are killed at identical times after mating. The variations in the rate of differentiation are particularly striking if the development of the attachment cones of the guinea pig embryos are observed in tissue culture. The attachment cones of each of the 2 to 3 blastocysts recovered from the cornua of the same animal may be in a different stage of development and may retain this difference throughout the period of cultivation.


The successful transplantation of eggs from animal to animal in certain rodents is feasible and may be the means whereby an experimental approach to the problem of spacing can be made. One or more fertilized eggs could be transferred to the oviducts of properly timed hosts and their sites of attachment observed. One of the problems in evaluating implantation grossly in transplantation e.xperiments is the possibility of inert objects (lint, clumps of cells, etc.) affecting the decidual response and mimicking imiilantation.


In normal, pregnant rats the embryos are more evenly spaced in cornu when the number of young is 5 or more. If the number of implanting blastocysts is less than 4, there is a tendency to occupy chiefly the caudal halves of the horns (Frazer, 1955).


Information is needed as to the manner in which eggs enter the cornua, i.e., whether they enter singly or as a group and what the relationship of the multiple eggs may be one to another during the several days that they lie free within the uterine lumen. It is ciuite clear that embryonic spacing in utero is more even than random. This raises the cjuestion as to what controls the size of the refractory area if the cornu is crowded by superovulation, transplantation of eggs, or more than normal numbers of eggs from compensatory hypertrophy in cases where one ovary has been removed.


It has long been known that in bicornuate uteri blastocysts may pass from one cornu into the other through the body of the uterus (Boyd, Hamilton and Hammond, 1944; Boyd and Hamilton, 1952; and many others). Bischoft' (1845) interpreted transuterine migration as a method by which the distribution of embiyos could be equalized in cases where there is a disparity in the number of eggs ovulated from each ovary. The means by which this migration is accomplished has been the subject of speculation and some investigation.

At present, there is no direct evidence that the unimplanted embryo has the power of independent movement. If this is true then the positioning of the blastocyst in utero and its orientation in relation to the endometrium must depend on chemical and/or physical forces. Markee and Hinsey (1933) suggested that alternate contractions of the cornu transport blastocysts from one to another. Krehbiel (1946) anastomosed the cornua of ovariectomized rats in a variety of ways and concluded that each uterine cornua retains its individuality in effecting: the distribution of embryos.


The role of the myometrium in the distribution and spacing of the blastocysts in utero has received considerable attention. Corner (1923) and Wislocki and Guttmacher (1924) found active myometrial contractions in the sow during the preimplantation period. Even though the postovulatory contractions occurred with greater frequency, they were greatly diminished in amplitude compared with those recorded during the estrous phase. The motility pattern of the myometrium changes gradually from day to day so that, by the time of implantation (12th or 13th day), the spontaneous contractions continue at a rate of 4 to 8 per minute, but their amplitude is so slight that the kymographic tracings are almost level. Similar observations were reported for the excised uterine horns of the rabbit (Knaus, 1927). Using a more refined technique and beginning their observations immediately after the muscle strips were put into the bath, Csapo and Corner (1951) and Csapo (1955) showed that uterine muscle under the dominance of progesterone displays a high state of irritability but poor conduction, and it develops spontaneously a state of "contracture" when it is first placed in the muscle bath. Spontaneous contractions begin after a short interval but they are of very low amplitude. The initial "contracture" is reversible and may be suspended by electrical stimulation or anoxia. Progesterone in some way alters the response of the myometrium to stimuli.


The motility pattern of the myometrium under the dominance of progesterone is certainly different from that when the animal is in estrus, but the nature of these differences is still puzzling (Reynolds, 1949; Csapo and Goodall, 1954). A strip from an estrous uterus placed in the bath relaxes immediately. After a short interval, spontaneous contractions begin and continue with increasing amplitude. Thereafter, contractions occur at intervals of 1 to 2 minutes followed by prompt relaxation. In contrast, similar relaxation was not observed in uterine strips under the influence of progesterone. Instead they slowly shorten.


Ivy, Hartman and Koff (1931j observed that muscular contraction waves in the monkey uterus originate from an area slightly ventral and cranial to the insertions of each of the oviducts and then proceed medially to meet in the midline. They concluded that in the monkey the area of the endometrium where implantation usually occurs is affected by contractions to a lesser extent than the remainder of the uterus.


Nicholas (1936) interposed a section of duodenum into the rat's uterus and found embryos in the lower uterine segment. Lim and Chao (1927) reversed the middle portion of one or both cornua of the rabbit and reported that pregnancy was not prevented.


Markee (1944) introduced sea urchin eggs, celloidin balls, and glass beads into the tubal ends of rabbit cornua and observed their distribution at varying intervals from estrus to 10 days after ovulation. He found that the sea urchin eggs were distributed most evenly in the uteri of cstrous rabbits, especially at the time of ovulation. Fairly good distribution was recorded at 5 days and poor distribution at 10 days after ovulation. As noted below, none of these inert objects or sea urchin eggs expand with time as do rabbit blastocysts before attachment. It is doubtful that the movements of these objects in utero could be considered as the normal state of affairs in the transport of blastocysts. In order to study this problem further, Markee observed uterine contractions directly through a glass window which had been sewn into the abdominal wall. Three types of contractions were observed during estrus and for 5 days after ovulation : (1) local ring-type contractions persisting for approximately 10 seconds, (2) peristaltic contractions proceeding throughout the length of the cornu, and (3) antiperistaltic waves of approximately the same intensity as the peristaltic contractions. After the 5th day, the peristaltic and antiperistaltic contractions decreased greatly in amplitude and in the length of their excursions.


Recent studies on the mechanisms contributing to the distribution of the implanting rabbit blastocysts have directed attention to the possibility that both physical and chemical interactions between the blastocyst and uterus are important (Boving, 1952a, b, 1954, 1956, 1959). Boving has found that by 7 days post coitum, rabbit blastocysts have achieved an almost even distribution, not only with reference to the space between them, but also with respect to the entire length of the uterine cornu (Fig. 14.18). If the number of blastocysts in utero varies, the spacing is nevertheless appropriate to their number. The cornua reacts to the presence of each blastocyst and positions it in relation to all other blastocysts present until a remarkably even distribution is achieved by the 7th day post coitum. There is evidence from the work on the rabbit at least that the movement and i)ositioning of blastocysts in utero coincide with their increase in size. Rabbit blastocysts of approximately 1-mm. size are propelled much more slowly than blastocysts or glass beads 3 to 6 mm. in diameter. Boving suggested that each blastocyst acts as a localized stimulus which initiates the propulsive muscular activity and that the size of the blastocysts determines the way in which the myometrium responds. Cessation of positioning is coincident with a local loss of uterine tone and a ballooning out of the antimesometrial wall to form a "dome."


The blastocysts of the leporid family of rodents, the carnivores, some insectivores, and bats undergo considerable expansion in the uterine cavity before and at the time of attachment. In these animals, then, the spacing of the blastocysts may be arranged according to Boving's theory that myogenic uterine contraction is the effector of both propulsion and spacing.

As mentioned earlier, during the 6th and 7th days after copulation in rabbits, the expanded blastocysts occupy a distended, antimesometrial "dome" caused by a local decrease in uterine muscle tone. From in and to-and-fro motion approximately every 30 seconds. This seems to be effected by a change in the tone of the muscles forming the uterine dome. The rotational motion could provide an orientational mechanism, because eventually all surfaces of the blastocyst would come in contact with the dome. In the in vivo observations, it seemed that the blastocyst is "grasped" by the muscular action of the uterus, and by the 7th day post coitum is confined along the antimesometrial border.



Fig. 14.18. Positions of rabbit blastocysts (dots) in utero (bars) from the 3rd to the 8th day post coitum. There is little change in position during days 3 and 4. Even distribution is achieved 6 to 7 days -post coitum. The crosses in the 8-day uterine horn represent the position of blastocyst models which had been in the uterus for 2 days (Boving, 1954).



As implied earlier, the orientation of the blastocyst with reference to the uterus and mesometrium varies considerably in different species. It may be mesometrial as in the Pteropodidae and Tarsiidae, antimesometrial as in most rodents and insectivores, or orthomesometrial as in the Centetes and Hemicentetes (Mossman, 1937).

The orientation of the embryonic disk within the uterus is remarkably constant in closely related species but varies greatly in different orders. Thus the inner cell mass at the time of attachment may be directed toward the mesometrium in the rodents, toward the antimesometrial side in the vesperilionid bats and some insectivores, or toward the lateral side as in the golden mole. With the possible exception of the rabbit and guinea pig, the role of the blastocyst in determining the pole of attachment is unknown.


Alden (1945) reversed the mesometrialantimesometrial axis of the uterus of the rat by surgical means and demonstrated that, regardless of the position of the altered segment, the implanting embryos were correctly oriented relative to the uterus. Apparently, gravity alone is not of great importance in determining the pole of attachment, at least not for the rat egg.


Before the cells of the trophoblast can come into contact with the uterine epithelium, either the tough and resistant zona pellucida must be removed or the cells of the living trophoblast must penetrate the zona. A number of investigators have thought chiefly in terms of the removal of the mucous coat and zona pellucida by uterine factors. As we will see, others have been impressed by the possibility of participation by the trophoblast.

In 1935 Hall presented evidence which seemed to support the former view. He found that in rats and mice the zonae pellucidae disappear rapidly when immersed in fluids of pH 3.7 or below. In less acid solutions (pH 4 to 5), they were affected much more slowly. Acidified Ringer's fluid at first caused a swelling of the zonae, and the ordinarily smooth outer contour became wavy and fringe-like. In measuring the hydrogen ion concentration of the fluids in the vicinity of deciduomata of the rat, values as low as pH 5.7 were recorded. Such values w^ere of sufficient acidity to effect the gradual softening of the zona pellucida. Pincus and Enzmann (1936) also measured the pH of uterine luminal fluids in pseudopregnant rats and at no time observed values below 6.7. From Hall's work it was concluded that "as the decidua develops around the implanting egg and as the metabolic activities of the dividing blastocyst increase, the fluid bathing the blastocyst may become sufficiently acid to be a factor in the removal of the oolemma." Fertilized mouse ova, transplanted to the anterior chamber of the eye, lost their zonae independently of a change in hydrogen ion concentration of the environmental fluids <Fawcett. Wislocki and


Waldo, 1917). Other factors which alter the physical properties of the secondary and tertiary membranes were described earlier. At this point, how^ever, it is important to direct attention to LutwakMann's (1959) recent comments on the toughness and resilience of the zona in the rabbit and the difficulty in dissolving it except by harsh enzyme and chemical means. Results obtained during work on the guinea pig and rabbit have prompted investigators to think of other methods by which the zona pellucida and other investing membranes might be shed. Remnants of the zona pellucida have been found adhering to the blastocyst wall in sections of early implanting guinea pig ova (von Spee, 1901; Maclaren and Bryce, 1933). This fact indicates that the zona pellucida is not uniformly lost by chemical action of the fluids of the uterus. In the guinea pig the abembryonal pole of the blastocyst first makes contact with the endometrial epithelium of the antimesometrial border of the cornu. In 1883 von Spee described an increase in the size and number of the abembryonal pole cells of the guinea pig shortly before implantation of the blastocyst and gave an account of the pseudol)odia-like processes of these cells penetrating the zona. These processes were regarded by other investigators as fixation artifacts or "as of the nature of a secretion" (Sansom and Hill, 1931). Recently, the early iml)lantation of the guinea pig has been reinvestigated and the observations of van Spee have been confirmed (Blandau, 1949b). It is remarkable that there should be so little change in the zona of the guinea pig egg during its 3 day sojourn in the cornu. The blastocyst completely fills the perivitelline sjiace and the abembryonal pole cells comprise but a single layer (Fig. 14.19). Within a few hours before the ovum attaches itself to the uterine epithelium, the abembryonal pole cells proliferate to form the implantation cone. The trophoblast cells lying next to the zona send numerous slender protoplasmic processes through it (Fig. 14.20) until the abembryonal pole is riddled with them. The cytoplasmic extensions increase rapidly in size and may extend as bulbous expansions of varying shape for some distance beyond the zona pellucida (Fig. 14.21). It is only in the region where the zona is perforated by the extension of the abembryonal pole cells that it gradually becomes thinner and disappears. The remainder of the zona pellucida has been observed in vitro to slough off from the attaching blastocyst, much as a grape skin is removed from the flesh of the grape. Attachment cones have been described in fixed preparations of a number of genera of grovnid squirrels and chipmunks (Lee, 1903; Mossman, 1937). Although the extension of conclusions based on the study of one species to other species is precarious, it is possible that the same relationship of the attachment cone to the zona pellucida exists in other forms (Mossman, 1937). Boving noted a change in the viscosity and adhesiveness of the rabbit egg investments at the time of implantation which he attributed to local alkalinity (pH 9) released from one or more regions of the abembryonic hemisphere. Following adhesion, the outer investments of the blastocyst in this area disintegrate. Inasmuch as remnants of the membranes are sometimes observed in the areas between the implanting blastocysts, their final removal apparently is similar to that described for the guinea pig. When the membranes have been shed the abembryonic trophoblast adheres to the uterine epithelium, particularly in areas where blood vessels are subjacent to the epithelium. The trophoblast penetrates the epithelium by displacement, and the invasion of the stroma at first is not destructive.



Fig. 14.19. Photomicrograph of a living guinea pig blastocyst removed on the 6th day after ovulation. The inner cell mass is directed towards the top of the page. Abembryonal cells form but a single layer (compare with Figure 14.17, 2). X 900.


FiG. 14.20. AiiiK-aiaiicc of li\iiig guinea pig l)la.'<tiicysi api>r()xiiiiaii'l\- oiic hour before attachment of the abembryonal pole to the endometrium. Note the increase in the number of the abembryonal pole cells and the cytoplasmic extensions of these cells through the zona pellucida. X900.



Fig. 14.21. Living guinea pig blastocyst removed appioximatel}' one-half hour before attachment to the endometrium. The blastocyst is slightly rotated to show the extensive protoplasmic projections at the abembryonal pole. X 900.



O. Blastocyst Expansion

In the guinea pig, rat, mouse, and hamster, the diameter of the blastocyst at the time of attachment is approximately the same as that of the tubal ova. In these species implantations are more or less regularly spaced but not invariably so, because placental fusion occurs frequently. Thus there does not seem to be the same purpose fill interplay between the embryos and cornua as described for the rabbit. The blastocysts of these rodents are definitely polarized in relation to the uterine epithelium at the time of attachment and invasion. Although it is universally stated that the blastocyst does not have the cal^acity for independent movement in utero, observations on the behavior of the guinea pig blastocyst in tissue culture and the cytologic descriptions of the attachment cones in the monkey, ground squirrels, and chipmunks suggest that the blastocyst plays an active role in its positioning at the time of attachment. This possibility would encourage one to examine more carefully the living blastocysts of various animals at the time of attachment. From some of the earlier investigations, it would seem that the expansion of the rabbit blastocyst is dependent on physiologic factors external to the egg itself (Pincus and Werthessen, 1938). Thus, blastocyst expansion is interfered with if ovariectomy is performed or estrogen is injected 3 to 5 days after mating. On the other hand, injections of progesterone can reverse the effect of estrogen (Burdick and Pincus, 1935; Pincus, 1936; Pincus and Kirsch, 1936). Allen and Corner (1929) showed that if progesterone is injected into rabbits ovariectomized shortly after fertilization, the fertilized eggs will implant normally. If fertilized rabbit ova are grown in watch glass cultures, they will ■ cleave normally, but they herniate and collapse during the blastocyst stage (Lewis and Gregory, 1929) . If crystalline progesterone is added to these cultures, there is no increase in the rate of cleavage nor is herniation or collapse prevented (Pincus and Werthessen, 1937). The same investigators have shown that regular expansion of the blastocyst is obtained if the morulae or blastocysts are cultured in homologous serum and the medium is continually circulated. Recently, Bishop observed that expansion is suppressed if the oviducts of rabbits are ligated soon after the blastocysts have entered the uterus. The implication is that some oviducal factor is necessary for expansion. The problem is complicated by the fact that the egg does not expand during its 3 day sojourn in the oviduct.


From the observations recorded above, it seems that in order to stimulate normal growth and expansion of the blastocyst, in the leporid family of rodents at least, progesterone must act in some way on oviducal and uterine metabolism since both parts of the genital tract are probably involved.


The specific physicochemical processes in blastocyst expansion are not known. A plausible explanation is that the expansion may be due simply to the processes of osmosis, the changes in size being related to ionic variations of the fluid within the blastocyst cavity and the surrounding environment. It is more likely, however, that comjMex processes of active transport are involved, and, if these are to be elucidated, help from the biochemist and physical chemist is essential.


One of the difficulties confronting investigators so trained is the small amount of material obtainable for study by the conventional chemical methods. This is particularly true in such laboratory animals as the mouse, rat, hamster, guinea pig, and monkey, in which the blastocyst undergoes very little expansion before implantation and in which uterine secretions are present in very minute amounts. Nevertheless the recent approaches to the study of embryo attachment and implantation in the rabbit, particularly those by Boving (1954), Bennett (1956) and Lutwak-Mann (1959), offer a methodological approach that is essential if the dynamic aspects of nidation are to be understood. Lutwak-Mann especially and her co-workers have been the most active in discerning the practical problems in the handling of early embryologic material for biochemical study and in devising sound methodologic approaches.


In 1938 Pincus and Werthessen described a crystalline deposit in the abembryonal membranes of certain blastocysts of rabbits removed on the 5th day after mating from females which had been ovariectomized 18 to 20 hours after copulation. Boving (1954) identified this crystalline material as calcium carbonate and noted that there is little or none present 3 to 4 days after mating, but that the deposit increases to a maximum at the 6th day post coitum. He suggested that the osmotic effect of the blastocyst fluid is increased by the ionization of the inherent calcium carbonate reserve. Deficient respiration of the free blastocyst may perhaps lead to the production of acids which react with the calcium carbonate reserve. At the time of uterine attachment, there is improved gas exchange due to the embryo's close proximity to subepithelial blood vessels. Thus the bound alkali is liberated, the ionic concentration of the fluid is decreased, and blastocyst turgidity is lessened.


In measuring the bicarbonate of the rabbit blastocyst cavity fluid, Lutwak-Mann and Laser (1954) found a remarkably high content in 6- and 7-day-old embryos. Thereafter, the level of bicarbonate fell rapidly so that on the 8th day, when implantation is completed, the level was somewhat below that for maternal blood. The occurrence of high concentrations of bicarbonate in the unattached blastocysts led to assays of carbonic anhydrase activity in extracts of pregnant and nonpregnant rabbit uterine mucosa. It was found that carbonic anhydrase activity was very low in the uteri from nonpregnant animals but very high in the uteri from pregnant individuals. The oviducts, endometrium, and placental tissues are the main loci of carbonic anhydrase activity in the female reproductive tract. There are, however, species differences in the extent and the time at w^hich the enzyme can be demonstrated. The endometria of pregnant or nonpregnant hamsters, rats, and guinea pigs do not contain measurable quantities of carbonic anhydrase. However, significant enzyme activity has been found in the maternal portions of the placenta of these animals (Lutwak-Mann, 1955).


It has been clearly established for the rabbit that the enzyme is hormone-dependent. Progesterone and progesterone-like compounds greatly increase the amounts of the enzyme measured in the endometrium and this increase is proportional to the dosage of the hormone injected. There is no concomitant increase of carbonic anhydrase in the blood (Lutwak-IVIann and Adams, 1957a, b).


There is a 10- to 30-fold increase in the weight of the blastocyst between the 5th and 6th days. Dry weight measurements have shown that this increase is due primarily to water. The enzyme system responsible for the active transport of water is as yet unknown, but is being actively sought. Concentrations of Na, K, and CI ions in the yolk sac fluid approach or, in the case of K, exceed that of the maternal serum. Glucose, on the other hand, is present in less than half the amount found in maternal blood on the 7th day and two-thirds the amount on the 8th day. Data are also available on total nitrogen, phosphorus, bicarbonate, and various vitamins, particularly the components of the B complex, in the unimplanted blastocyst (Kodicek and Lutwak-Mann, 1957; Lutwak-Mann, 1959). Obviously the opportunities for utihzing isotopes for transfer studies in the fresh and implanting blastocysts are many indeed, and one may confidently expect a rapid unravelling of the manifold functional aspects of implantation if these techniques are employed by competent investigators.


P. Embryo-Endometrial Relationships

The interrelationship between the blastocyst and the endometrium at the time of attachment and implantation is not only exceedingly complex but also highly variable in different species. Irrespective of the complexity of the attachment, each type has as its purpose the apposition or intimate fusion of the fetal membranes to the maternal endometrial epithelium or stroma so that adequate physiologic exchange can take place.

Earlier studies on the experimental production of deciduomas by mechanical stimulation of the sensitized endometrium, and the dependence of implantation on the proper hormonal stimulation of the uterine mucosa, had the effect of swinging the pendulum of opinion toward the endometrium as being the most active agent in the process of nidation (Huber, 1915; Kirkham, 1916; Selye and McKeown, 1935; Krehbiel, 1937; Rossman, 1940). More recently, however, the observations ( 1 ) on the development of the attachment cone in some specific area of the trophoblastic wall just before attachment, (2) the changes in the viscosity and adhesiveness of the egg envelopes at the time of attachment, and on the developmental potentialities of ova transplanted to the anterior chamber of the eye and other sites have swung the pendulum back to the embryo and the role that it may l)lay in nidation (Asshcton, 1894; von Spec, 1901; Schoenfeld, 1903; Mossman, 1937; Fawcett, Wislocki and Waldo, 1947; Runner, 1947; Blandau, 1949a; and Boving, 1954, 1961).


The extensive i)rolil'eralion and differentiation in the endometrium of certain animals after ovulation undoubtedly arc iml)ortant in the nourishment and maintenance of the ovum in utero and in providing a suitable implantation site. The considerable growth and differentiation which the blastocysts of many animals undergo before they make contact with the uterine mucosa would indicate that more nutrients are required than are stored in the ooplasm of most mammalian eggs. The widespread occurrence of glucose, glycogen, lipids, phosphatases, iron, calcium, and many other substances, including vitamins and enzymes, in the endometrium may provide the necessary nourishment during the very early stages of implantation ( Wislocki and Dempsey, 1945) . Bloch (1939) described the secretion of an osmol)hilic substance by the uterine epithelium which is thought to be absorbed by the free mouse blastocyst. The work of Daron (1936), Markee (1940), Phelps (1946), Parry (1950), and Boving (1952a, 1961) has demonstrated that there is an increased blood supply immediately below the uterine epithelium at about the time of blastocyst attachment. The increased vascularity may not only provide nutrition to the uterine epithelium, but more importantly it provides blood vessels for specific physicochemical reactions between the trophoblast and endometrium (Boving, 1959a) . A similar increase in the blood supply in the antimesometrial area has been observed in the guinea pig (Bacsich and Wyburn, 1940). This is the area in which implantation invariably occurs in this species, and the localized hyperemia is considered to be a factor in tlie antimesometrial implantation.

It is well established that the presence of an actively secreting corpus luteum is essential if implantation is to be complete and successfully maintained. In rabbits progesterone is necessary, not only for the nutrition of the free blastocyst in utero, but also for implantation (Fraenkel, 1903; Corner, 1928b; Corner and Allen, 1929; Hafez and Pincus, 1956a, b) . Histochemical and quantitative tests have indicated that lipids are present in the endometrium in greater amounts during the luteal phase of the reproductive cycle than at any other time (Krehl)iel, 1937; van Dyke and Chen, 1940; Alden, 1947).

It is clear that the presence of an embryo in the cornu exerts a significant effect on the secretion of luteotrophic hormone and on the functional life of the corpus luteum. How these effects are producecl remains a challenging problem. We need to determine whether direct invasion of the endometrium is essential or whether mere expansion of the embryo can act as a trigger mechanism. Nalbandov and St. Clair (1958) have shown that if plastic beads of more than 2 mm. in diameter are inserted into the cornua on the 8th day of the estrous cycle in sheep, the cycle is significantly lengthened. Denervation of the cornu containing the beads prevented this change in length.

It has been found repeatedly that endometrial sensitivity to the formation of deciduomata is limited normally to the period of implantation and placentation (Loeb, 1908; Allen, 1931; Selye and McKeown, 1935; Krehbiel, 1937; Greenwald, 1958b). The traumatizing substances were physical, chemical, and electrical stimuli. From these studies, three facts were revealed: (1) The formation of the "maternal placenta" can be induced in the complete absence of the blastocyst (Krehbiel, 1937; Mossman, 1937; Dawson and Kosters, 1944). (2) Even though tissue destruction in the endometrium can be brought about by specific and nonspecific stimuli and even though the endresult may appear similar, the mechanisms producing the changes do not necessarily stem from the same basic stimulus. (3) All of the stimuli used are presumed to have as the basis of their action some kind of tissue injury.^ Notwithstanding, the histologic transformations of the deciduomas correspond exactly to those occurring normally in early pregnancy. Krehbiel (1937) found, for example, in the experimentally induced deciduomas of the rat that glycogen and lipids appeared intracellularly in cells which cytologically seemed identical with those of the normal endometrium of pregnancy.


  • The passage of an electric current of sufficient magnitude through the endometrium to induce the decidual response gives no evidence of tissue damage that can be detected microscopically. This of course does not eliminate the possibility^ that cellular injury has not occurred.


It would be interesting to know whether the same intensity of artificial stimulus would induce the decidual response in the uteri of a variety of animals. In the rat, for example, the slightest pressure against the superficial uterine epithelium, at the proper time after ovulation, is sufficient to initiate the decidual response. Thus a bit of lint, small clumps of cells, and glass or paraffin beads the approximate size of eggs effect an endometrial response identical with the response to the normally implanting embryo (Blandau, 1949a). In this species the very earliest changes in the subepithelial stroma begin when the blastocyst is attached only very tenuously to the uterine epithelium (Fig. 14.22). From this response of the endometrium, perhaps localized pressure is sufficient to induce the decidual reaction. Equally impressive is the fact that the decidual response begins before there is any alteration in the superficial uterine epithelium detectable by microscopic means. Thus, any stimulus from living eggs or inert objects within the lumen is transmitted to the underlying stroma directly through the intact lining epithelium. Wimsatt (1944), in describing the earliest phases of implantation in the bat, came to the conclusion that the changes in the epithelium of the pocket into which the blastocyst comes to rest is "an expression of a localized physiologic reaction of the uterus to some chemical stimulus of unknown nature liberated by the ovum, which may produce this effect by acting locally on the epithelium or by inducing a local relaxation in the uterine muscle."



Fig. 11.22. I.MiigiiiKhii.il ^.riinn 1 hrough the antimesometiial wall of a pregnant rat killed on the 5th day. The loosely attached rat blastocyst has initiated the subepitheUal decidual response. There is no detectable alteration in the superficial epithelium. X 450.



It is important to recall again that the destruction and removal of the uterine epithelium by the trophoblastic cells of the rat blastocyst do not begin until the embryo lies deeply within the decidual crypt and a sizable decidual response has been elicited (Alden, 1948). Therefore, the initiation of the decidual reaction and the active invasion of the endometrium by the trophoblast are two distinctly different phenomena separated by a considerable interval of time. In the guinea pig, rabbit, monkey, man, and possibly other mammals, the normal decidual response is not elicited until the embryo has effected the removal of the sujierficial uterine epithelium. Recently, it has been shown that there is a definite species difference in the response of the endometrium to glass or paraffin beads inserted into the uterus of properly timed females (Blandau, 1949a). In the rat, the beads initiated the decidual response and were implanted in a manner similar to blastocysts. In the guinea pig, the beads did not effect the removal of the uterine epithelium, and only occasionally was a minimal decidual response induced. Thus it would appear for the guinea pig, at least, not only that the .stimulus must be a direct one to the underlying stroma but that a certain amount of tissue injury or invasion is necessary before the decidual response can be initiated.


As we suggested earlier, the initiation of the decidual reaction may be the result of a localized pressure exerted by the blastocyst, or of the action of some chemical substance secreted by the egg, which is transmitted to a properly sensitized subepithelial stroma. Recently, Shelesnyak (1952, 1954, 1959a, 19591) I undertook to investigate the nature of the non-specific stimulus required to initiate the deciduomas by determining the effects of histamine and histamine antagonists on the endometrium. He theorized that some degree of injury was a common factor to all methods of uterine stimulation, that a histamine or histamine-like substance was present at the site of injury, and further, that at the time of blastocyst attachment there is an "estrogen surge" which acts to release histamine from the endometrium and which in turn initiates the decidual cell response. Evidence for the role of histamine in deciduoma production also includes the depletion of the mast cell population of the endometrium just before attachment. On this basis, after instilling diphenhydramine hydrochloride or other antihistamines into one horn, both cornua of pseudopregnant rats were stimulated to induce deciduoma development. Definite inhibition of deciduoma was noted in the cornu receiving the antihistamine, particularly if the drug was instilled before the transformation of endometrial cells to decidual cells. Consistent with this finding are the indications from extensive tests that drugs having a specific histamine antagonism are effective in suppressing the decidual cell reaction when introduced into the uterine lumen of rats and mice during pseudopregnancy. On the other hand, antihistamines injected subcutaneously in these animals ordinarily fail to prevent implantation. Species differences must also be considered. Boving (1959) was unable to find mast cells associated with rabbit trophoblast invasion.


The theory that some mechanism of histamine release is responsible for initiating the decidual cell reaction would logically imply that the blastocyst is an active histamine secretor or that it indirectly effects a rise of "free" histamine in the cornua, or interferes with its destruction. In all of the work that has been reported in the attempt to establish histamine as the primary evocator in the decidual cell response and implantation, the blastocyst has been ignored. There has been no attempt to examine the living blastocyst itself and to determine the effects of the various drugs used on it. Consequently, the conclusions drawn as to the failure of implantation are equivocal because the condition of the implanting agent in the experiment has not been evaluated. Also relevant is the fact mentioned earlier that the decidual response in the rat and mouse is evoked, not only by living embryos, but also by many inert objects inducing the response without evidence of epithelial destruction. The mechanism of histamine release under these conditions must be based on some unknown factor.


The appearance of implantation cones, just before and durine attachment of the blastocyst to the endometrium in guinea pigs, rabbits, squirrels, chipmunks, and probably primates, raises the question as to whether the embryo may not initially send protoplasmic extensions between the epithelial cells lining the lumen and thus secrete some substance which not only initiates the decidual response, but also effects the removal of underlying endometrial tissue (compare Figs. 14.23, 14.24 and 14.25) (Mossman, 1937; Wislocki and Streeter, 1938; Boving, 1954, 1959a j. It is interesting that during this initial invasion in the guinea pig, rabbit, and man, there is a negligible amount of endometrial necrosis. In the description of the implantation stages of the macaque, Wislocki and Streeter also emphasized that during the earliest phases of embryonic attachment to the uterine epithelium, the subepithelial mucosa shows no reaction whatever. When joined, these observations remind us that as yet there is no conclusive evidence that the implanting embryo secretes cytolytic enzymes but may secrete other substances.


Fig. 14 23. Section of a guinea pig blastocyst showing the \eiy earliest stage in the attachment of the abembiyonal pole cells to the maternal endometrium. X 500.


Fig. 14.24. The (•.-Illy M.-itic of attacliiucnt of the O-day maca.iuc M im.hxm The .Miihryonic pole is directed towards the uterine epithelium (Wislocki and Streeter, 1938).



Fig. 14.25. A section through an implanting rabbit blastocyst showing an unusually narrow trophoblast invasion of the uterine epithelium. There is no evidence of epithelial debris within the trophoblast cells. A group of clumped uterine epithelial nuclei surrounded by pale cytoplasm lies to the left of the invading foot (Boving, 1959a). Fixation: Sousa, Azan stain. X600.



The most imaginative experimental approach to the problems of embryo spacing, attachment, and implantation is the work of Boving (1959a, b and c, 1961) on the rabbit. He has clearly shown that, in this animal, invasion is promoted by a chemical substance elaborated witliin the blastocyst and transferred to the maternal circulation. The invasion-promoting substance has been characterized as being in the form of bicarbonate which induces a localized high 1)H. Circulating progesterone increases the level of endometrial carbonic anhydrase and accelerates the removal of bicarbonate from the embryo by catalyzing the formation of carbonic acid. The carbonic acid is converted to carbon dioxide which is removed by the maternal circulation. The local pH rises and the various blastocysts' membranes become very sticky, particularly at the site of attachment. The physicochemical interrelationship of the trophoblast and endometrial epithelium effects a dissociation of the epithelium, thus opening a path for the trophoblast.


At this writing, the precise roles of the egg and endometrium during implantaton are unknown and remain a challenging problem. The numerous modifications of the implantation processes in the different mammalian families create difficulties of interpretation in what is already an unusually complex problem. As more detailed descriptions of the embryo-endometrial relationships appear, it seems clear that neither the ovum nor the endometrium is primarily responsible for implantation, but that both play mutual and overlapping roles. One of the greatest gaps in our knowledge of implantation for any animal is a detailed description of the process itself and the precise timing of the events in this phenomenon. The various experimental approaches to the physiologic and biochemical mechanisms of implantation have quickened our interest and broadened our view of the complex metabolic processes required if implantation is to be successful, but our efforts to interpret correctly the data from biochemical, physiologic, and pharmacologic investigations will be limited until more accurate information has been obtained bearing on the morphologic features of the process itself.


V. References

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Pages where the terms "Historic" (textbooks, papers, people, recommendations) appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms, interpretations and recommendations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)
Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex
Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction
The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions
Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation
Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds
Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior


Reference: Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.


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