Fertilization of Mammalian Eggs in Vitro

Fertilization of Mammalian Eggs

in Vitro

C. R. AUSTIN’ Division of E.zprrirrzeatn1 Biology, Nafional Institlife f o r Medical Research, Mill Hill, Lolidon, Eizylaiid P17gC

I. Introduction ........................................... 11. Observatioiis on Nonmammalian Germ Cells . . . . . . . . . . . . . 111. Observations on Mammalian Germ Cells ...... A. Practicability of Fertilization ir, Vitro ........................... B. Specific Studies Associated with Fertilization im Vitro . . . . . . . . . . . . . ..................................... IV. Conclusions ......... References .........................................................

340 345 356 356

I. Introduction I n this review, the word “fertilization” is used to denote the entire process-the entry of the spermatozoon, the formation, growth, and syngamy of the pronuclei, and the union of material and paternal chroniosome groups on the first cleavage spindle. During the course of these events, the egg is said to be “penetrated” or “undergoing fertilization” ; a “fertilized egg” is one that has progressed at least to the nietaphase of the first cleavage mitosis or else is undergoing cleavage. The qualification “in viko” implies that the fertilization process takes place outside the animal body and under controllable conditions in the laboratory. Ideally, the coming together of the germ cells and the union of their nuclei occur under full and continuous observation. Studies made in vitro are complementary to investigations based on the living animal ; potentially, at least, they support a conception of fertilization as a progressive and continuing process, where otherwise it can be known only as a succession of isolated events. Studies in vitro may also be supplementary to those ilz vivo, for the effects can be observed of environmental variations quite impossible to establish in vivo. This article is mainly about mammalian germ cells ; some work involving nonmammalian material is also discussed, but briefly and only for its comparative interest. 11. Observations on Nonmammalian Germ Cells The penetration of the spermatozoon into the egg was first watched by Nelson (1851) in Ascuris and Newport (1853) in the frog. Since then, reports have been published by many other observers, and on the germ cells of many other animals. Some idea of the range of animal species that have provided eggs for direct observations on fertilization may be had from the number of purely marine forms that have been successfully used in the ~~~

1 Present address : Department of Physiology of Reproduction, Physiological Laboratory, Cambridge, England. 337

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Woods Hole area (Costello et al., 1957). The list includes species among the coelenterates, nemertines, annelids, molluscs, echinoderms, enteropneusts, tunicates, and teleosts. In some of these animals, fertilization is normally internal, as in the annelid Platynereis megalops, and the molluscs Ensis directus, Teredo navalis, and Loligo pealii. As a general rule, the fertilization of these animals’ eggs under laboratory conditions involves merely placing eggs arid spermatozoa together, in sen water, in a suitable receptacle. There are, naturally, certain precautions that must be taken if normal fertilization is to occur. For example, the sperm suspension must not be too dense or polyspermy is apt to follow. Often, the eggs as recovered are not ready for fertilization and time must be allowed after they have been placed in sea water for maturation to take place. On the other hand, some eggs, such as those of the mollusc Cmssostrea virginica, rapidly become stale and must be seminated immediately on release if they are to be fertilized. Commonly, it is recommended that contamination of the germ cells with tissue fluids is to be avoided because of their capacity to interfere with fertilization. In some instances, as with Platynereis megalops, no success is had if eggs are diluted with more than an equal volume of sea water, and best results are obtained with “dry” eggs and spermatozoa. Nonmammalian germ cells provide incomparable opportunities for the study of fertilization, and since both eggs and spermatozoa can be obtained in enormous numbers it is not surprising to find that a vast amount of work has been done with this material. Detailed investigations have been possible on the interaction of sperm and egg substances, on the mechanism of sperm penetration, on the immediate reactions of the egg to sperm penetration, on the metabolic changes associated with activation, on the time relations and cytology of fertilization, on the effect of inany chemical, physical, and mechanical factors on sperm penetration and fertilization, and on the possibilities and consequences of fertilization by alien spermatozoa. Nonmammalian germ cells are not, of course, without their shortcomings-there are still many varieties that normally take part in external fertilization but that have not yet been used successfully in the laboratory ; the number of species exhibiting internal fertilization and of which the eggs can he fertilized in vitro is still a very small fraction of the total number with internal fertilization. Nevertheless, this class of material has proved far more amenable to experimentation than its mammalian counterpart, and it is of interest to consider briefly some of the information that has been gathered on problems analogous to those appertaining to mammalian germ cells in vitro. It has long been thought that the often remarkable efficiency with which spermatozoa are able to reach eggs over distances and in an open environ-

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ment must depend upon the release of sperm-attracting substances from rhe eggs. Orientation of sperm motility in this way is known as cheniotaxis and is established as a real phenomenon in certain primitive p!ants, but its existence in the animal kingdom has yet to be demonstrated unequivocally (see Tyler, 19.55 ; Rothschild, 1956). Numerous ingenious experiments have been made, but none appears to have excluded fully the possibility that the observed accumulation of spermatozoa was in reality due to some form of trapping action. A chemical agent of another kind is the component of the jelly coat of echinoderm and other eggs known as “fertilizin,” which can react with a component of the spermatozoon called “antifertilizin.” Tested in vitro with extracts of jelly coat or with “egg water,” spermatozoa become agglutinated to each other, and the same thing happens in the vicinity of eggs; the agglutination is not permanent, but when the spermatozoa become free again they are found to have lost their fertilizing power. Under normal conditions, in the sea, the function of the fertilizin-atitifertiliziti reaction is possibly to ensure attachment of spermatozoa to the egg surface as a preliminary to penetration. The reaction has a high order of species specificity and may in some instances help to prevent cross fertilization. Full information on fertilizin and antifertiliziti is given in reviews by Metz (1957, 1961). Another result of the approach of spermatozoa to eggs is the acrosonie reaction. This can involve, in different species, one or both of two processes: the release from the sperm head of an egg-membrane lysin and the development of an acrosome filament. Lysins can be extracted from spermatozoa and their ability to dissolve certain egg membranes supports the idea that their function is to assist sperm penetration through the membranes. The acrosome filament which often takes the form of an extremely long thin thread, capable of extending across the full thickness of the egg jelly coat, seems clearly to arise from a structure normally covered by an overlying part of the acrosome which breaks down in the initial phase of the reaction. The filament makes contact with the egg cytoplasm ahead of the rest of the spermatozoon and its function seems to be concerned with the final step in penetration, the passage into the cytoplasm, but the precise mechanisms involved are unknown. Further data are given by Dan (1956) and Colwin and Colwin (1957). I n many species, contact of the sperm head or of the acrosonie filament with the vitellus evokes a receptive reaction on the part of the egg, taking the form of a localized elevation of the cytoplasm. This is the “fertilization cone” and it assumes a variety of different shapes in different species, ranging from a small simple hillock to a high and complex flamelike structure. Sometimes its full development is reached only after the sperma-

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tozooti has disappeared into the vitellus, but, where an acrosome filament is involved, it grows out around the filament to meet the approaching spermatozoon, and subsides as the spermatozoon is engulfed (see Wilson, 1928 ; Colwin and Colwin, 1957). Somewhat later structural reactions of the egg to sperm entry are the emission of one or two polar bodies (depending on the state of maturation of the egg) and the contraction of the vitellus. These are not specific reactions to sperni penetration, however, but denote rather the occurrence of activation. Activation provoked by stimuli other than that of sperm penetration can lead to far-going parthenogenetic development. Eggs of a variety of animals have been activated by many kinds of artificial stimuli including heat, cold, acids, bases, hypo- and hypertonic solutions, alkaloids, fat solvents, bile salts, soaps, ultraviolet radiation, radium emanations, mechanical agitation, and electric currents (see Tyler, 1955 ; Austin and Ilralton, 1960). In few nonmammalian animals can the course of fertilization, involving the development of pronuclei, be observed with ease in the living egg, owing to the large numbers of granular or vesicular elements and other inclusions within the cytoplasm, but it is often possible to make out the general form of the pronuclei and the paths taken in their movements through the cytoplasm, and to determine fairly accurately the time of their union. Cross fertilization between the germ cells of a large number of different species is possible, generally between closely related forms, but even interphyletic crosses are known. In other instances, sperni penetration fails altogether or, if it does occur, the male pronucleus is not formed or regresses at some stage in the course of fertilization. The chances of penetration by alien spermatozoa can often be increased by various means : making the medium more alkaline or altering its concentration, removing the jelly coat, treating the eggs with trypsin, leaving the gametes for a longer time together, or using abnormally high concentrations of spermatozoa. [See reviews by Wilson (1928) and Rothschild ( 19%) .]

111. Observations on Mammalian Germ Cells in ydr0 A. PRACTICA4BILITY O F FERTILIZATION 1 . The Dificulty of Finding Appropriate Conditions Eggs have been recovered from the follicles or Fallopian tubes of u11mated rats, mice, and rabbits, and transferred to the Fallopian tubes of mated or artificially inseminated animals for fertilization i n vizw (Chang, 19.52, 195313, 1955a,b; Xoyes, 1952: Runner and Palm, 1953 : Lin ~f nl., 1957; Sherman and Lin, 19.58, 1959). The results of these experiinents

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show that eggs can tolerate hours of exposure in vitro to temperatures down to freezing point and even short periods of vitrification, and can endure suspension in simple salt solutions, or solutions containing glycerol, without losing their capacity for normal fertilization in vivo. With such a robust nature, eggs should certainly be capable of undergoing fertilization in vitro in any of the common physiological media; perusal of published reports indicates, however, that this has not proved to be so (see the next section), and the high rate of failure suggests that the process of fertilization must depend upon rather special experimental conditions. Eggs iron: mated rats, mice, and rabbits have been recovered while undergoing fertilization and have then been maintained in culture for different periods (Lewis and Gregory, 1929a,b ; Defrise, 1933 ; Smith, 1949, 1953). The rabbit eggs developed well, progressing as far as the blastocyst. Mouse and rat eggs were also able to complete their fertilization in vitro and pass through one or two cleavage divisions. Evidently, fertilization begun in viva is not likely to be halted by conditions encountered in recovery and culture. Further indications that this is so are provided by the results of experiments to be described in Section 111, B, 4, Cytology of Fertilization, which show that phases of the fertilization process, beginning as early as the entry of the spermatozoon into the vitellus, are resistant to a highly abnormal environment in vitro. These two sets of data indicate that well-recognized methods are available for keeping eggs in vitro in a fertile state and also for maintaining in vitro the process of fertilization, once this has been initiated. The central problem in the study of fertilization in vitro, therefore, concerns the conditions required for initiation, or, in other words, the conditions needed for obtaining the petletration of the spermatozoon into the egg in vitro. The likely nature of these conditions is considered later, in Section 111, B.

2. The Dificidty of Fipzdiiag Appropriate Criteria Establishment of the practicability of fertilization in vitro has taxed the ingenuity of many investigators. There are two main reasons for this: (a) the membranes and investments surrounding eggs so interfere with direct observation that no one has yet been able to make a satisfactory record of actual sperm entry; and (b) so many of the cytological features of fertilization can be evoked by influences other than sperm entry. ( a ) Fertile eggs of the rodents and the rabbit are still surrounded by dense masses of follicle cells when recovered from the Fallopian tube, and spermatozoa can be clearly seen moving among these cells only if the cell niass is well compressed between cover glass and slide. The requisite degree of compression grossly deforms the egg and often hreaks it. Even

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if care is taken not to break the egg, the considerable stretching of the zona pellucida that occurs may well preclude its penetration by the sperniatozoon. There is evidence that spermatozoa iia vivo can penetrate into denuded eggs (Austin, 195la; Chang, 1953b) arid so it night prove possible to observe the course of sperm entry in vitro after the eggs have been stripped of follicle cells by treatment with hyaluronidase (aided hy teasing, for the rabbit egg). Difficulties of direct observation have driven workers to adopt a post Izoc procedure, whereby several hours are allowed for penetration to occur and the eggs are then examined for contained spermatozoa. In denuded living eggs, recovered from mated animals, any spermatozoa in the perivitelline space can be seen quite clearly. Rabbit eggs normally display several to numerous perivitelline spermatozoa, at least with fertilization in vlvo, but the other laboratory animals are not so accommodating: oidy about 20% of rat and mouse eggs, and extremely few golden hamster eggs, normally have supplementary spermatozoa, and the fertilizing sperniatozoon spends only about one-half hour in the perivitelline space on its way into the vitellus (Odor and Blandau, 1949; Braden et al., 1954; Austin, 1956b ; Austin and Braden, 1956 ; Strauss, 1956). The presence of perivitelline spermatozoa constitutes the most readily demonstrable evidence that sperm penetration has occurred and that the eggs are probably undergoing fertilization-provided, of course, that experimental treatment has not been such as to cause breaks in the continuity of the zona pellucida, which appears to have happened in some investigations (Shettles, 1953). I n eggs fixed and examined histologically, it is possible to discern spermatozoa in the vitellus as well as in the perivitelline space, but special care must be taken to avoid artifacts. An important hazard lies in the ease with which sperm heads can be moved from one part of a histological section to another, either in the cutting of the sections or during the staining and mounting procedures. This kind of artifact has received particular attention from Vojtiskova (1956). Sperm heads may thus come to lie upon some part of the section of the egg, within the area defined by the zona pellucida, and be interpreted as heads of penetrating spermatozoa. Judging from appearances presented by the published photographs, several authors seem to have been misled by such artifacts (for example, Yamane, 1935 ; Moricard, 1949 ; Moricard and Bossu, 1949a). [The pictures published later by Moricard ( 19S4b) showed perivitelline spermatozoa in whole unfixed eggs and were much more convincing, despite the author’s practice of touching up his photographs.] Another difficulty with histological material is that of distinguishing the sperm head, which is often the only part of the spermatozoon visibly stained, from other mall dense

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basophilic bodies which have been described in the eggs of several animals (see Kremer, 1924). Staining by the Feulgen method would no doubt overcome this trouble, though confusion could still arise with the nuclei of leucocytes which are prone to enter eggs in culture (Dauzier and Thibault, 1956j . (b) The more notable changes that occur in eggs as a result of sperm entry include the contraction of the vitellus, the expulsion of the second polar body, the formation of two pronuclei, and the initiation of cleavage, but none of these changes individually is specific to fertilization. They are in fact the result of activation ; this can take p!ace “spontaneously” (when the stimulus is not known) or be attributab!e to various recognizable stimuli of which sperm entry is only one form. One qualification should, however, be made : excluding the occurrence of rare abnormalities, expulsioii of the second polar body associated with development of two pronuclei is possible only after sperm penetration. Activation by other stimuli constitutes the start of parthenogenesis and in this process the formation of two pronuclei depends upon suppression of the second polar body; if the polar body is emitted, only a single nucleus develops. Suppression of the second polar body does not necessarily lead to formation of two pronuclei ; if the second meiotic division is suppressed at a sufficiently early stage, all the chromosomes become housed within a single nucleus. Spontaneous activation (as distinct from fragmentation) is most uncommon in rabbit, rat, and niouse eggs in o h o , but has been observed in as many as 80% of hamster eggs (Austin, 1956a; Chang and FernandezCaiio, 1958j . Even in the hamster, however, development seemed most unlikely to pass much beyond the 2-cell stage. Cold shock (subjection of the eggs to a temperature near 0” C.) causes activation in most rat eggs but not in mouse eggs (Thibault, 1949; Austin and Braden, 1954b; Xraden and Austin, 1 9 5 4 ~ ) .Mouse eggs, by contrast, are activated by heat shock (41 to 43” C.) . Kabbit eggs can be activated by culture in vitro and especially by heat shock, and by treatment in vifro with hypo- and lippertonic solutions and solutions of butyric acid (Pincus, 1930, 1936a ; €’incus and Enzmann, 1934 ; Thibault, 1919 ; Smith, 1949j . Probably, there are many other agents that would have this effect, to judge from the wide range of stimuli that have been shown to activate nonniammalian eggs. Artificially activated rat and mouse eggs often reach the 2-cell stage and sometimes the 4-cel1, but seem rarely to develop further (Austin and Braden, 1 9 5 4 ~ ;Braden and Austin, 1 9 5 4 ~ ) . Kabbit eggs, on the other hand, can form nornial-looking blastocysts and even implant (Thibault, 1949 ; Chang, 1954, 1957h). Pincus and Enzmann (1935, 1936) claimed that sonie rabbit eggs activated in vitro and transferred to host animals

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are capable of completing embryonic development and giving rise to normal young, but this work has yet to be confirmed. The parthenogenetic rabbit blastocysts that have been specifically examined were found to be diploid (Chang, 1954j , their regulation to diploidy probably involving suppression of the second meiotic division and formation of a single nucleus. Beatty (1957), who deals in detail with the possibilities and mechanisms of parthenogenesis, points out that there is no reason why spontaneous diploid parthenogenones could not occasionally be born and survive; the fact that they are so far unrecorded may inerely reflect the difficulty of their recognition. They could be normal functional females, but not iiornial males, since the niamnialian egg contains no male-determining ( Y ) chromosome. From the foregoing, it is obvious that in any instalice of presumed in vitro fertilization, great care is necessary to eliminate the possibility of partlienogenesis. The need for caution is further indicated by the finding of Dauzier and Thibault (1956) that evidence of activation (without sperm penetration) was shown by more eggs when they were incubated in the presence of spermatozoa, especially when these had been recovered from the uterus of a rabbit mated 12 hours previously, and could be assumed to be capable of penetration. If it has not been found possible to establish sperm entry and the formation of a male as well as a female pronucleus, subsequent cleavage of eggs in culture or development after transfer to a host cannot in themselves serve as alternative proof of fertilization. Nevertheless, if young are born and some of these are males, parthenogenetic development (of the males, at least) is automatically excluded. Venge (1953) reported some males among the young born in his experiments, but, for the reasons given in the next paragraph, it is unlikely that the young developed from eggs fertilized in vitro. Of interest in connection with the risk of parthenogenetic development with studies on fertilization in vitro is Smith’s ( 1949j observation that cleavage of unfertilized rabbit eggs in culture is much less common if the eggs have been incubated for a few hours with scrapings of Fallopian-tube mucosa. IVith the transfer of eggs to a host after treatment with spermatozoa in vitro, further difficulties are introduced. Spermatozoa can remain attached to the outside of the eggs, despite vigorous washing, and effect their entry when the eggs reach the Fallopian tubes of the host. The host animal must be brought into a suitably receptive state if the transferred eggs are to implant. This is done (in the rabbit, which has been the esperimental animal of choice in this work) by inducing ovulation in the host by mating it with a vasectoniized male or by hormone injection. Vasectomy operations are not always properly done, so that it is possible for spermatozoa from an operated male to fertilize the host’s eggs, and

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the resulting embryos (or young born) are mistakenly interpreted as deriving from the transferred eggs. Hornione injection eliminates this possibility, but there reinailis the risk that Spermatozoa inadvertently introduced with the transferred eggs fertilize the host’s eggs. At least some of the young born in the experiments of Pincus and Enzmann (1934) and of Venge (1953) almost certainly arose from eggs fertilized in vlvo under one or other of these circumstances. On the other hand, Chang (1959) seems to have avoided these pitfalls ; he induced ovulation by hormone injection, both in the donor and the host, and cultured the eggs for 18 hours after semitiation (by which time they had reached the 4-cell stage), The eggs were transferred to hosts 12 hours after ovulation had been induced in these animals. I t is conceivable that the spermatozoa might have retained their fertilizing capacity for 22 hours ( 4 - h o ~ rseniinatioii period plus 1 s hours culture) and have fertilized the host’s 12-hour-old eggs ; but this explanation is unlikely and is inconsistent with the fact that the coat color of the young showed no influence from the host. Clearly, experiments on the fertilization of eggs in vitro (Table I ) need to be very carefully planned if they are to yield unequivocal results, and, because this was not fully appreciated earlier, it is only some of the work of the past decade that can be regarded as providing good evidence for the practicability of the procedure with mammalian material (Smith, 1951 ; Moricard, 1954a,b; Dauzier et d.,1954; Dauzier and Thibault, 1959; Thibault et al., 1954; Thibault and Dauzier, 1960; Chang, 1959). Nevertheless, many of the reports, including earlier ones, yielded useful data ; in addition, there have been investigations on eggs and spermatozoa in vitro that were not designed with the object of obtaining fertilization but which have added to knowledge in this sphere of gametology. It s e e m most profitable to consider here any work that helps to throw light upon problenis attending the process of fertilization in vitro.

B. SPECIFIC STUDIES A s s o c i ~ ~ rWITH m FERTILIZATION in Vitro 1. Capacitation of Spermatozoa Before 1951, it was customary to treat eggs in vitro with either ejacu-

lated or epididymal semen and expect fertilization to occur. Then evidence was produced to show that, at least in vivo, spermatozoa had to undergo some kind of physiological change within the female genital tract before they were capable of passing through the zoiia pellucida of eggs (Chang, 1951a; Austin, 1951a). This observation was well supported by further studies on conditions in vivo (Austin, 1952; Noyes, 1953; Austin and Braden, 1954a ; Chang, 1955c, 1957a, 1958 ; Strauss, 1956 ; Noyes et ad., 1958) ; the need for capacitation was demonstrable in the rabbit, rat, and hamster (but not in the mouse; Braden and Austin, 1954b),

TABLE I REPORTSOK EXPEHIMEKTS DESIGNED TO OBTAINTHE FERTILIZATION OF MAMMALIAN EGGSin Vitro Authors Schenk (1878)

Test species

Type of eggs

Type of spermatozoa Epididymal

Observations Polar-body cleavage

emission

Notes

and

Eggs in follicular fluid and uterine mucus or 011 piece of uterine mucosa

Guinea pig and rabbit

Follicular

Onanoff (1893)

Guinea pig and rabbit

“Uterine”

Long (1912)

Rat

Tuhal

Epididymal

Polar-body emission

Yarnane (1930, 1935, 1937)

Rabbit, rat, and horse

Tuba1

Ejaculated and epididymal

Sperms in eggs, polar-body emission

Sperms of all three species said to be effective on rabbit eggs

I’incus (1930, 1936a, 1939) ; Pincus and Enzmann (1934, 1935, 1936)

Rabbit, rat, guinea pig, and man

Follicular and tuba1

Ejaculated and epididymal

Sperm entering, sperms in eggs, shrinkage of vitellus, polar-body emission, pronuclei, cleavage, birth of young

Rabbit eggs used throughout

Froinmolt (1934)

Rabbit

Icrassovskaja (1934, 1935a) ; Krassovskaja and Diomdova (1934) ; Diotnidova and Kusnetzova (1935)

Rabbit

Krassovskaja (19331)

Rabbit, rat, guinea pig, and dog

Rock and Menkiti (1944) ; Menkin and Rock (1948)

Man

Developing embryos

-

Only conclusions published

Shrinkage of vitellus

Epididymal

Polar-body emission, shrinkage of vitellus, pronuclei, cleavage

Tuhal

Epidid ymal

Polar-body emission, pronuclei, cleavage

Follicular

Ejaculated

Sperms in eggs, shrinkage of vitellus. cleavage

Rat and guinea pig spermatozoa, but not dog spermatozoa, said to fertilize rabbit eggs

T A B L E I (Continued) Authors

Test species

Type of eggs

Type of spermatozoa

Observations

Notes

Moricard (1949, 1950, 1954a, b ) ; Moricard and Bossu (1949a)

Rabbit

Tubal

Ejaculated and uterine

Sperms in eggs, shrinkage of vitellus, pronuclei, cleavage

Ejaculated sperms added to eggs in Fallopian tube in vitro; uterine sperms added to eggs in watch glass undcr oil

Smith (1951, 1953)

Rabbit

Tubal

Ej aculatcd

Sperm entering, sperms in eggs, pronuclei, cleavage

Scrapings of Fallopian-tube mucosa in medium

Shettles (1953)

Man

Follicular and tubal

Ejaculated

Sperms in eggs

Fragments of Fallopian-tube mucosa in medium

Venge (1953)

Rabbit

Follicular and tubal

Ejaculated

Birth of youug

Dauzier et al. (1954) ; Thibault et a1. (1954) ; Dauzier and Thibault (1956, 1959) ; Thibault and Dauzier (1960)

Rabbit and sheep

Tubal

Uterine

Sperms in eggs, shrinkagc of vitellus, polar-body emission, pronuclei, cleavage

Eetter results when uterine sperms recovered in thc mucus than when flushed out of uterus, and whcn eggs washed before semination

Chang (1959)

Rabbit

Tubal

Uterine

Sperms in eggs, pronuclei, cleavage, birth of young

The best supported case for fertilization irt vitro

-

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and it was shown that capacitatioii could occur, in part at least, in organs other than the genital tract. Support was also forthcoming from reports on fertilization in vitro, authors maintaining that success was obtained with spermatozoa recovered from the Fallopian tubes or uterus but not with spermatozoa from freshly ejaculated semen. All this tended to throw serious doubt upon claims that sperm penetration in zhro occurred with the direct use of epididymal or ejaculated semen and these make up the majority of the reports (Table I ) , Categorical denial of the claims is unjustified, however, for possibilities exist that have not been properly investigated. The design of some of the experiments involved the presence in the medium of follicular, tubal, or uterine secretions, or of fragments of Fallopian tube (Schenk, 1878; Smith, 1951, 1953; Shettles, 1953), and it cannot be denied that under these conditions capacitation of some spermatozoa might have occurred. Noyes et al. (195S), working with fertilization in vivo, obtained evidence that some capacitation could take place in the excised uterus of the rabbit, and there was a suggestion of its occurrence when spermatozoa were incubated in a saline solution in which strips of uterine endometrium were suspended. Neither these observations nor those of Schenk, Smith, and Shettles, were conclusive in themselves but, taken together, they offer an indication that spermatozoa might undergo capacitation in vitro, in the presence of tubal or uterine secretions, and in the absence of living tissues. The possibility certainly deserves further study. Observations on the likely mechanism of capacitation are discussed at the end of the sectiou on hyalurotiidase (p. 349).

2. Sperwz and Egg Interacting Substances

a. Chentotaxis. No new data on this problem have been obtained in any of the investigations on the fertilization of mammalian eggs in vitro, but Schwartz et al. (1958) have stimulated some interest with their work on human spermatozoa. They found that the spermatozoa tended to gather in regions containing high concentrations of ovarian cyst fluid or of egg white, in spite of increased motility which was also provoked by these substances. They give reasons for discarding explanations other than that involving chemotaxis. Implications that cheinotaxis of spermatozoa might be a normal process in inanimals arise also in recent studies in vivo: Braden (1959) pointed out that the observed spatial distribution of spermatozoa about the eggs in mice could be explained by invoking chemotaxis, and this explanation could also be offered in genetic studies on peculiarities of the segregation ratio at the T locus (Braden, 1960). If some kind of chemotactic effect does, in fact, exist, it may well be

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difficult to demonstrate clearly owing to interference from other agents that mammalian eggs seem to release (see p. 351, “Fertilizin”). b. Hyaltrronidasc. Schenk (1878) placed follicular guinea pig and rabbit eggs on a glass slide in a drop of follicular fluid to which some uterine mucus was added, or on the mucosal surface of an excised piece of uterus, maintaining the preparations at body temperature and taking precautions against evaporation. H e observed that when a small drop of a suspension of epididynial spermatozoa was deposited beside the eggs the surromiding mass of follicle cells broke up and the cells became dispersed by the movements of the spermatozoa. Some of the eggs retained a layer of adherent cells while other eggs were denuded; Schenk considered that only the denuded eggs were mature and these he used for his attempts to obtain fertilization in vitro. Schenk’s observations on the breakup of the follicle-cell mass by sperm suspensions have been confirmed on innumerable occasions since that time, and it is now common knowledge that the effect is due to the solvent action of free hyaluronidase upon the gelatinous matrix of the follicle-cell mass. It soon came to be assumed that denudation of eggs was a necessary preliminary to their fertilization and, as a corollary, that large numbers of spermatozoa must normally be present about the eggs for sufficient hyaluronidase to be released to produce this effect. This led to the use of comparatively dense suspensions of spermatozoa by workers wishing to procure fertilization in ztitro. Indeed, Pincus and Enzmann (1934) maintained that the proportion of eggs that could be fertilized in v i t r o was directly related to the concentration of spermatozoa in the medium. Then Lewis and Wright (1935) reported that rat eggs recovered from mated animals had been penetrated by spermatozoa, although the enveloping follicle-cell mass was apparently quite intact. Evidently, under normal conditions, breakup of the cumulus does not have to precede sperm entry into the egg and spermatozoa are able to pass through the investment individually ; this has since been confirmed in the rat and rabbit (Leonard et al., 1947; Austin, 1948a; Moricard and Bossu, 1949b, 1951 ; Bowman, 1951: Chang, 1951b; Odor and Blandau, 1951). It was found, too, that very few spermatozoa could be recovered from the site of in vizio fertilization : usually less than a hundred in the rat, mouse, and hamster (Austin, 1948b, 1956b; Blandau and Odor, 1948, 1949; Moricard and Bossti, 1951; Braden and Austin, 1954a), a few hundred in the field vole (Austin, 1957) and of the order of a thousand in the ralhit (Austin, 194%; Moricard and BOSSU,194913; Chang, 1951b; Braden, 1953) and sheep (Braden and Austin, 1954a), Appropriately, the trend in experiments on fertilization in vitro in the last decade has involved the use of much lower concentrations of spermatozoa than those employed formerly. Smith ( 19511 added

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“a minute quantity” of semen to her preparations. Suspensions of uterine spermatozoa obtained by flushing or direct sampling (Table I ) are unavoidably comparatively dilute. Chang remarked that the proportion of penetrated eggs found was not related to sperm concentration. Two further points of interest in Schenk’s (1S78) work are ( a ) that he regarded the failure to become denuded as a mark of immaturity in an egg, and (b) that denudation of rabbit as well as guinea pig eggs took place in the presence of uterine secretions. ( a ) Failure to lose the follicle-cell investment seenis quite likely to be related to the greater density of packing of follicle cells that has been observed to characterize some freshly ovulated eggs in the rat and mouse and which is thought to delay sperm penetration until, through some kind of ripening process, the follicle cells become less closely adherent to the zotia pellucida (Austin and Braden, 1954a ; Braden and Austin, 1954b ; Braden, 1958, 1959). Blandau (1959) reported that, when the freshly ovulated rat cumulus is maintained for several hours in vitro, follicle cells can be observed to migrate actively out of the matrix. It can be inferred that rat and mouse eggs are more suitable for studies on fertilizatioii in vitro if recovered after the lapse of 3 or 4 hours from the time of ovulation. (b) Rabbit eggs in vitro generally cannot be denuded by treatment with sperm suspensions or hyaluronidase solutioiis alone. Swyer ( 1947) showed clearly that, while treatment of rabbit eggs with solutions of hyaluronidase caused the breakdown and removal of most of the folliclecell mass, the immediately surrounding cells remained attached ; these were soon dislodged, however, if the eggs were returned to the Fallopian tube. The “tubal factor” was thought to be associated with the activity of cilia with which the mucosa is richly provided. Bradeii (1952) found that the closely adherent follicle cells could be removed in zdro by vigorously propelling the eggs with their suspending medium into and out of a fine pipet. The iinplication from these two reports is that some form of niechanical action is needed. However, Smith ( 1949j found that if unfertilized rabbit eggs surrounded by follicle cells were incubated for 2 or more hours with scrapings of Fallopian-tube mucosa, the follicle cells usually became detached from the zona pellucida ; in the absence of the scrapings, a layer of cells remained about the eggs. Later, Smith (1951 j and Shettles (1953) noted that rabbit and human eggs, respectively, are denuded by treatment with sperm suspension when small pieces of Fallopian tube or of tubal niucosa are present in the medium, but not when these tissues are lacking. There is evidence, therefore, that the tubal factor resides in a component of the tubal secretions, rather than in mechanical action of any kind, and that it can work with the hyaluronidase released by spermatozoa to

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produce the complete denudation of the eggs. Denudation, as already noted, is not a requirement for sperm penetration, but the observed action of the tubal factor may well reflect some change of a lesser degree that is necessary before the spermatozoa can pass through. Smith ( 1953) stated that without Fallopian tube in the preparation she never found spermatozoa within the bounds of the zona pellucida. Possibly a similar tubal factor is responsible for the changes observed in the density of packing of the follicle-cell mass in freshly ovulated rodent eggs, though this can perhaps be adequately explained as the consequence of migration of follicle cells. It was stated at the beginnitig of the previous section that capacitation enabled the spermatozoon to pass through the zona pellucida-this was the first effect for which evidence was obtained. Since then it has been found that, in those species in which the acrosome can readily be seen, the morphological concomitant of capacitation is a change in the optical properties of this structure, leading to its elevation and ultimate loss from the sperm head. Observations also indicate that the visible changes are associated with release of hyaluronidase from the acrosome. If appropriate precautions are taken, it can be demonstrated in vitvo that sperniatozoa obtained from epididynial or ejaculated semen are unable to penetrate the gelatinous matrix of the cumulus, whereas spermatozoa from the Fallopian tube penetrate readily. It is therefore concluded that capacitation has a twofold effect : ( 1 alteration of the acrosome, releasing hyaluronidase and thus enabling the spermatozoon to pass through the cumulus, and (2) removal of the acrosome, thus exposing the perforatorium, which is apparently the organelle directly concerned in sperm penetration of the zona pellucida (Austin and Bishop, 195Sa,b; Austin, 1960, 1961). c. “FPvtilr’zin.” Bishop and Tyler (1956) observed that when eggs and spermatozoa were placed together in vitro the spermatozoa became agglutinated, head to head, far more often in the vicinity of the eggs than further away. The effect spread out from the eggs with the passage of time. An agglutinating solution could be prepared by incubating eggs in acid or hypertonic saline solutions ; the agglutinating agent could be inactivated 11y treatment with sodium periodate solution. No effect was seen when mucin-coated rabbit eggs were used. Tests were made with rabbit, mouse, arid cow eggs, and rabbit, mouse, bull, and human sperniatozoa : some indication of species specificity was detected in cross tests. The authors considered that their data showed the presence of a substance similar to the fertilizins of many nonmammalian eggs ; they believed that it emanated from the zona pellucida. The nornial function of the agent was thought to be concerned with the attachment of the spermatozoon to the zona pellucida and with its penetration through that membrane.

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The main implication in this work for studies on fertilization in vitro seems to be that the eggs to be seminated should be washed before use and not suspended in too small a volume of medium; otherwise the concentration of “fertilizin” in the medium may be enough to agglutinate all spermatozoa before they reach the eggs. Among nonmammalian gametes, spermatozoa agglutinated by “fertilizin” lose their ability to become attached to and enter eggs, even though they may become free and still be vigorously motile. A mammalian “fertilizin” has also been reported by Thibault and Dauzier ( 1960). They found that the frequency of sperm penetration into rabbit eggs in vitro was much increased if the eggs were held iii vifro for 2 to 3 hours, or else washed several times, before semitiation with uterine spermatozoa. Alternatively, equally good results could be obtained by seminatiiig with an undiluted suspension of uterine spermatozoa, used as recovered in uterine mucus. The authors infer that an agent, which they term “fertilizin,” is released by the freshly ovulated eggs and has the effect of inhibiting the fertilizing ability of spermatozoa, without actually agglutinating them (actions similar to those of some invertebrate fertilizins) . The secretions of the female genital tract are held to oppose or neutralize the “fertilizin.” An “antagglutin” which counteracts or abolishes the spontaneous headto-head agglutination that spermatozoa undergo in various circumstances has been described in the secretions of the female genital tract (Lindahl a i d Kihlstrom, 1952; Liiidahl and Nilsson, 19.54; Lindahl et a)., 39S6; Ingelinan-Sundberg and Lindahl, 1958 ; Lindahl, 1960). Whether it is this or a different agent that opposes Thibault and Dauzier’s “fertilizin” is not known.

3. Sperm Penetrution nizd the Iiwzediate Reactions of the Egg I t might have been expected that studies on fertilization in v i f r o would have been an important source of data on sperm penetration and the immediate reactions of the egg. This has not as yet proved to be so, however, chiefly because of the difficulties of observation already mentioned, particularly under conditions likely to favor survival and fertilization of eggs. Pincus (1930, 1936a) claimed to have seen the actual entry of a spermatozoon into a rabbit egg, and described at the point of entry the elevation of part of the cytoplasm, resembling the “fertilization cone” well known in nonmammalian eggs. [Observations on fertilization in vivo in the mouse, rat, and rabbit indicate that a cytoplasmic elevation, probably analogous to the fertilization cone, is formed at the point where the sperm head sinks into the vitellus (Sobotta, 1895 ; Lams and Doorme, 1908;

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Pincus and Enzmann, 1932; Austin arid Braden, 1956) .] Smith (1953) also recorded that she had seen the spermatozoon go through the zona pellucida and into the vitellus in the rabbit egg; she remarked that the process seemed to be a good deal slower than it is in vivo, and that the difference may have been attributable to the experimental conditions. Eggs recovered soon after ovulation in mated rats, mice, and hamsters sometimes display the fertilizing spermatozoon with its head attached to the surface of the vitellus and still within the perivitelliiie space. The last phase of penetration-passage through the vitelline membrane-can then be observed in vitro. In all the recorded instances, the spermatozoon head lay flat upon the vitellus and attachment involved chiefly the anterior region of the head, possibly implying that attachment is first made by the perforatorium ; residual motility of the spermatozoon showed that the posterior part of the head could move independently of the vitelline cytoplasm whereas movements of the anterior end were associated with corresponding movements of cytoplasmic granules. Progressively the whole head becomes attached to and passes through the vitelline surface which closes over it without any sign of a break in continuity, and the midpiece becomes gradually absorbed in the same manner (Austin, 1951b, 1956b; Austin and Braden, 1956). Dauzier and Thibault (19%) noted that in rabbit eggs seminated in vitro the sperm head comes to lie flat upon the vitelline surface and enters the vitellus from this position. Polar-body emission and shrinkage of the vitellus have been reported by several investigators to occur soon after semination in vitro (see Table I) though it is likely, in some of these instances, that the changes should have been ascribed to artificial activation and not to that resulting from sperm entry. Even in those experiments in which sperm penetration may be held responsible, actual entry was not seen and detailed observations on the structural changes, and on associated features such as time relations, were not made.

4. Cytology of Fertilization Most investigations on fertilization in vitro have been made with the

rabbit egg, in which the vitellus is optically unsuitable for the study of internal detail in eggs in the fresh state; human, sheep, dog, and guinea pig eggs suffer from the same drawback. T o be sure, the eggs can be fixed and examined histologically, but this would be to neglect one of the main advantages sought from work in vitro, namely the opportunity to make continuous observation of changes occurring in living cells. Despite the difficulties with rabbit eggs, Smith (1951) noted that, with the aid of phase-contrast microscopy, the sperm head and the developing male pronucleus, as well as the female pronucleus, could be seen within the vitellus.

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More eggs examined in the early stages showed the male pronucleus than both pronuclei, and so it can be inferred that the male pronucleus of the rabbit egg develops before the female. This is consistent with observations on rabbit eggs fertilized in vivo (Pincus and Enzmann, 1932) and distinguishes the rabbit egg from that of the rat in which generation of pronuclei is approximately synchronous (Austin, 195lc) , Rat eggs are much better for study as living material, but they have evidently proved very refractory: the only claim to have obtained the fertilization of rat eggs in zritro is that of Long (1912) who noted no more than breakup of the follicle-cell mass and emission of a polar body. Some of the difficulties with rat eggs can be avoided by adopting a compromise: eggs recovered a few hours after ovulation from mated rats often contain spermatozoa that have passed through the zona pellucida and have either become attached to the surface of the vitellus or have just passed through the vitelline surface, and these eggs can be used for the study in vitro of the remainder of the fertilization process. When the eggs are set up on a slide and partially compressed so that internal strnctures are clearly visible, the fertilization changes continue for a period despite the abnormal environment (Austin, 1950). Best results are obtained when the eggs are dissected from the Fallopian tube under a layer of liquid paraffin and transferred to a slide in the fluid that emerges with them from the tube. Some paraffin should be transferred too, as this surrounds the droplet of tuba1 fluid on the slide and prevents evaporation. Observations are best made in a warm box at 35 to 37" C. Ry this means, it was found possible to ohserve clearly and at high magnification (2-mm. phase-contrast objective) the passage of the sperm head through the vitelline surface, the metamorphosis of the sperm head into a male pronucleus, the course of the second meiotic division from metaphase onwards and including the rotation of the spindle, the initial development of a fissure which would have been responsible, in the uncompressed egg, for the formation of the polar body, and the nietatnorphosis of the egg chromosome group into a female pronucleus (Austin, 1951b). Phases in the growth of the pronuclei could also be studied and continuous observation was possible of the progress of syngamy-the diminution of the pronuclei and their replacement by chromosome groups which became arranged as the metaphase plate of the first cleavage spindle. [One series of photographs illustrating the course of syngamy appeared in the report just referred to, and another series was published later (Austin and Walton, 1960),] Under the conditions of the experiments, the first cleavage division would proceed to late anaphase or early telophase, but cytoplasmic division of the egg was evidently precluded. These observations were set on record by taking photographs at ap-

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propriate intervals with a plate camera. A better record could have been made by the preparation of a time-lapse cinematographic film, but this was not attempted. A film of this kind, showing tlie development of a male pronucleus from the sperm head has been prepared by R. J. Blandau of Seattle. The egg was not much compressed in this study, nor was high microscopic magnification employed, so that only the grosser morphological features were discernible, but tlie iiioving picture provided a much fuller conception of pronucleus formation as a living process. More recently, diniinution of pronuclei in the ear'y stages of syngamy has been recorded for the hamster egg as it occurred in vitro (Ohnuki,

1569).

5 . Heterologotts Fertilization The effects on eggs of treatment with alien spermatozoa in vitro have heen studied many times and Yatiiatie (1930), Krassovskaja ( 1935b), and Pincus ( 1939) maintained that penetration or fertilization by alien spermatozoa could occur. Yamane tested rat and horse spermatozoa on rabbit eggs, but his only evidence for penetration was the emission of the second polar body. Krassovskaja reported tlie results of semitiating rabbit eggs with rat, guinea pig, and dog spermatozoa. With rat spermatozoa, she noted the emission of the second polar body at 1 to 2 hours after semination, tlie foriiiatioii of two pronuclei at 5 to G hours, and the cleavage of tlie egg at 15 to 16 hours [times that are similar to though a little longer than those estiinated for homologous fertilization of rabbit eggs tn vivo (see Austin and Walton, 1960)l. Despite these favorab'e signs, Krassovskaja did not mention finding spermatozoa within the eggs, and her results were almost certainly attributable to artificial activation. With guinea pig spermatozoa, too, polar bodies and pronuclei were said to be formed, but no cleavage occurred; with dog spermatozoa, no signs of activation were seen. Pincus, 011 the other hand, maintained that rat sperniatozoa did not take part in fertilization in the rabbit egg nor cause polar-body emission, though they were said to penetrate to various degrees, even into the superficial cytop'asm of tlie vitellus. According to Pincus, there was no evidence of penetration or activation with guinea pig or human spermatozoa. Studies made in vivo, involving mating or artificial insemination, have shown in a number of instances that viable tnammalian hybrids can be derived from animals belonging to different species within the same genera, but intergeneric crosses usually fail (see Gray, 1954). Failure is often to be ascribed to death of embryos, so that heterologous fertilization in vivo is of wider incidence than full hybridization. Since tlie bar to fertilization in viva between the germ cells of distantly related animals may well

356

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reside in part in factors precluding the meeting of eggs and spermatozoa, it is reasonable to suppose that heterologous fertilization in vitro could have even fewer limitations, and is well worthy of further study.

IV. Conclusions The germ cells of many nonmammaliaii animals have proved to be excellent material for the study of fertilization in vitro, and the knowledge that has accumulated can provide valuable clues in related problems of mammalian fertilization in vitro. I t has been shown repeatedly that rabbit, rat, and mouse eggs can tolerate wide variations in their environment without losing the capacity for subsequent normal fertilization in vivo, and that eggs recovered in the course of fertilization in vivo can complete the process in vitro. Difficulties encountered in obtaining the whole course of fertilization in vitro are associated chiefly with the need for capacitation of the speriiiatozooii a i d for removal or neutralization of egg “fertilizin.” Most published reports on the fertilization of mammalian eggs ifz vitro are inconclusive because the possibility cannot be excluded that observed phenomena were attributable to the commission of technical errors, the misinterpretation of artifacts, or the accidental induction of parthenogenesis. Success seems most likely to have been attained by Smith (1951), Dauzier et aE. (1954), Dauzier and Thibault (1959), Thibault eC al. (1954), Thibault and Dauzier (1960), Moricard (1954a,b), and, particularly, Chang ( 1959). Problems associated with fertilization in vifvo that have been the subject of active research in the last decade are principally : the roles of mammalian “fertilizins” and supposedly cheniotactic agents, the mechanisms of capacitation and sperm penetration, and the cytological changes of polar-body formation, pronuclear development, and synganiy.

REFERENCES Austin, C. R. (1948a) Nature 162, 63. Austin, C. R. (1948b) Nature 162, 534. Austin, C. R. (1950) Nature 166, 407. Austin, C. R. (1951a) Australian I . Sci. Research B4, 581. Austin, C. R. (1951b) J. Roy. microscop. SOC.71, 295. Austin, C. R. (1951~) Nature 168, 558. Austin, C. R. (1952) Nature 170, 326. Austin, C. R. (1956a) J . Exptl. Biol. 33, 338. Austin, C. R. (195613) J. Roy. Microscop. SOC.75, 141. Austin, C. R. (1957) .I. Anat. 91, 1. Austin, C. R. (1960) J . Reproduct. Fertility 1, 310. Austin, C. R. (1961) Proc. Innfern. Congr. Airinid Reproduct. 4th Cougr. The Hague, 1961 (in press).

FERTILIZATION OF MAMMALIAN EGGS

in Vitt‘O

357

C. R., and Bishop, M. W. H. (1958a) Proc. Roy. Soc. B149, 234. C. R., and Bishop, M. W. H. (1958b) Proc. Ro y Soc. B149, 241. C. R., and Braden, A. W. H. (1954a) Azutraliaic J . Biol. Sci. 7, 179. C. R., aiid Braden, A. W. H. (1954b) Ausfralian J . Biol. Sci. 7, 195. C. R., and Braden, A. W. H. (1954~) Nature 173, 999. C. R., and Braden, A. W. H. (1956) J . Exptl. Biol.33, 358. C. R., and Walton, A. (1960) I n “Marshall’s Physiology of Reproduction” (A. S. Parkes, ed.), 3rd ed., Vol. 1, Pt. 2. Longmans, Green, New York. Beatty, R. A. (1957) “Parthenogenesis and Polyploidy in biammalian Development.” Cambridge Univ. Press, London aiid New York. Bishop, D. W., and Tyler, A. (1956) J. Exptl. 2001. 132,575. Blandau, R. J. (1959) Film and report presented at Conference on Physiological Mechanisms Concerned with Conception under the auspices of the Planned Parenthood Federation of America Inc. and World Population Council at West Point, New York. Blandau, R. J., and Odor, D. L. (1948) h a t . Record 100, 733. Blandau, R. J., and Odor, D. L. (1949) Anat. Record 103,93. Bowman, R. H. (1951). Proc. SOC.Exptl. Biol. Med. 76,129. Braden, A. W. H. (1952) Australian I . Sci.Research B5,460. Braden, A. W. H. (1953) Australiaiz J . Biol. Sci.6,693. Braden, A. W. H. (1958) J. Getzet. 56, 37. Braden, A. W. H. (1959) Proc. Iwtern. S y t i ) o s i z m Exptl. Biol. Spullanzaizi, Pa7r’a. (in press). Braden, A. W. H. (1960) .1. Cellular Comp. Physiol. 56 (Suppl. I ) , 17. Braden, A. W. H., and Austin, C. R. (195h) Aiisfralian J. Biol. Sci. 7, 543. Braden, PI. W. H., and Austin, C. R. (1954b) Aztstraliait J . Biol. Sci. 7, 552. Braden, A. W. H., and Austin, C. R. (1954~) Exptl. Cell Research 7,277. Braden, A. W. H., Austin, C . R., and David, H. A. (1934) Airstraliatt J . Biol. Sci. 7, 391. Chang, M. C. (1951a) Natiirc 168, 697. Chang, M. C. (1951b) ‘ h z . ostet. e ginecol. 74 918. Chang, M. C. (1952) J . Exptl. Zool. 121, 351. Chang, M. C. (1953a) Nature 172, 333. Chang, hi. C. (1953b) Ztt “Mammalian Germ Cells” (G. E. W. Wolstenholnie, ed.), p. 226. Churchill, London. Chang, M. C. (1954) J . Exptl. Zool. 125, 127. Chang, M.C. (1955a) J . Exptl. 2001.128, 379. Chang, M. C. (1955b) Science 121, 867. Chang, M. C. (1955~) Nature 175, 1036. Chaiig, M. C. (1957a) Nature 179, 258. Chang, hl. C. (19.5713) Zn “The Beginnings of Eiiibryonic Development” (A. Tyler, R. C. voii Borstel, and C. B. Metz, eds.), pp. 109-134. ,4inerican Association for the Advancement of Science, Washington, D.C. Chang, M. C. (1958) Eiadocrinology 63, 619. Chang, M. C. (1959) Nature 184, 467. Chang, M. C., and Fernandez-Cano, L. (1958) A n a f . Record 132, 307. Colwin, A. L., and Colwin, L. H. (1957) Zit ”The Beginnings of Embryonic Development” (A. Tyler, R. C. yon Borstel, and C. B. Metz, eds.), p. 135. American Association for the Advancement of Science, Washington, D.C. Austin, Austin, Austin, Austin, Austin, Austin, Austin,

35s Costello, D. P., Davidson,

C. R. AUSTIN

M. E., Eggers, -4., Fox, M. H., and Henley C. (1957) “Methods for Obtaining and Handling Marine Eggs and Embryos.” Marine Biological Laboratory, Woods Hole, Massachusetts. Dan, J. C. (1956) Iiiferit. Rev. Cytol. 5, 365. Dauzier, L., and Thibault, C. (1956) 3rd Corigr. Proc. Iirtrrri. Coitgr. Ariiiirnl Reprodiicl. Cambridge. 1956, Section 1, p. 58. Dauzier, L., and Thibault, C, (1959) Compt. relzd. acad. sci. 248, 2655. Dauzier, L., Thibault, C., and Wintenberger, S. (1954) Coiiipt. rciid. w a d . sci. 238, 844. Defrise, 4. (1933) Anat. Record 57, 239. Diomidova, H. A., and Kusnetzova, N. A. (1935) Z. B i d . 4, 250. Frommolt, G. (1934) Zeiztr. Gynakol. 58, 7. Gray, A. P. (1954) “Mammalian Hybrids.” Commonwealth Agr. Bureaux, Farnham Royal, England. Ingelman-Suiidberg, 4., and Lindahl, P. E. (1958) J. Obstet. Gjvzaecol. Brif. Empire 65, 493. Krassovskaja, 0. V. (1934) d r c h . ntss. aitnt. hisfol. rt criibryol. 13, 415. Krassovskaja, 0. V. (1935a) Biol. %eiitr. 4, 251. Krassovskaja, 0. V. (193%) Acta Zool. 16, 449. Krassovskaja, 0. V., and Diomidova, H. A. (1934) Z. Riol. 3, 19. Kremer, J. (1924) Z. niikroskop. uiiut. Forsch. 1, 353. Lams, H., and Doorme, J. (1908) Arch. Biol. Paris 23, 259. Leonard, S. L., Perlnian, P. L., and Icurzrok, R. (1947) Proc. Soc. Exptl. Biol. Med. 66, 517. Lewis, W. H., and Gregory, P. M. (1929a) Anat. Record 42 (Suppl.), 27. Lewis, W. H., and Gregory, P. M. (1929b) Scieiice 69, 226. Lewis, W. H., and Wright, E. S. (1935) Carizegie Inst. Coiitribs. Embryol. 25, 113. Tin, T. P., Sherman, J. K., and Willett, E. L. (1957) J . Exptl. Zool. lM,275. Lindahl, P. E. (1960) J. Reproduct. Fertility 1, 3. Lindahl, P.E., and Kihlstrom, J. E. (1952) Proc. Iritcrrr. C o ~ i g r Ariiiiial . Reproduct. 2nd Congr. Copenhagen, 1952 1, 70. 1-indahl, P. E., and Nilsson, ’4. (1954) A r k i v Zool. 7, 223. Lindahl, P. E., Ingelman-Sundberg, A., Furuhjelm, A,, and Nilsson, -4. ( 1956) J. Obstct. Gyiiaccol. Brit. Eiripire 63,363. Long, J. A. (1912) Uitiv. Calif. (Berkeley) Pzhbls. Zool. 6, 1. Menkin, M. F., and Rock, J. (1948) Ant. J . ODstet. G p e c o l . 55, 440. hletz, C. B. (1957) 11% “The Beginnings of Embryonic Development” (A. Tyler, R. C. voii Borstel, and C. B. Metz, eds.), p. 23. American Association for the Advancement of Science, Washington, D.C. hletz, C. B. (1961) I i i f m z . Rrv. Cyfol. 11, 219. lloricard, R. (1949) Compt. rriid. soc. friiizg. gynkcol. 19, 226. Moricard, R. (1950) Compt. rewd. ussoc. 4rtut. Loiczwin No. 63, 337. hloricard, R. (1954a) Nati~re173, 1140. Moricard, R. (1954b) Compt. rend. SOC. bid. 148, 423. Moricard, R., and Bossu, J. (1949a) Compt. rend. acad. mid. 33, 659. Moricard, R., and Bossu, J. (194913) Bull. ussoc. gynicol. luizgzre fraizg. 1, 30. Moricard, R., and Bossu, J. (1951) Fertility and Sterility 2, 260. Nelson, H. (1851) Proc. Roy. Soc. B6, 86. Newport, G. (1853) Phil.Trans. Roy. Soc. Loitdon 143,233. Noyes, R.W. (1952) Fertility arid Sterility 3, 1.

FERTlLIZATION OF MAMMALIAN EGGS it1

VitrO

359

Noyes, R. W. (1953) Western J . Swg. Obstet. Gynecol. 61, 342. Koyes, R. W., Walton, A., and Adams, C. E. (1958) Nattcre 181, 1209. Odor, D. L., and Blandau, R. J. (1949) Aizat. Record 104, 1. Odor, D. L., and Blandau, R. J. (1951) Am. J. Anat. 89, 29. Ohnuki, Y. (1959) Cytologia ( T o k y o ) 24, 348. Onanoff, J. (1893) Compt. rend. SOC. biol. 46, 719. Pincus, G. (1930) Proc. Roy. SOC.B107, 132. Pincus, G. (1936a) “The Eggs of Mammals.” Macmillan, New York. Piiicus, G. (19361,) Anat. Record 67 (Suppl. I ) , 34. Pincus, G. (1939) J . Exptl. 2001.82, 85. Pincus, G., and Enzmann, E. V. (1932) 1.Exptl. Bid. 9, 403. Pincus, G., and Enzmann, E. V. (1934) Proc. Natl. Acnd. Sci. U S . 20, 121. Piticus, G., and Etizniann, E. 17. (1935) J . E x p f l . Med. 62,665. Pincus, G., and Enzmann, E. V. (1936) .I. Exptl. 2001.73, 195. Rock, J., and Menkin, A t . F. (1944) Scicme 100, 105. Rothschild, Lord ( 1956) “Fertilization.” hfethuen, London ancl Wiley, New I’ork. Runner, 11. N., antl Palm, J. (1953) .I. Exptl. 2001.124, 303. Schenk, S. L. (1878) Mitt. Entbryol. Zmt. K.K. Uniz. Wieu 1, 107. Schwartz, R., Brooks, W., and Zinsser, H. H. (1958) Fertility und Sterility 9, 300. Sherman, J. I<., and I h , T. P. (1958) Proc. SOL.Exptl. Biol. Med. 98, 902. Sherman, J. I<., and Lin, T. P. (1959) Fertility mid Sterility 10, 384. Shettlcs, L. B. (1953) Anz. J . ODstet. Gynecol. 66,235. Smith, .4. U. (1949) Nature 164, 1136. Smith, A. U. (1951) Biochem. SOC.Sjrnzposia (CatizBvidgc, Engl.) 7,3. Smith, A. U. (1953) I n Discussion after paper by Venge (1953). Sobotta, J. ( 1895) Arch. naikroskop. AFrat. 11. Entzwicl~ltingsit~rch. 45, 15. Straws, F. (1956) .I. embryol. Exptl. Morphol. 4, 42. Swyer, G. 1. M. (1947) N n f w e 159, 873. Thibadt, C. (1949) Aim. sci. m t . 2001.ct biol. atziinale 11, 136. Thibault, C., antl Dauzier, L. (1960) Conzpt. rend. acad. sci. 260, 1358. Thibault, C., Dauzier, L., and Wintenberger, S. (1954 j Cotnpt. reizd. soc. biol. 148, 789. Ttler, A. (1955) A h “Analysis of Development” (B. H. Willier, P. A. Weiss, and V. Hamburger, eds.), p. 170. Saunders, Philadelphia, Pennsylvania. Venge, 0. (1953) Zfi “Mammalian Germ Cells” (G. E. W. Wolstenl~oline,ed.), p. 243, Churchill, London. Vojtiskova, M. (1956) Folia Biol. (Prague) 2, 245. Wilson, E. B. (1928) “The Cell in Development and Heredity.” Macmillan, New York. Yamane, J. (1930) Cytologia ( T o k y o ) 1, 394. Yamanc, J. (1935) Cytologia ( T o k y o ) 6, 233. Yamane, J. (1937) Cytologia ( T o k y o ) 6, 583.

ADDENDUM A review of the work of C. Thibault and L. Dauzier on fertilization in vitro has recently been published ( A m . Biol. A&n. Biocltent. Biophys. 1, 277, 1961). This gives a detailed account of these authors’ important work during the past seven years and presents also additional data, including observations on the birth of young rabbits from eggs transferred after fertilization t n vifro.