Reprod. Fertil. Dev., 1992, 4 , 459-66
Capacitation and its Sequelae*
C. R. Austin 47 Dixon Road, Buderim, Qld 4556, Australia.
The year 1951 was important, for that was when capacitation was conceived! The details were published by M. C. Chang and me independently (Austin 1951a; Chang 1951). In June this year, the news came through of Chang's death, marking the end of a truly remarkable research career; he was indeed a gentle genius. I had a glimmer of the need for sperm residence in the female tract two years earlier, during a working visit to the National Institute for Medical Research in London (Austin 1949). Further enquiry had to await my return home, but the resulting coincidence of Chang's and my papers strengthened our joint case. In addition, while in London, I had been seduced by phase-contrast microscopy, which had only just been introduced. This system made a huge difference to the detail that could be observed in living cells, and so was of great value for the informed study of maturation, fertilization and cleavage in the rat (Austin and Smiles 1948; Austin 1951b). My co-author for the first publication on these subjects was John Smiles, a very accomplished technician, who had actually played a key role in the development of the phase-contrast microscope at the optical firm of Cooke, Troughton and Simms. Since that time, the phase-contrast optical system had been largely replaced by the interference system which does give a better rendering in highly refractile tissues. Soon after my return to Sydney, Allan Braden came to work with me at the McMaster Laboratory. This was in the period when Dudley Gill was in charge of the laboratory, and Lionel Bull was Chief of the CSIRO Division of Animal Health. The Chairman of CSIRO then was Ian Clunies Ross, whose handsome visage now adorns the back of our $50 notes. Allan and I made further observations on capacitation (Austin 1952; Austin and Braden 1952, 1954a), as also did Chang, and I think by 1954 it all added up to quite a convincing case. The word 'capacitation' was my invention, and has since earned itself two entries in the Encyclopaedia Britannica. Things could have been better expressed there, though, for one statements reads: 'Maturation of the spermatozoon in the female tract is called "capacitation"; little is known about it'. I should point out that at two recent Serono Symposia, on Gamete Physiology and Fertilization in Mammals held in Boston, no less than 34 of the main papers contained significant contributions on capacitation. Back in the 1950s, though capacitation was always in our minds, what more especially preoccupied us were those 'sequelae' referred to in my title, namely, the acrosome reaction, sperm penetration of zona and vitellus, reactions of the egg to sperm entry, nuclear events in fertilization and cleavage, and, for comparison, parthenogenesis and so-called 'somatic fertilization'. We duly attacked these, though not in the order just mentioned. *The 1991 James R. Goding Memorial Lecture given at the 23rd Conference of the Australian Society for Reproductive Biology, University of Sydney, in October 1991. 1031-3613/92/050459$05.00
C. R. Austin
To begin with, we were able to do some ultra-violet microscopy, with invaluable advice from W. P. ('Buddy') Rogers, then at the McMaster Laboratory. Thus aided, we obtained the first U.V. photomicrographs of living rat eggs in various stages of maturation, fertilization and cleavage; these were taken at a wavelength of 260 mp, the absorption maximum for DNA and RNA (Austin 1953; Austin and Braden 1953a). The equipment consisted of reflecting optics based on spherical mirrors and was loaned to us by the Standards Laboratory of Sydney University; for a source of radiation we adapted an ultra-violet spectrophotometer, normally used for assay work. We found a very similar distribution of the nucleic acids by histochemical methods (Braden and Austin 1953). Then we received a letter from Lord Rothschild who was well known for his work on fertilization in marine invertebrates (summarized in his book: Rothschild 1956). In his letter to us, Rothschild pointed out that there was very little known about polyspermy in mammals, in contrast to the many studies in non-mammals, and he suggested we look into it. So we set about what was in fact the first detailed study of polyspermy in the rat, mouse and hamster, finding the incidence to be around 1 or 2%, under normal circumstances (Austin and Braden 1953b). In the rat, with the induction of hyperthermia, the incidence reached over 30% (Austin 1955, 1956b, 1960). Later observations showed that more than 90% can occur in immature oocytes of pigs (Hunter et al. 1976). When we published our first results, we had a letter from Richard Blandau in the States, who was already well known for his studies on early development in the rat, and he told us that at that time (the middle 1950s) he had seen hardly any cases of polyspermy. It turned out that some strains of rats are genetically much more prone to polyspermy than are others, and we had been lucky to have had a very sensitive strain. One finding we made which we thought was of special interest was that mammalian eggs cleave in a normal manner even if polyspermic, we imagined because mammalian sperm centrioles play no vital role in the induction of cleavage spindles, whereas with invertebrate polyspermy the extra sperms introduce additional division centres, leading to chaotic cleavage. Concern with polyspermy led on naturally to a study of one of the 'defence' mechanisms of the egg, namely the 'zona reaction' in rat and mouse eggs (Braden et al. 1954). This was our name for the change that we were able to establish by mathematical analysis, with the invaluable guidance of Helen Newton Turner. The fact that the zona reaction is a propagated change could be inferred from the distribution of sperm penetration points through the zona, the persistence of which I had in fact first reported in 1951 (Austin 1951a). When two sperms enter an egg, the holes they make are much more commonly in opposing hemispheres. The time taken by the zona reaction to reach completion we estimated to lie somewhere between 10 min and 2 h. It is comforting to find that a much more detailed assessment made some 18 years later matched our shorter estimate very closely (Barros and Yanagimachi 1972). We believed that the mechanism probably began with a response in the vitelline surface of the egg at the point of sperm attachment and spread thence over the entire surface, the zona being influenced by something released from the vitellus. This idea has been developed much more fully by later investigators (see Cherr and Ducibella 1990). Our further study revealed that species differ in the nature of their protection against polyspermic fertilization, with appropriate modification in the properties of zona, or vitelline surface, or both (Austin and Braden 1956). Then there was the problem of 'rudimentary parthenogenesis', which often includes a significant amount of apparently normal cleavage (Braden and Austin 1954). Allan and I did not take this investigation far, but some 30 years later my first Ph.D. student in Cambridge, Matt Kaufman, made parthenogenesis his major project and subsequently published a book on that subject, which is still regarded as the prime reference (Kaufman 1983). The problems of parthenogenesis, and the related states of gynogenesis and androgenesis, also became the hobby of another of our erstwhile Cambridge Ph.D. students,
Capacitation and its Sequelae
namely Azim Surani who, with his research group at the Institute for Animal Physiology and Genetics at Babraham near Cambridge, now leads the field with his theory of genomic imprinting (see Surani 1991). Back in the 1950s, Allan Braden and I published almost 20 papers during our 2-3 years' collaboration, and then unfortunately we had to go our several ways, me to London to join Alan Parkes's group at the National Institute for Medical Research. Allan Braden also went overseas and spent two or three highly productive years, with Charles Waddington in Edinburgh and Salome Gluecksohn-Waelsch in the States, studying the genetic influence of the T locus on male fertility in mice. Through this research, he was able to show for the first time that genes can express themselves in the phenotype of mammalian gametes and so affect function in a non-Mendelian manner (Braden 1960, 1972). At the National Institute in London, I began a collaboration with Marcus Bishop. He was the first person to recognize the mammalian sperm acrosome reaction, which he observed in a hamster sperm that had penetrated the zona and was lying within the perivitelline space; this is a rare event, and we set off on a diligent head hunt, in hamsters and several other species as well. There was no doubt about it: all sperms that were traversing the zona or had entered the perivitelline space had slimmer heads than corresponding ejaculated sperms. We inferred that 'loss' of the acrosome was sequential to capacitation and was an essential step in the process of sperm entry into eggs (Austin and Bishop 1958a, 1958b). A second and more immediate consequence of capacitation was recognized some years later when Yanagimachi described the sudden increase in sperm motility, a 'frantic' and undirected tail lashing, which he termed 'hyperactivation' (Yanagimachi 1981). In 1967, Claudio Barros and I, along with Mike Bedford and Luther Franklin, were able to show clearly for the first time that the mammalian acrosome reaction involves vesiculation of plasma and outer acrosomal membranes (Barros et al. 1967). The idea of acrosomal vesiculation derived from rather fragmentary evidence provided earlier by Lajos Piko and Albert Tyler (1964). Recently, Mate and Rodger (1991) have described the remarkable stability of the marsupial acrosome as seen in the brush-tailed possum and the tammar wallaby; in marsupial sperms, it seems, the acrosome reaction is a much more subtle process than in eutherian sperms. Back in 1958, Marcus Bishop and I collaborated for a few more projects. One included the use of acridine orange for fluorescent staining, which dramatically displayed the distribution of DNA in living rat eggs (Austin and Bishop 1959~). One thing I particularly envied about Marcus was the way he could combine business and pleasure. Some years back, a certain barnacle called Elminius modestus, which originated in Australia, was proving a serious problem-not only so successful in competition that it was expanding its range world-wide and had entered the Mediterranean, but also it was devastating oyster beds as it went. Marcus had a background in marine biology, and he had come upon Elminius quite by accident one summer while on vacation in the eastern Mediterranean. As a result, the Royal Society commissioned him to track the barnacle as it migrated around the coasts of Italy, Spain and France. The migration occurred mostly in summer months, so that meant that Marcus had no option but be paid to spend his entire summer holidays at the seaside-but he never complained. Returning to 1956: in that year, with the aid of phase-contrast microscopy, I had managed to spot cortical granules in unpenetrated hamster eggs and they were, appropriately enough, largely lacking from penetrated eggs (Austin 1956). Cortical granules had previously only been seen in the eggs of marine invertebrates. I could not discern them in other mammalian eggs by light microscopy, but a few years later Dan Szollosi (1962), using electron microscopy, reported them in rat and rabbit eggs, and he added many other interesting ultrastructural details. Cortical granules have subsequently been the subject of a good deal of research, much of which is discussed in a recent publication by Cherr and Ducibella (1990).
C. R. Austin
The summer of 1961 was for me the first of nine summers as a member of the Fertilization and Gamete Physiology Research Training Program (known affectionally as FERGAP) at the Marine Biological Laboratory in Woods Hole, Massachusetts, an opportunity to observe and wonder at a whole new world of fertilization and development as seen in marine invertebrates. It was also a chance to become acquainted with several of the prominent personalities in that area of research. These included Charles Metz, the organizer of FERGAP, who was well known for his studies on mating types in Paramecium and on invertebrate and vertebrate gamete surface receptors. Others who regularly or occasionally contributed to FERGAP were: Albert Tyler with his work on 'fertilizin', parthenogenesis, immunity, and so on; Laura and Arthur Colwin and Jean Dan, the trio who did most to sort out the fabulous acrosome reactions of marine sperms; and Alberto Monroy, an authority on the biochemistry of fertilization. Then were was Leonard Nelson and the contractile proteins of sperm tails, Baccio Baccetti and the evolution of sperm, Don Fawcett and the ultrastructure of sperm, eggs, fertilization and a good deal else, Giovanni Giudice and nucleo-cytoplasmic interactions in development, and so on. For two and a half months every summer, the MBL was, and I am sure still is, a veritable Mecca for assorted biologists, coming both from various American States and from other countries, several hundred workers converging there for research and discussion instead of indulging in aimless summer vacations. The gathering commonly includes several Nobel Laureates, and in fact one year we had no less than seven of these characters in residence! For my part, I take pride in the fact that a trainee in my group one summer was Ralph Brinster who, with his team, was later to achieve world renown by transferring rat growth hormone genes to mice, thus inducing large heritable increases in body weight (Palmiter et al. 1982). In point of fact, though, several of our trainees went on to do outstanding things. Lynn Fraser, for instance, who currently chairs the Society for the Study of Fertility in the UK, was one of our earliest trainees, and she has also become an authority on capacitation (see, for example, Fraser 1990). Likewise, Pat Olds-Clarke with her observations on sperm motility and capacitation (see, for example, Olds-Clarke 1990). Two other former trainees have shown that capacitation-like interactions occur also in the sperm of some non-mammals: Alec Shivers and a colleague for the frog and Michael O'Rand for the coelenterate Campanularia (see O'Rand 1979). (Similar interactions have also been observed in the housefly Musca by other workers: Leopold and Degrugillier 1973.) The MBL is a great place to spend the summer, with well equipped laboratories, a first-rate library open day and night, and memorable after-work parties. In the mid 1960s, my wife and I became 'permanent' residents in America, during which time I had charge of a research group at the Delta Regional Primate Research Center in Covington, Louisiana, my department being known as the Genetic and Developmental Disorders Research Program. While I was at the Delta, Claudio Barros came from Chile to work with me for his Ph.D. Amongst other things we were able to obtain fertilization in vitro with hamster follicular oocytes and epididymal sperms, thus excluding any role for the bulk of male and female tract secretions (Barros and Austin 1967). Recognition of the need for capacitation paved the way of course for the fertilization of mammalian eggs in vitro, and several people strove to reach this goal early on, but all to no avail. Except perhaps in the case of Audrey Smith, who was another member of Alan Parkes's group at the National Institute for Medical Research; she could possibly have achieved success as early as 1951 when she reported seeing sperm heads and pronuclei within the vitellus and the progress of one egg through cleavage divisions up to the 8-cell stage (Smith 1951). She was a meticulous worker, famous for her pioneering investigations on the biological effects of sub-zero temperatures, but she never pursued the fertilization goal. Undoubtedly, IVF was demonstrated really convincingly for the first time by Chang (1959); he announced his success during a Conference held at, of all places, the West Point
Capacitation and its Sequelae
Military Academy in upper New York State, and he showed slides of the resulting young rabbits. In his summing-up, the Chairman, Solly Zuckerman, named Chang's paper as the outstanding contribution of the entire Conference. A well deserved accolade, and it was to be 19 years before the climax of this drama was reached, with the birth of the first 'test-tube baby' . . . but more of this later. Intriguingly enough, it is not only eggs that get penetrated by sperms in the female genital tract. Back in the first half of this century, several authors claimed that in histological sections sperms could be discerned within the tissues of the female tract as well as in those of developing embryos, and this seemed to support the concept of so-called 'somatic fertilization'. Not everyone agreed, however, and one of the most outspoken dissenting voices was that of Marta Vojtiskova (1956) in Prague, who strongly disparaged the claims, maintaining that they were based on artefacts. After examining some mouse and rat material, I fully agreed with Marta, and duly said so in a short paper (Austin 1957). She then wrote me a nice letter saying she was happy to read of our concurrence. Only after that did I get around to examining histological sections of a number of different species, generously supplied by Ruth Deanesly, and I was disconcerted to find several incontrovertible instances of sperm heads within Fallopian tube mucosal cells. So of course this required another publication, flatly contradicting my previous one (Austin 1959), which must have been sad news for Marta! In 1967 I was appointed to the Charles Darwin Chair of Animal Embryology at the Physiological Laboratory in Cambridge. I found that my new department, the Marshall Laboratory, contained just two members of teaching staff, namely Dennis New and Bob Edwards, a couple of technicians and a Secretary. During the ensuing years we managed to enlarge the group to around forty. In research, Dennis was concerned with the culture in vitro of whole rat fetuses, being a true pioneer in this line of work, which he continues (New 1991). Bob and I had first met some 20 years previously, when he was a Ph.D. student in Edinburgh under the supervision of Alan Beatty. At that time, in the early 1950s, Bob was inseminating mice with a hypodermic syringe, but really his big ambition in life was to fertilize human eggs in vitro; in 1967 he was really giving this a go, but so far without any success, despite very many attempts. Fortunately, we were soon joined by Barry Bavister who had come to work for his Ph.D., and it was not long before he was fertilizing hamster eggs in vitro (Bavister 1969), following the method of Yanagimachi and Chang (1964). Then, David Whittingham, who was one of our group for a few years, became the first person to fertilize mouse eggs in vitro (Whittingham 1968); however, the hamster proved to be the better model for the human subject. Bob tried the same procedure on human eggs and to everyone's delight it worked (Bavister et al. 1969; Edwards et al. 1969). Subsequently, Barry Bavister and his research group in Madison, Wisconsin, succeeded in producing the first IVF non-human primate, a rhesus monkey called 'Petrie' (Bavister et al. 1984). Human IVF appeared in 1969 to be well in hand, and the test-tube baby goal seemed assured, but that took another nine years to achieve. Before then, innumerable healthylooking embryos were returned to their mothers but consistently failed to implant. Eventually the reason was identified-the hormonal treatment that was being used to induce ovulation of a useful number of eggs at a convenient time was interfering with the normal course of preimplantation changes in the uterus and thus precluding establishment of pregnancy. So dependence had instead to be placed on the natural cycle, with the use of the 'Hi-Gonavis' test to detect the urinary LH peak, followed by laparoscopic recovery of what was nearly always only one ripe oocyte. Despite the difficulties, the first enduring pregnancy was soon established, culminating in 1978 with the birth of Louise Brown (Steptoe and Edwards 1978; Edwards and Steptoe 1980). The fact that the pregnancy was well under way could not be kept secret and the Press were onto it with breath-taking enthusiasm. The competition to come up with the latest information, and, if possible, photographs of
C. R. Austin
the pregnant mother and later the baby, was so intense that reporters from newspapers all over the world stormed the hospital trying every trick t o be ahead of competitors-they dressed up as various members of hospital staff, and even as window cleaners perched on gantries and armed with telephoto cameras. The atmosphere got painfully intense and Mrs Brown came up with some disturbing sequelae-and eventually developed eclampsia. Finally, the delivery, of a baby girl, had to be made somewhat prematurely and by Caesarian section. It was an enormous relief to have that episode successfully completed. Now, of course, this is ancient history; the Browns had a second baby by the same method, a son this time, and all are doing fine. During these dramatic events, I hovered in the wings, involved in teaching, attending t o the needs of Ph.D. students, and finding research funds. In addition, I became involved for eleven years in the Human Reproduction Programme at the W H O in Geneva, with a group who were much involved with population control and contraception, being convinced that you can have too much capacitation. In those days we appreciated greatly many invaluable discussions with Brian Heap. However it was the Ph.D. students in our group who represented my most rewarding duty. Matt Kaufman, Azim Surani and Barry Bavister I have already mentioned. Other students who went on to make names for themselves included Richard Gardner with his fascinating embryo manipulations, Martin Johnson and Alan Handyside with the genetic aspects of early development, Carol Readhead with the immunobiology of early embryos, Roger Gosden and the potentials of primordial follicles, Simon Fishel with his improvements to the IVF technique, Vithaya Yodyingyuad and the blocks t o embryonic development, and so on. My last research student before retirement 10 years ago was Kamal Ahuja from India, who has done conspicuously well in the test-tube baby field and now runs the IVF clinic at the Cromwell Hospital in London. But, academic activities are not yet entirely over for me, for there is a worthy cause u p our way called the University of the Third Age, which takes up quite a lote of time; 'mature' students of 70 plus can ask some very searching questions-and not just about the sequelae of capacitation! References Austin, C. R. (1949). Fertilization and the transport of gametes in the pseudopregnant rabbit. J. Endocrinol. 6, 63-70. Austin, C. R. (1951~).Observations on the penetration of the sperm into the mammalian egg. Aust. J. Sci. Res., Series B 4, 581-96. Austin, C. R. (1951b). The formation, growth and conjugation of the pronuclei in the rat egg. J. R. Micros. SOC.71, 295-306. Austin, C. R. (1952). The 'capacitation' of mammalian sperm. Nature (Lond.) 170, 326. Austin, C. R. (1953). Nucleic acids associated with the nucleoli of living segmented rat eggs. Exp. Cell. Res. 4, 249-51. Austin, C. R. (1955). Polyspermy after induced hyperthermia in rats. Nature (Lond.) 175, 1038. Austin, C. R. (1956~).Cortical granules in hamster eggs. Exp. Cell Res. 10, 533-40. Austin, C. R. (1956b). Activation of eggs by hypothermia in rats and hamsters. J. Exp. Biol. 33, 338-47. Austin, C. R. (1957). Fate of spermatozoa in the uterus of the mouse and rat. J. Endocrinol. 14, 335-42. Austin, C. R. (1959). Entry of spermatozoa into the Fallopian-tube mucosa. Nature (Lond.) 183, 908-9. Austin, C. R. (1960). Anomalies of fertilization leading to triploidy. J. Cell. Comp. Physiol. 56, (Suppl. I), 1-15. Austin, C. R., and Bishop, M. W. H. (1958~).Some features of the acrosome and perforatorium in mammalian spermatozoa. Proc. R. Soc. Lond. B. Biol. Sci. 149, 234-40. Austin, C. R., and Bishop, M. W. H. (1958b). Role of the rodent acrosome and perforatorium in fertilization. Proc. R. Soc. Lond. B. Biol. Sci. 149, 241-8.
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Austin, C. R., and Bishop, M. W. H. (1959~).Differential fluorescence of living rat eggs treated with acridine orange. Exp. Cell Res. 17, 35-43. Austin, C. R., and Bishop, M. W. H. (1959b). Presence of spermatozoa in the uterine-tube mucosa of bats. J. Endocrinol. 18, viii-ix. Austin, C. R., and Braden, A. W. H. (1952). Passage of the sperm and the penetration of the egg in mammals. Nature (Lond.) 170, 919-21. Austin, C. R., and Braden, A. W. H. (1953a). The distribution of nucleic acids in rat eggs in fertilization and early segmentation. I. Studies on living eggs by ultraviolet microscopy. Aust. J. Biol. Sci. 6, 324-33. Austin, C. R., and Braden, A. W. H. (1953b). An investigation of polyspermy in the rat and rabbit. Aust. J. Biol. Sci. 6, 674-92. Austin, C. R., and Braden, A. W. H. (1954~).Time relations and their significance in the ovulation and penetration of eggs in rats and rabbits. Aust. J. Biol. Sci. 7, 179-94. Austin, C. R., and Braden, A. W. H. (1954b). Nucleus formation and cleavage induced in unfertilized rat eggs. Nature (Lond.) 173, 999-1000. Austin, C. R., and Braden, A. W. H. (1956). Early reactions of the rodent egg to spermatozoon penetration. J. Exp. Biol. 33, 358-65. Austin, C. R., and Smiles, J. (1948). Phase-contrast microscopy in the study of fertilization and early development of the rat egg. J. R. Microsc. Soc. 68, 13-19. Barros, C., and Austin, C . R. (1967). In vitro fertilization and the sperm acrosome reaction in the hamster. J. Exp. Zool. 166, 317-24. Barros, C., and Yanagimachi, R. (1972). Polyspermy-preventing mechanisms in the golden hamster. J. EXP. ZOO^. 180, 251-66. Barros, C., Bedford, J. M., Franklin, L. E., and Austin, C. R. (1967). Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell. Biol. 34, C1-C5. Bavister, B. D. (1969). Environmental factors important for in-vitro fertilization in the hamster. J. Reprod. Fertil. 18, 544-5. Bavister, B. D., Edwards, R. G., and Steptoe, P. C. (1969). Identification of midpiece and tail of the spermatozoon during fertilization of human eggs in vitro. J. Reprod. Fertil. 20, 159-60. Bavister, B. D., Boatman, D. E., Collins, K., Dierschke, D. J., and Eisele, S. G. (1984). Birth of rhesus monkey infant following in vitro fertilization and non-surgical embryo transfer. Proc. Natl Acad. Sci. USA 81, 2218-22. Braden, A. W. H. (1960). Genetic influences on the morphology and function of the gametes. J. Cell. Comp. Physiol. 56, (Suppl. I), 17-29. Braden, A. W. H., and Austin, C. R. (1953). The distribution of nucleic acids in rat eggs in fertilization and early segmentation. 11. Histochemical studies. Aust. J. Biol. Sci. 6, 665-73. Braden, A. W. H., and Austin, C. R. (1954). Reaction of unfertilized mouse eggs to some experimental stimuli. Exp. Cell Res. 7, 277-80. Braden, A. W. H., Austin, C. R., and David, H. A. (1954). The reaction of the zona pellucida to sperm penetration. Aust. J. Biol. Sci. 7 , 391-409. Chang, M. C. (1951). Fertilizing capacity of spermatozoa deposited into the Fallopian tubes. Nature (Lond.) 168, 697-8. Chang, M. C. (1959). Fertilization of rabbit ova in vitro. Nature (Lond.) 184, 466-7. Cherr, G . N., and Ducibella, T. (1990). Activation of the mammalian egg: cortical granule distribution, exocytosis and the block to polyspermy. In 'Fertilization in Mammals'. (Eds B. D. Bavister, J. Cummins and R. S. Roldan.) pp. 309-30. (Serono Symposia: Norwell, Massachusetts, USA.) Edwards, R. G., and Steptoe, P. C. (1980). 'A Matter of Life.' (Hutchinson: London.) Edwards, R. G., Bavister, B. D., and Steptoe, P . C. (1969). Early stages of fertilization in vitro of human oocytes matured in vitro. Nature (Lond.) 221, 632-5. Fraser, L. R. (1990). Sperm capacitation and its modulation. In 'Fertilization in Mammals'. (Eds B. D. Bavister, J. Cummins and R. S. Roldan.) pp. 141-53. (Serono Symposia: Norwell, Massachusetts, USA.) Hunter, R. H. F., Cook, B., and Baker, T. G. (1976). Dissociation of response to injected gonadotrophin between Graafian follicle and oocyte in pigs. Nature (Lond.) 260, 156-7. Kaufman, M. H. (1983). 'Early Mammalian Development: Parthenogenetic Studies.' (Cambridge University Press: Cambridge.)
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Leopold, R. A., and Degrugillier, M. E. (1973). Sperm penetration of housefly eggs: evidence for involvement of a female accessory secretion. Science 181, 555-7. Mate, K. E., and Rodger, J. C. (1991). Stability of the acrosome of the brush-tailed possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) in vitro and after exposure to conditions and agents known to cause capacitation or acrosome reaction of eutherian spermatozoa. J. Reprod. Fertil. 91, 41-8. New, D. A. T. (1991). The culture of postimplantation embryos. Human Reprod. (Oxford) 6 , 58-63. Olds-Clarke, P. (1990). Variation in the quality of sperm motility and its relationship to capacitation. In 'Fertilization in Mammals'. (Eds B. D. Bavister, J. Cummins and E. R. S. Roldan.) pp. 91-9. (Serono Symposia: Norwell, Massachusetts, USA.) O'Rand, M. G. (1979). Changes in sperm surface properties correlated with capacitation. In 'The Spermatozoon'. (Eds D. W. Fawcett and J. M. Bedford.) pp. 195-204. (Urban and Schwarzenberg: Baltimore and Munich.) Palmiter, R. D., Brinster, R. L., Hammer, R. E., Trumbauer, M. E. Rosenfeld, M. G., Birnberg, N. C., and Evans, R. M. (1982). Dramatic growth of mice that develop from eggs microinjected with metallothionine-growth hormone fusion genes. Nature (Lond.) 300, 611-15. Piko, L., and Tyler, A. (1964). Fine structural studies of sperm penetration in the rat. Proceedings of the Vth International Congress on Animal Reproduction and Artificial Insemination, Trento 2, pp. 372-7. Rothschild, Lord (1956). 'Fertilization.' (Methuen: London.) Smith, A. U. (1951). Fertilization in vitro of the mammalian egg. Biochem. Soc. Symp. No. 7, p. 3. Steptoe, P. C., and Edwards, R. G. (1978). Birth after the reimplantation of a human embryo. Lance Surani, M. A. (1991). Influence of genome imprinting on gene expression, phenotypic variations and development. Hum. Reprod. (Oxford) 6 , 45-51. Szollosi, D. (1962). Cortical granules: a general feature of mammalian eggs? J. Reprod. Fertil. 4, 223-4. Vojtiskova, M. (1956). The question of the participation of non-fertilizing sperms in the sexual process. Folia Biol. (Prague) 2, 245-8. Whittingham, D. G. (1968). Fertilization of mouse eggs in vitro. Nature (Lond.) 220, 592-3. Yanagimachi, R. (1981). Mechanisms of fertilization in mammals. In 'Fertilization and Embryonic Development In Vitro'. (Eds L. Mastroianni, Jr and J. D. Biggers.) pp. 81-182. (Plenum: New York and London.) Yanagimachi, R., and Chang, M. C. (1964). In vitro fertilization of golden hamster ova. J. Exp. 2001. 156. 361-76.
Manuscript received and accepted 9 October 1991
Capacitation and its Sequelae*
C. R. Austin 47 Dixon Road, Buderim, Qld 4556, Australia.
The year 1951 was important, for that was when capacitation was conceived! The details were published by M. C. Chang and me independently (Austin 1951a; Chang 1951). In June this year, the news came through of Chang's death, marking the end of a truly remarkable research career; he was indeed a gentle genius. I had a glimmer of the need for sperm residence in the female tract two years earlier, during a working visit to the National Institute for Medical Research in London (Austin 1949). Further enquiry had to await my return home, but the resulting coincidence of Chang's and my papers strengthened our joint case. In addition, while in London, I had been seduced by phase-contrast microscopy, which had only just been introduced. This system made a huge difference to the detail that could be observed in living cells, and so was of great value for the informed study of maturation, fertilization and cleavage in the rat (Austin and Smiles 1948; Austin 1951b). My co-author for the first publication on these subjects was John Smiles, a very accomplished technician, who had actually played a key role in the development of the phase-contrast microscope at the optical firm of Cooke, Troughton and Simms. Since that time, the phase-contrast optical system had been largely replaced by the interference system which does give a better rendering in highly refractile tissues. Soon after my return to Sydney, Allan Braden came to work with me at the McMaster Laboratory. This was in the period when Dudley Gill was in charge of the laboratory, and Lionel Bull was Chief of the CSIRO Division of Animal Health. The Chairman of CSIRO then was Ian Clunies Ross, whose handsome visage now adorns the back of our $50 notes. Allan and I made further observations on capacitation (Austin 1952; Austin and Braden 1952, 1954a), as also did Chang, and I think by 1954 it all added up to quite a convincing case. The word 'capacitation' was my invention, and has since earned itself two entries in the Encyclopaedia Britannica. Things could have been better expressed there, though, for one statements reads: 'Maturation of the spermatozoon in the female tract is called "capacitation"; little is known about it'. I should point out that at two recent Serono Symposia, on Gamete Physiology and Fertilization in Mammals held in Boston, no less than 34 of the main papers contained significant contributions on capacitation. Back in the 1950s, though capacitation was always in our minds, what more especially preoccupied us were those 'sequelae' referred to in my title, namely, the acrosome reaction, sperm penetration of zona and vitellus, reactions of the egg to sperm entry, nuclear events in fertilization and cleavage, and, for comparison, parthenogenesis and so-called 'somatic fertilization'. We duly attacked these, though not in the order just mentioned. *The 1991 James R. Goding Memorial Lecture given at the 23rd Conference of the Australian Society for Reproductive Biology, University of Sydney, in October 1991. 1031-3613/92/050459$05.00
C. R. Austin
To begin with, we were able to do some ultra-violet microscopy, with invaluable advice from W. P. ('Buddy') Rogers, then at the McMaster Laboratory. Thus aided, we obtained the first U.V. photomicrographs of living rat eggs in various stages of maturation, fertilization and cleavage; these were taken at a wavelength of 260 mp, the absorption maximum for DNA and RNA (Austin 1953; Austin and Braden 1953a). The equipment consisted of reflecting optics based on spherical mirrors and was loaned to us by the Standards Laboratory of Sydney University; for a source of radiation we adapted an ultra-violet spectrophotometer, normally used for assay work. We found a very similar distribution of the nucleic acids by histochemical methods (Braden and Austin 1953). Then we received a letter from Lord Rothschild who was well known for his work on fertilization in marine invertebrates (summarized in his book: Rothschild 1956). In his letter to us, Rothschild pointed out that there was very little known about polyspermy in mammals, in contrast to the many studies in non-mammals, and he suggested we look into it. So we set about what was in fact the first detailed study of polyspermy in the rat, mouse and hamster, finding the incidence to be around 1 or 2%, under normal circumstances (Austin and Braden 1953b). In the rat, with the induction of hyperthermia, the incidence reached over 30% (Austin 1955, 1956b, 1960). Later observations showed that more than 90% can occur in immature oocytes of pigs (Hunter et al. 1976). When we published our first results, we had a letter from Richard Blandau in the States, who was already well known for his studies on early development in the rat, and he told us that at that time (the middle 1950s) he had seen hardly any cases of polyspermy. It turned out that some strains of rats are genetically much more prone to polyspermy than are others, and we had been lucky to have had a very sensitive strain. One finding we made which we thought was of special interest was that mammalian eggs cleave in a normal manner even if polyspermic, we imagined because mammalian sperm centrioles play no vital role in the induction of cleavage spindles, whereas with invertebrate polyspermy the extra sperms introduce additional division centres, leading to chaotic cleavage. Concern with polyspermy led on naturally to a study of one of the 'defence' mechanisms of the egg, namely the 'zona reaction' in rat and mouse eggs (Braden et al. 1954). This was our name for the change that we were able to establish by mathematical analysis, with the invaluable guidance of Helen Newton Turner. The fact that the zona reaction is a propagated change could be inferred from the distribution of sperm penetration points through the zona, the persistence of which I had in fact first reported in 1951 (Austin 1951a). When two sperms enter an egg, the holes they make are much more commonly in opposing hemispheres. The time taken by the zona reaction to reach completion we estimated to lie somewhere between 10 min and 2 h. It is comforting to find that a much more detailed assessment made some 18 years later matched our shorter estimate very closely (Barros and Yanagimachi 1972). We believed that the mechanism probably began with a response in the vitelline surface of the egg at the point of sperm attachment and spread thence over the entire surface, the zona being influenced by something released from the vitellus. This idea has been developed much more fully by later investigators (see Cherr and Ducibella 1990). Our further study revealed that species differ in the nature of their protection against polyspermic fertilization, with appropriate modification in the properties of zona, or vitelline surface, or both (Austin and Braden 1956). Then there was the problem of 'rudimentary parthenogenesis', which often includes a significant amount of apparently normal cleavage (Braden and Austin 1954). Allan and I did not take this investigation far, but some 30 years later my first Ph.D. student in Cambridge, Matt Kaufman, made parthenogenesis his major project and subsequently published a book on that subject, which is still regarded as the prime reference (Kaufman 1983). The problems of parthenogenesis, and the related states of gynogenesis and androgenesis, also became the hobby of another of our erstwhile Cambridge Ph.D. students,
Capacitation and its Sequelae
namely Azim Surani who, with his research group at the Institute for Animal Physiology and Genetics at Babraham near Cambridge, now leads the field with his theory of genomic imprinting (see Surani 1991). Back in the 1950s, Allan Braden and I published almost 20 papers during our 2-3 years' collaboration, and then unfortunately we had to go our several ways, me to London to join Alan Parkes's group at the National Institute for Medical Research. Allan Braden also went overseas and spent two or three highly productive years, with Charles Waddington in Edinburgh and Salome Gluecksohn-Waelsch in the States, studying the genetic influence of the T locus on male fertility in mice. Through this research, he was able to show for the first time that genes can express themselves in the phenotype of mammalian gametes and so affect function in a non-Mendelian manner (Braden 1960, 1972). At the National Institute in London, I began a collaboration with Marcus Bishop. He was the first person to recognize the mammalian sperm acrosome reaction, which he observed in a hamster sperm that had penetrated the zona and was lying within the perivitelline space; this is a rare event, and we set off on a diligent head hunt, in hamsters and several other species as well. There was no doubt about it: all sperms that were traversing the zona or had entered the perivitelline space had slimmer heads than corresponding ejaculated sperms. We inferred that 'loss' of the acrosome was sequential to capacitation and was an essential step in the process of sperm entry into eggs (Austin and Bishop 1958a, 1958b). A second and more immediate consequence of capacitation was recognized some years later when Yanagimachi described the sudden increase in sperm motility, a 'frantic' and undirected tail lashing, which he termed 'hyperactivation' (Yanagimachi 1981). In 1967, Claudio Barros and I, along with Mike Bedford and Luther Franklin, were able to show clearly for the first time that the mammalian acrosome reaction involves vesiculation of plasma and outer acrosomal membranes (Barros et al. 1967). The idea of acrosomal vesiculation derived from rather fragmentary evidence provided earlier by Lajos Piko and Albert Tyler (1964). Recently, Mate and Rodger (1991) have described the remarkable stability of the marsupial acrosome as seen in the brush-tailed possum and the tammar wallaby; in marsupial sperms, it seems, the acrosome reaction is a much more subtle process than in eutherian sperms. Back in 1958, Marcus Bishop and I collaborated for a few more projects. One included the use of acridine orange for fluorescent staining, which dramatically displayed the distribution of DNA in living rat eggs (Austin and Bishop 1959~). One thing I particularly envied about Marcus was the way he could combine business and pleasure. Some years back, a certain barnacle called Elminius modestus, which originated in Australia, was proving a serious problem-not only so successful in competition that it was expanding its range world-wide and had entered the Mediterranean, but also it was devastating oyster beds as it went. Marcus had a background in marine biology, and he had come upon Elminius quite by accident one summer while on vacation in the eastern Mediterranean. As a result, the Royal Society commissioned him to track the barnacle as it migrated around the coasts of Italy, Spain and France. The migration occurred mostly in summer months, so that meant that Marcus had no option but be paid to spend his entire summer holidays at the seaside-but he never complained. Returning to 1956: in that year, with the aid of phase-contrast microscopy, I had managed to spot cortical granules in unpenetrated hamster eggs and they were, appropriately enough, largely lacking from penetrated eggs (Austin 1956). Cortical granules had previously only been seen in the eggs of marine invertebrates. I could not discern them in other mammalian eggs by light microscopy, but a few years later Dan Szollosi (1962), using electron microscopy, reported them in rat and rabbit eggs, and he added many other interesting ultrastructural details. Cortical granules have subsequently been the subject of a good deal of research, much of which is discussed in a recent publication by Cherr and Ducibella (1990).
C. R. Austin
The summer of 1961 was for me the first of nine summers as a member of the Fertilization and Gamete Physiology Research Training Program (known affectionally as FERGAP) at the Marine Biological Laboratory in Woods Hole, Massachusetts, an opportunity to observe and wonder at a whole new world of fertilization and development as seen in marine invertebrates. It was also a chance to become acquainted with several of the prominent personalities in that area of research. These included Charles Metz, the organizer of FERGAP, who was well known for his studies on mating types in Paramecium and on invertebrate and vertebrate gamete surface receptors. Others who regularly or occasionally contributed to FERGAP were: Albert Tyler with his work on 'fertilizin', parthenogenesis, immunity, and so on; Laura and Arthur Colwin and Jean Dan, the trio who did most to sort out the fabulous acrosome reactions of marine sperms; and Alberto Monroy, an authority on the biochemistry of fertilization. Then were was Leonard Nelson and the contractile proteins of sperm tails, Baccio Baccetti and the evolution of sperm, Don Fawcett and the ultrastructure of sperm, eggs, fertilization and a good deal else, Giovanni Giudice and nucleo-cytoplasmic interactions in development, and so on. For two and a half months every summer, the MBL was, and I am sure still is, a veritable Mecca for assorted biologists, coming both from various American States and from other countries, several hundred workers converging there for research and discussion instead of indulging in aimless summer vacations. The gathering commonly includes several Nobel Laureates, and in fact one year we had no less than seven of these characters in residence! For my part, I take pride in the fact that a trainee in my group one summer was Ralph Brinster who, with his team, was later to achieve world renown by transferring rat growth hormone genes to mice, thus inducing large heritable increases in body weight (Palmiter et al. 1982). In point of fact, though, several of our trainees went on to do outstanding things. Lynn Fraser, for instance, who currently chairs the Society for the Study of Fertility in the UK, was one of our earliest trainees, and she has also become an authority on capacitation (see, for example, Fraser 1990). Likewise, Pat Olds-Clarke with her observations on sperm motility and capacitation (see, for example, Olds-Clarke 1990). Two other former trainees have shown that capacitation-like interactions occur also in the sperm of some non-mammals: Alec Shivers and a colleague for the frog and Michael O'Rand for the coelenterate Campanularia (see O'Rand 1979). (Similar interactions have also been observed in the housefly Musca by other workers: Leopold and Degrugillier 1973.) The MBL is a great place to spend the summer, with well equipped laboratories, a first-rate library open day and night, and memorable after-work parties. In the mid 1960s, my wife and I became 'permanent' residents in America, during which time I had charge of a research group at the Delta Regional Primate Research Center in Covington, Louisiana, my department being known as the Genetic and Developmental Disorders Research Program. While I was at the Delta, Claudio Barros came from Chile to work with me for his Ph.D. Amongst other things we were able to obtain fertilization in vitro with hamster follicular oocytes and epididymal sperms, thus excluding any role for the bulk of male and female tract secretions (Barros and Austin 1967). Recognition of the need for capacitation paved the way of course for the fertilization of mammalian eggs in vitro, and several people strove to reach this goal early on, but all to no avail. Except perhaps in the case of Audrey Smith, who was another member of Alan Parkes's group at the National Institute for Medical Research; she could possibly have achieved success as early as 1951 when she reported seeing sperm heads and pronuclei within the vitellus and the progress of one egg through cleavage divisions up to the 8-cell stage (Smith 1951). She was a meticulous worker, famous for her pioneering investigations on the biological effects of sub-zero temperatures, but she never pursued the fertilization goal. Undoubtedly, IVF was demonstrated really convincingly for the first time by Chang (1959); he announced his success during a Conference held at, of all places, the West Point
Capacitation and its Sequelae
Military Academy in upper New York State, and he showed slides of the resulting young rabbits. In his summing-up, the Chairman, Solly Zuckerman, named Chang's paper as the outstanding contribution of the entire Conference. A well deserved accolade, and it was to be 19 years before the climax of this drama was reached, with the birth of the first 'test-tube baby' . . . but more of this later. Intriguingly enough, it is not only eggs that get penetrated by sperms in the female genital tract. Back in the first half of this century, several authors claimed that in histological sections sperms could be discerned within the tissues of the female tract as well as in those of developing embryos, and this seemed to support the concept of so-called 'somatic fertilization'. Not everyone agreed, however, and one of the most outspoken dissenting voices was that of Marta Vojtiskova (1956) in Prague, who strongly disparaged the claims, maintaining that they were based on artefacts. After examining some mouse and rat material, I fully agreed with Marta, and duly said so in a short paper (Austin 1957). She then wrote me a nice letter saying she was happy to read of our concurrence. Only after that did I get around to examining histological sections of a number of different species, generously supplied by Ruth Deanesly, and I was disconcerted to find several incontrovertible instances of sperm heads within Fallopian tube mucosal cells. So of course this required another publication, flatly contradicting my previous one (Austin 1959), which must have been sad news for Marta! In 1967 I was appointed to the Charles Darwin Chair of Animal Embryology at the Physiological Laboratory in Cambridge. I found that my new department, the Marshall Laboratory, contained just two members of teaching staff, namely Dennis New and Bob Edwards, a couple of technicians and a Secretary. During the ensuing years we managed to enlarge the group to around forty. In research, Dennis was concerned with the culture in vitro of whole rat fetuses, being a true pioneer in this line of work, which he continues (New 1991). Bob and I had first met some 20 years previously, when he was a Ph.D. student in Edinburgh under the supervision of Alan Beatty. At that time, in the early 1950s, Bob was inseminating mice with a hypodermic syringe, but really his big ambition in life was to fertilize human eggs in vitro; in 1967 he was really giving this a go, but so far without any success, despite very many attempts. Fortunately, we were soon joined by Barry Bavister who had come to work for his Ph.D., and it was not long before he was fertilizing hamster eggs in vitro (Bavister 1969), following the method of Yanagimachi and Chang (1964). Then, David Whittingham, who was one of our group for a few years, became the first person to fertilize mouse eggs in vitro (Whittingham 1968); however, the hamster proved to be the better model for the human subject. Bob tried the same procedure on human eggs and to everyone's delight it worked (Bavister et al. 1969; Edwards et al. 1969). Subsequently, Barry Bavister and his research group in Madison, Wisconsin, succeeded in producing the first IVF non-human primate, a rhesus monkey called 'Petrie' (Bavister et al. 1984). Human IVF appeared in 1969 to be well in hand, and the test-tube baby goal seemed assured, but that took another nine years to achieve. Before then, innumerable healthylooking embryos were returned to their mothers but consistently failed to implant. Eventually the reason was identified-the hormonal treatment that was being used to induce ovulation of a useful number of eggs at a convenient time was interfering with the normal course of preimplantation changes in the uterus and thus precluding establishment of pregnancy. So dependence had instead to be placed on the natural cycle, with the use of the 'Hi-Gonavis' test to detect the urinary LH peak, followed by laparoscopic recovery of what was nearly always only one ripe oocyte. Despite the difficulties, the first enduring pregnancy was soon established, culminating in 1978 with the birth of Louise Brown (Steptoe and Edwards 1978; Edwards and Steptoe 1980). The fact that the pregnancy was well under way could not be kept secret and the Press were onto it with breath-taking enthusiasm. The competition to come up with the latest information, and, if possible, photographs of
C. R. Austin
the pregnant mother and later the baby, was so intense that reporters from newspapers all over the world stormed the hospital trying every trick t o be ahead of competitors-they dressed up as various members of hospital staff, and even as window cleaners perched on gantries and armed with telephoto cameras. The atmosphere got painfully intense and Mrs Brown came up with some disturbing sequelae-and eventually developed eclampsia. Finally, the delivery, of a baby girl, had to be made somewhat prematurely and by Caesarian section. It was an enormous relief to have that episode successfully completed. Now, of course, this is ancient history; the Browns had a second baby by the same method, a son this time, and all are doing fine. During these dramatic events, I hovered in the wings, involved in teaching, attending t o the needs of Ph.D. students, and finding research funds. In addition, I became involved for eleven years in the Human Reproduction Programme at the W H O in Geneva, with a group who were much involved with population control and contraception, being convinced that you can have too much capacitation. In those days we appreciated greatly many invaluable discussions with Brian Heap. However it was the Ph.D. students in our group who represented my most rewarding duty. Matt Kaufman, Azim Surani and Barry Bavister I have already mentioned. Other students who went on to make names for themselves included Richard Gardner with his fascinating embryo manipulations, Martin Johnson and Alan Handyside with the genetic aspects of early development, Carol Readhead with the immunobiology of early embryos, Roger Gosden and the potentials of primordial follicles, Simon Fishel with his improvements to the IVF technique, Vithaya Yodyingyuad and the blocks t o embryonic development, and so on. My last research student before retirement 10 years ago was Kamal Ahuja from India, who has done conspicuously well in the test-tube baby field and now runs the IVF clinic at the Cromwell Hospital in London. But, academic activities are not yet entirely over for me, for there is a worthy cause u p our way called the University of the Third Age, which takes up quite a lote of time; 'mature' students of 70 plus can ask some very searching questions-and not just about the sequelae of capacitation! References Austin, C. R. (1949). Fertilization and the transport of gametes in the pseudopregnant rabbit. J. Endocrinol. 6, 63-70. Austin, C. R. (1951~).Observations on the penetration of the sperm into the mammalian egg. Aust. J. Sci. Res., Series B 4, 581-96. Austin, C. R. (1951b). The formation, growth and conjugation of the pronuclei in the rat egg. J. R. Micros. SOC.71, 295-306. Austin, C. R. (1952). The 'capacitation' of mammalian sperm. Nature (Lond.) 170, 326. Austin, C. R. (1953). Nucleic acids associated with the nucleoli of living segmented rat eggs. Exp. Cell. Res. 4, 249-51. Austin, C. R. (1955). Polyspermy after induced hyperthermia in rats. Nature (Lond.) 175, 1038. Austin, C. R. (1956~).Cortical granules in hamster eggs. Exp. Cell Res. 10, 533-40. Austin, C. R. (1956b). Activation of eggs by hypothermia in rats and hamsters. J. Exp. Biol. 33, 338-47. Austin, C. R. (1957). Fate of spermatozoa in the uterus of the mouse and rat. J. Endocrinol. 14, 335-42. Austin, C. R. (1959). Entry of spermatozoa into the Fallopian-tube mucosa. Nature (Lond.) 183, 908-9. Austin, C. R. (1960). Anomalies of fertilization leading to triploidy. J. Cell. Comp. Physiol. 56, (Suppl. I), 1-15. Austin, C. R., and Bishop, M. W. H. (1958~).Some features of the acrosome and perforatorium in mammalian spermatozoa. Proc. R. Soc. Lond. B. Biol. Sci. 149, 234-40. Austin, C. R., and Bishop, M. W. H. (1958b). Role of the rodent acrosome and perforatorium in fertilization. Proc. R. Soc. Lond. B. Biol. Sci. 149, 241-8.
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Austin, C. R., and Bishop, M. W. H. (1959~).Differential fluorescence of living rat eggs treated with acridine orange. Exp. Cell Res. 17, 35-43. Austin, C. R., and Bishop, M. W. H. (1959b). Presence of spermatozoa in the uterine-tube mucosa of bats. J. Endocrinol. 18, viii-ix. Austin, C. R., and Braden, A. W. H. (1952). Passage of the sperm and the penetration of the egg in mammals. Nature (Lond.) 170, 919-21. Austin, C. R., and Braden, A. W. H. (1953a). The distribution of nucleic acids in rat eggs in fertilization and early segmentation. I. Studies on living eggs by ultraviolet microscopy. Aust. J. Biol. Sci. 6, 324-33. Austin, C. R., and Braden, A. W. H. (1953b). An investigation of polyspermy in the rat and rabbit. Aust. J. Biol. Sci. 6, 674-92. Austin, C. R., and Braden, A. W. H. (1954~).Time relations and their significance in the ovulation and penetration of eggs in rats and rabbits. Aust. J. Biol. Sci. 7, 179-94. Austin, C. R., and Braden, A. W. H. (1954b). Nucleus formation and cleavage induced in unfertilized rat eggs. Nature (Lond.) 173, 999-1000. Austin, C. R., and Braden, A. W. H. (1956). Early reactions of the rodent egg to spermatozoon penetration. J. Exp. Biol. 33, 358-65. Austin, C. R., and Smiles, J. (1948). Phase-contrast microscopy in the study of fertilization and early development of the rat egg. J. R. Microsc. Soc. 68, 13-19. Barros, C., and Austin, C . R. (1967). In vitro fertilization and the sperm acrosome reaction in the hamster. J. Exp. Zool. 166, 317-24. Barros, C., and Yanagimachi, R. (1972). Polyspermy-preventing mechanisms in the golden hamster. J. EXP. ZOO^. 180, 251-66. Barros, C., Bedford, J. M., Franklin, L. E., and Austin, C. R. (1967). Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell. Biol. 34, C1-C5. Bavister, B. D. (1969). Environmental factors important for in-vitro fertilization in the hamster. J. Reprod. Fertil. 18, 544-5. Bavister, B. D., Edwards, R. G., and Steptoe, P. C. (1969). Identification of midpiece and tail of the spermatozoon during fertilization of human eggs in vitro. J. Reprod. Fertil. 20, 159-60. Bavister, B. D., Boatman, D. E., Collins, K., Dierschke, D. J., and Eisele, S. G. (1984). Birth of rhesus monkey infant following in vitro fertilization and non-surgical embryo transfer. Proc. Natl Acad. Sci. USA 81, 2218-22. Braden, A. W. H. (1960). Genetic influences on the morphology and function of the gametes. J. Cell. Comp. Physiol. 56, (Suppl. I), 17-29. Braden, A. W. H., and Austin, C. R. (1953). The distribution of nucleic acids in rat eggs in fertilization and early segmentation. 11. Histochemical studies. Aust. J. Biol. Sci. 6, 665-73. Braden, A. W. H., and Austin, C. R. (1954). Reaction of unfertilized mouse eggs to some experimental stimuli. Exp. Cell Res. 7, 277-80. Braden, A. W. H., Austin, C. R., and David, H. A. (1954). The reaction of the zona pellucida to sperm penetration. Aust. J. Biol. Sci. 7 , 391-409. Chang, M. C. (1951). Fertilizing capacity of spermatozoa deposited into the Fallopian tubes. Nature (Lond.) 168, 697-8. Chang, M. C. (1959). Fertilization of rabbit ova in vitro. Nature (Lond.) 184, 466-7. Cherr, G . N., and Ducibella, T. (1990). Activation of the mammalian egg: cortical granule distribution, exocytosis and the block to polyspermy. In 'Fertilization in Mammals'. (Eds B. D. Bavister, J. Cummins and R. S. Roldan.) pp. 309-30. (Serono Symposia: Norwell, Massachusetts, USA.) Edwards, R. G., and Steptoe, P. C. (1980). 'A Matter of Life.' (Hutchinson: London.) Edwards, R. G., Bavister, B. D., and Steptoe, P . C. (1969). Early stages of fertilization in vitro of human oocytes matured in vitro. Nature (Lond.) 221, 632-5. Fraser, L. R. (1990). Sperm capacitation and its modulation. In 'Fertilization in Mammals'. (Eds B. D. Bavister, J. Cummins and R. S. Roldan.) pp. 141-53. (Serono Symposia: Norwell, Massachusetts, USA.) Hunter, R. H. F., Cook, B., and Baker, T. G. (1976). Dissociation of response to injected gonadotrophin between Graafian follicle and oocyte in pigs. Nature (Lond.) 260, 156-7. Kaufman, M. H. (1983). 'Early Mammalian Development: Parthenogenetic Studies.' (Cambridge University Press: Cambridge.)
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Leopold, R. A., and Degrugillier, M. E. (1973). Sperm penetration of housefly eggs: evidence for involvement of a female accessory secretion. Science 181, 555-7. Mate, K. E., and Rodger, J. C. (1991). Stability of the acrosome of the brush-tailed possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) in vitro and after exposure to conditions and agents known to cause capacitation or acrosome reaction of eutherian spermatozoa. J. Reprod. Fertil. 91, 41-8. New, D. A. T. (1991). The culture of postimplantation embryos. Human Reprod. (Oxford) 6 , 58-63. Olds-Clarke, P. (1990). Variation in the quality of sperm motility and its relationship to capacitation. In 'Fertilization in Mammals'. (Eds B. D. Bavister, J. Cummins and E. R. S. Roldan.) pp. 91-9. (Serono Symposia: Norwell, Massachusetts, USA.) O'Rand, M. G. (1979). Changes in sperm surface properties correlated with capacitation. In 'The Spermatozoon'. (Eds D. W. Fawcett and J. M. Bedford.) pp. 195-204. (Urban and Schwarzenberg: Baltimore and Munich.) Palmiter, R. D., Brinster, R. L., Hammer, R. E., Trumbauer, M. E. Rosenfeld, M. G., Birnberg, N. C., and Evans, R. M. (1982). Dramatic growth of mice that develop from eggs microinjected with metallothionine-growth hormone fusion genes. Nature (Lond.) 300, 611-15. Piko, L., and Tyler, A. (1964). Fine structural studies of sperm penetration in the rat. Proceedings of the Vth International Congress on Animal Reproduction and Artificial Insemination, Trento 2, pp. 372-7. Rothschild, Lord (1956). 'Fertilization.' (Methuen: London.) Smith, A. U. (1951). Fertilization in vitro of the mammalian egg. Biochem. Soc. Symp. No. 7, p. 3. Steptoe, P. C., and Edwards, R. G. (1978). Birth after the reimplantation of a human embryo. Lance Surani, M. A. (1991). Influence of genome imprinting on gene expression, phenotypic variations and development. Hum. Reprod. (Oxford) 6 , 45-51. Szollosi, D. (1962). Cortical granules: a general feature of mammalian eggs? J. Reprod. Fertil. 4, 223-4. Vojtiskova, M. (1956). The question of the participation of non-fertilizing sperms in the sexual process. Folia Biol. (Prague) 2, 245-8. Whittingham, D. G. (1968). Fertilization of mouse eggs in vitro. Nature (Lond.) 220, 592-3. Yanagimachi, R. (1981). Mechanisms of fertilization in mammals. In 'Fertilization and Embryonic Development In Vitro'. (Eds L. Mastroianni, Jr and J. D. Biggers.) pp. 81-182. (Plenum: New York and London.) Yanagimachi, R., and Chang, M. C. (1964). In vitro fertilization of golden hamster ova. J. Exp. 2001. 156. 361-76.
Manuscript received and accepted 9 October 1991
