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MIN CHUEH CHANG October ~ 0, ~ 908-fune S. ~ 991 BY ROY O. GREEP THE LIFE WORK OF Min Chueh Chang centered on a dis- crete portion of the mammalian reproduction process, the part that begins with the existence of male ant! female free living gametes and ends with their successful union, fertilization. In this sphere Chang was a world leacler, a giant of his time. That, however, is only part of the fame that is conjured up by the mention of his name. Actually, Chang is best known in the public minct for his work on the development of the oral contraceptive, "the Pill." The lat- ter benefited millions of women and fomented a social/ sexual revolution. This freeing of the sexual act from the threat of conception lecl to major changes in the way men ant! women live together. To bring these two related but very different aspects of Chang's research into perspective, it is important to note that of his forty-five years in research only five ~1951 through 1956) were spent in proving the effectiveness of certain steroids in controlling fertility in laboratory mammals when aciministered orally. This was his greatest contribution in pragmatic terms. This brief departure from Chang's abiding interest in eggs ancl sperm themselves is in keeping with his recogni- tion of the critical neec! for better means of controlling 45

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46 BIOGRAPHICAL MEMOIRS human fertility. It is noteworthy that Chang's work on de- veloping the Pill utilized fundamental information already available. Except for the mode of administering the contra- ceptive steroids, little was acicled to existing knowledge. On the contrary, Chang's monumental work on fertilization was purely an exercise in basic science for the purpose of gain- ing new insight into the mechanism of fertilization. That this turner! out to have great practical significance was, of course, a personal satisfaction, but it was the plaudits of the scientific community that pleasecI him most. His life career is a story of triumph and disappointments, perseverance and major accomplishments, accolades, inter- national recognition, and, lastly, an element of what Peter Meciawar recognized as chance. It was largely by chance that Chang often found himself the right man at the right place at the right time. This was especially true at Cam- bridge University ~ ~ 939-45) and again at the Worcester Foun- ciation for Experimental Biology (1951-561. Chang was aware of these favoring circumstances. In an unpublished manu- script titled "Reminiscences on the Study of Animal Repro- duction en cl Association with Reproductive Biologists," Chang wrote extensively, forthrightly, and illuminatingly on the many preceptors and counselors to whom he was greatly in(lebtecl for their material help, guidance, and encouragement. Lastly, note need be made of the fact that Chang grew up, as it were, with a newly founder! institution that pro- viclec! him with the opportunity and the facilities to carry out his extended program of research and to attain preemi- nence in the world of science. Chang returnee! this favor by leaving to the Worcester Foundation for Experimental Biol- ogy a proud legacy of prestige and renown. Not the least of what made Chang a notable and enclear- ing character is that he was every inch a kind, generous, fair-mincled, anc! gentle person whose integrity was a given.

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MIN CHUEH CHANG PERSONAL HISTORY 47 Min Chuch Chang was born in Tai Yuan (Shanxi prov- ince), China, ~ ~^ ~^^~ ~~ ~ ~ on ()ctober 1(), 19()~. His lather, a magistrate, was able to provide him with a quality education, inclucling in 1933, a bachelor's degree in animal psychology from Tsing Hua University in Peking. Over the next few years of turbulent times in China, Chang stayed at the university as a teacher and made some original observations on the stain- ing of nerve cells that gained publication in a prestigious American journal. Chang's brilliance of mind and unbounded! curiosity dicI not go unnoticed. In 1938 he was encouraged to compete in a national examination for a few much-prized fellow- ships to study abroacl and he won. He opted for a year of study in agricultural science at Edinburgh University. At year's ens! the chilly climate and his perception of some bias against foreigners were not to his liking. An appealing invitation from Arthur Walton to join him in research on ram spermatozoa at Cambridge University was glaclly ac- cepted. This was in keeping with Chang's newfound! inter- est in reproductive biology, a departure from his initial in- tent on a career in behavioral psychology. There uncler Walton's tutelage ant! association with such other greats as Sir John Hammonc! and F. H. A. Marshall, Chang became engrossed in research. On the basis of his multiple observa- tions on the effect of testicular cooling and various hor- monal treatments on the respiration, metabolism, anti sur- vival of sperm in rabbits and some farm animals, Chang was awardect a Ph.D. degree in animal breeding by Cambridge University in ~ 961. The options available to Chang at that time included returning to China and sharing in the suffering of his par- ents anc! friends. Fortunately, counselors at Cambridge pre-

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48 BIOGRAPHICAL MEMOIRS veiled upon him to remain there. With the exigencies im- posed by WorIcl War II, the best that could be provided was maintenance support en cl limiter! opportunities for research. At war's end Chang again was torn between returning to China or finding elsewhere an outlet for his study of fertil- ity. He sought and was granted a one-year fellowship with Gregory Pincus to learn the technique of in vitro fertiliza- tion before returning home. At the time of Chang's arrival in the United States, Pincus was at Clark University with Hudson Hoaglanct, ant! the two of them were in the process of founding the Worcester Foundation for Experimental Biology in Shrewsbury, Massachusetts, just outside Worces- ter. Chang was given a room at the newly creates! founcia- tion, and he often toil with some clelight how he server! as night watchman. It was soon evident to Pincus ant] Chang that they were an effective team with many common inter- ests in the broacI field of reproductive biology. Moreover, they almost immecliately formed a warm, personal, and en- during relationship. It was there that Chang wouIc! spencI the remainder of his illustrious and rewarding career in research on matters relating to mammalian fertility. As funds for support of research on reproduction be- came increasingly available after micI-century, Chang's labo- ratory began to attract a cacire of highly competent young investigators who today are clistinguished leaders in basic ant! clinical research on reproduction. In Chang's labora- tory they were mainly left to their own crevices except that Chang was always at hand for helpful guidance ant! advice when needled. Among the group of approximately 100 fel- lows and associates, singling out any for mention risks the sin of omission, but mention of a few will illustrate the ctistinction of the group as a whole: J. M. Beclforcl, C. R. Austin, R. Yanagimachi, M. R. J. Harper, Y. Toyo(lo, R. H. F. Hunter, i. H. Marston, T. Twamatsu, and H. Miyamoto. With

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MIN CHUEH CHANG 49 this concentration of expertise in Chang's special fielcT, his laboratory became an international crossroad. An unend- ing influx of distinguishecl visitors was a significant factor in establishing the Worcester Foundation for Experimental Biology as an important biomedical research center. Chang's work habits were incredible. He personified what is clubbed a workaholic. His quest for better understanding of the intricate series of sequential physiological mecha- nisms involved in the fertilization of mammalian ova was the dominant en c! consuming factor in his life. Some mea- sure of the intensity of his labors will be evident from the fact that at the peak of his productivity he was publishing up to nineteen papers annually all in first-rate, peer-re- viewed journals en cl all reporting substantive findings. Chang was by his own admission a patient and persevering type of investigator. He hac! long-range goals toward which he planned his experimentation assicluously. The strong likeli- hood of gaining substantive new information from each carefully clesigned experiment was a contributing factor to his prolific procluctivity. Chang's bibliography lists 347 pa- pers, of which he was sole author of Il2 and senior author of another 38. Most scientists will agree that such procli- gious effort comes at the expense of time with the family, cultural pursuits, and reflections on broader issues within and outsit e science. Shortly after Chang arrived in the United States he mar- riect an American-born Chinese woman, Isabelle Chin, whom he met by chance in the Yale University library. Their three chiTciren inclucle two daughters, Claudia Chang Tourtellotte, head of the anthropology department at Sweet Briar Col- lege in Sweet Briar, Virginia; Pamela O'Malley Chang, an architect and civil engineer in San Francisco, California; and a son, Francis Hugh Chang, director of a health center in Boston, Massachusetts.

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50 BIOGRAPHICAL MEMOIRS Chang was neither a family man in the usual sense nor a closing father. In his private life he was a Confucian scholar and held to the principles of strict discipline for himself ant! his son and mate dominance of the marital relation- ship. Much credit must be given to Chang's talented wife for her willing acquiescence in the role of a Confucian wife as her part in enabling Chang to develop his full potential unhindered by domestic concerns. On Chang's behalf it can be saict that he followed the cultural traditions of his Asian background in a Western setting yet retained the pro- founcl respect of his family. In his later years Chang traveled extensively to many parts of the world to participate in meetings devoted to his spe- cial fielcI of investigation. Such attendance was almost al- ways as an invitec! speaker. His distinguished accomplish- ments were otherwise recognizes! by numerous honors ant! awards. A partial list includes the Albert Lasker Awarc! (1954), Ortho Mecial and Award by the American Fertility Society (1961), Hartman Award by the Society for the Study of Fertility (1971), Frances Amory Prize by the American Acad- emy of Arts and Sciences (1975), Wippman Scientific Re- search Awarc! by the Planned Parenthood Federation of America ~ ~ 987), and election to membership in the Na- tional Academy of Sciences in 1990. PROFESSIONAL HISTORY A cletailed account of Chang's experimental work as cle- pictecl in nearly 350 publications is far beyond accommocla- tion here. A look at some of his major accomplishments must suffice. Chang's life work involved a series of highly interrelated projects. The first dealt with the metabolism, motility, and fertilizing capacity of ram sperm. This was closely tier! to a concurrent attempt to improve the effec- tiveness of artificial insemination in farm animals. It being

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MIN CHUEH CHANG 51 wartime this had the prospect of increasing food produc- tion. Once the war ended and Chang had moved to the United States, he was able to take up a quest that he had had in mind for some time namely, fertilization of ova outside the mammalian body (in vitro fertilization). To that end he sought first to understand why sperm from the epi- didymis or ejaculate were motile but incapable of penetrat- ing ova. Chang's competence in reproductive biology was occa- sioned by having to understand, and to manipulate, the reproductive status of the host animals from which he ob- tained male and female gametes. Tt was with this background that he was eminently qualified to meet the challenge of evaluating, on a virtually emergency basis, a wide range of steroidal compounds as potential orally active antifertility agents in the early ~ 950s. In his initial studies on eggs and sperm Chang carried out a variety of experiments mainly to acquire expertise in the techniques involved and to gain a thorough knowledge of the field. He examined the motility and fertilizing capac- ity of sperm taken from different areas of the mate repro- ductive tract, with special attention to sperm from various parts of the epididymis. Out of this came the finding that cooling by simply applying ice to the scrotum caused severe disintegration of sperm from the Tower end of the epididy- mis. This is now a generally recognized phenomenon known as cold shock. It occurs in a critical range of temperatures (13-0C) and results in a breakdown of membrane struc- ture and function. Chang showed that sperm subjected to deep freezing must be protected by a cryoprotective agent found in egg yolk. Chang's original observation on cooling led to a massive study of cold shock. Obversely, Chang found that exposure of unfertilized rabbit ova to elevated tem- peratures destroyed their fertilizability.

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52 BIOGRAPHICAL MEMOIRS Early in his career Chang was intrigued by the prodigal- ity of sperm production and macle several observations on the effect of the number of sperm on fertilization of ova. He once estimated that the human male produces about ~ billion sperm for every egg releaser! by the female gonads. He found that of approximately 200 million sperm clepos- ited in the rabbit vagina by ejaculation or artificial insemi- nation barely ~ percent make it past the cervical barrier to the uterine cavity and only about 5,000 find their way past the utero-tubal junction. Fewer still reach the site of fertili- zation in the outer segment of the oviduct. Earlier literature cIaimec! that fertilization required the presence of what were termec! swarms of sperm in contra- distinction to vanguards. The belief was that large numbers of sperm were necessary to release a Tytic agent that would dispel the follicular cells surrounding the oocyte, the cu- muTus oophorus, ant! corona radiate. Chang showed that it is the physiological integrity of an incliviclual sperm that is important for fertilization. He also found that a single sperm can penetrate the cumulus mass of cells and reach the zone pellucida, a thick mucoprotein membrane enveloping the ovum. Each sperm head carries an attacher! packet, the acrosome, containing hyaluronidase, which is releasect by the acrosome at the site of fertilization and was believer! to effect the clispersal of cumulus cells. Chang found that the number of sperm at the site was far too few to accomplish this event. Adcling hyaluroni(lase to sperm suspensions clic! not prove to be beneficial. Chang also clisproved an alleger! claim that phosphorylatec3 hespericlin, a hyaluroniciase in- hibitor, had an antifertility action when aciministerecl orally. Since large numbers of sperm are of no benefit to fertili- zation, their production in astronomical numbers through- out reproductive life posed a challenge to Chang. He pos- ited that every population of sperm is comprised of some

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MIN CHUEH CHANG 53 that are strong and others weak, morphologically defective, or aged. Since only the strongest of the strong reach the site of fertilization, Chang held that the more sperm enter- ing the female reproductive tract the more this would pro- vide for greater variation in the recombination of genes. Chang also did a large amount of work on the local mi- lieu of sperm. He wanted to understand the effect of the very different environmental factors to which sperm are exposed during their Passage through the enididYmis and ~ . ~ ~ ~ .. am. ~ ... . ascent ot the female reproductive tract. the tert~l~z~ng ca- pacity of rabbit epididymal sperm was not benefited by sus- nension in rabbit seminal Plasma as compared to Rin~er's or Tyrode's solution. On the matter of osmolaTity, Chang and Thorsteinsson found that rabbit sperm could tolerate without ill effect on either motility or fertility half the strength or twice the strength ot longer s solution at neutral pH. They also found that sperm couIcl survive a wide range of pH at isotonicity namely, from 5.57 to 10.94! That is for- tuitous since sperm often encounter a wide range of pH in the human vagina. Sperm deposited in the rabbit vagina on mating reach the fallopian tubes within minutes and await the arrival of ova for fertilization ten to twelve hours later. In a fateful experiment Chang deposited ejaculated sperm in the tubes to coincide with the arrival of ova. Fertilization failed. Test- ing his speculation that the waiting period was the crucial factor, Chang next deposited sperm in the tubes six to eight hours before the arrival of ova and obtained fertilization. This finding that soerm must undergo an incubation ne- ,1 , ,1 r ~~ ~ 1 ,~ 0 1 OCR for page 45
54 BIOGRAPHICAL MEMOIRS sense of disappointment. A year later Austin named this phenomenon sperm capacitationa term now in wide use by reproductive biologists ant! clinicians. Chang and his associates proceeded to show that capaci- tation was a general phenomenon occurring among all mam- maTian species stucliect. They found that the duration of the waiting perioc! varies! somewhat among species: rabbit, five to six hours; rat, four to five hours; mouse, one hour; goIclen hamster, two to three hours; and sheep, one and one-half hours. They also fount! that sperm could acquire capacita- tion in the uterus as well as the tubes. An exciting exten- sion of capacitation came with the discovery by Chang in 1957 that capacitated sperm exposed to either seminal plasma or bloocl serum from the same species or from other spe- cies lost their capacitation, an event termed decapacitation. This factor was found by Bedford ant! Chang to be a high- molecular-weight substance that adheres to the surface of sperm and is removable by centrifugation at ~ 05,000 times g. Taking this one step further, they found that clecapacitatec3 sperm could be recapacitated by placing them back in the uterus or tubes. In a 1958 study of the possible influence of the hormonal status of the female rabbit reproductive tract on the capaci- tation of sperm, Chang found that ejaculated sperm placed in the uteri of pseuclopregnant or progesterone-treated rabbits failecl to become capacitated. In striking contrast, sperm placed in the fallopian tubes of these rabbits diet become capacitated. Capacitation was also achiever! in the uteri of immature or ovariectomizec! rabbits with or without estro- gen treatment. Chang's mastery of capacitation did not prove to be the Holy Grail. He could not have been unaware that by the discovery of capacitation he was one step closer to the achieve- ment of in vitro fertilization. Back in 1945 Chang's main

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MIN CHUEH CHANG 55 purpose in coming to the Worcester Foundation was to learn the technique of in vitro fertilization from Gregory Pincus. As early as 1935 Pincus claimed to have obtained living young from rabbit eggs fertilizer! in vitro and returnee! to the floe. Doubts as to the authenticity of this report lin- gerecI, and Chang working in Pincus's laboratory was not able to repeat those inklings. This opener! an intense and competitive search for a solution to this important prob- lem. In 1954 Thibault and associates reported early embry- onic clevelopment in eggs fertilized in vitro. Chang's crown- ing achievement came in 1959 with his demonstration that eggs from a black rabbit fertilized in vitro by capacitated sperm from a black male ant! transferred to a white female resulted in the birth of a litter of black young. This evi- clence seemed beyond question, and was, but some skepti- cism persisted for a while. The circumstances that allowocl in vitro fertilization in rabbits proved to be species specific. For many years, Chant en c! his students continued to define the varying specific -r ~ ~ . v ._ conditions required for in vitro fertilization in several spe- cies. To wit, in 1963, Yanagimachi ant! Chang reported the first successful fertilization of golden hamster eggs in vitro. They user! Tyrocle's solution, containing glycine and sperm capacitated in the uterus. Next came the in vitro fertiliza- tion of mouse ova incubated in bovine follicular fluicI, as reported by Iwamatsu and Chang in 1969, and in the same year Pickworth and Chang succeeder! in fertilizing Chinese hamster eggs in vitro. In 1973-74 Miyamoto ant! Chang and Toyoda and Chang reported fertilization of rat eggs in vitro, ant! in ~ 978 Hanocia et al. accomplishec! the same for deermouse eggs. In an extension of this study of in vitro fertilization, Yanagirnachi and Chang (1963) found penetration of rab- bit ova by sperm taken from the epidiclymis, thus showing

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56 BIOGRAPHICAL MEMOIRS that capacitation had occurred in vitro. In 1973 Miyamoto and Chang observed fertilization of mouse eggs by in vitro capacitated sperm, and a year later Toyodo and Chang made similar observations on rat sperm capacitated in a chemi- cally defined medium. It was on the basis of this animal data that Steptoe, Bravister, and Edwards were able to achieve in vitro fertilization of human ova. Later, the landmark birth on July 25, 1978, of the world's first test-tube baby, Louise Joy Brown, in Oldham, England, was recorded by Steptoe and Edwards. From this overview of the entire spectrum of Chang's Investigational program, it is evident that the central and constant objective was understanding the detailed circum- stances involved in the process of sperm penetration and fertilization of mammalian ova. Such was the fabric of his illustrious career. In the twilight of his career Chang became disturbed by the confusion and controversy appearing in the literature as a result of disagreement as to what chances are to be ~ , . . . .. . . a considered part ot the capacitation process. Capacitation as ori~inallv clefined both by Austin and Chant in 1951 in- O . . ~ . . eluded all the changes that enable sperm to penetrate eggs. Over the years a variety of structural and chemical changes in the sperm during the capacitation were described, some being considered as components of the capacitation pro- cess, others not. The bulk of the controversy centered on whether the acrosome reaction was a separate feature or a part of capacitation. Among those holding that capacita- tion was simply a preliminary that enabled the acrosome reaction to occur were several of Chang's former colleagues, most notably Bedford' Austin, and Yanagimachi. In a review article on capacitation published in ~ 984, Chang argued forcefully at some length as to why the original definition' which includes all the changes that enable a sperm to pen-

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MIN CHUEH CHANG 57 etrate the egg, should be retained. He dealt strictly with the facts on a totally impersonal basis. It was an elegant appeal to await full understanding of what happens to sperm dur- ing that still ever so puzzling waiting period. PERSONAL COMMENTS BY THE AUTHOR Chang was an easily recognized figure, tall and slimly built with a copious head of dark hair tinged with gray. His twinkling eyes and ready smile were prominent features of his friendly greetings. Despite being a man of illustrious international stature, the impression he gave was that of a genuinely modest and somewhat humble man. He always seemed to look up to whomever he met on a casual basis. Albeit meeting Chang was always a welcome and delightful experience. Despite this self-effacement, Chang had a nor- mal healthy ego and took justifiable pride in his own ac- complishments. One of Chang's most endearing attributes was his won- derful sense of humor. Before an audience his quick wit and facile repartee often had his audience in stitches. This was aided by an unusual feature in his manner of speaking, a rapid motion of the lower jaw. This made it appear that his often pithy and sometimes pungent quips were being ushered out with gnashing of teeth. Chang was sensitive to any personal slight or any over- sight of his scientific work. The aftermath of the discovery of the oral contraceptive was particularly nettlesome. As have indicated elsewhere Journal of Andrology, Nov.-Dec. 1992), "Chang sometimes figured in the series of clinical reports on these field trials but not to the extent that seemed deserving. Certainly there was no intent to downplay his role in this remarkable development and with the passage of time Chang came into his rightful dues as a co-founder of the Pill. It is much to Chang's credit that he never wa-

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58 BIOGRAPHICAL MEMOIRS vered in his admiration ant! respect for his benefactor, Gre- gory Pincus." In a final review summarizing his extensive studies, Chang noted rather pointedly and with some understatement that, "We have achieved a good deal towards the unclerstancting of mammalian fertilization by simple biological experimen- tation." In this age of high technology and molecular prob- ing, this may give encouragement to some who would as- pire to extent! the frontiers of knowledge by other means. Chang was by any measure an extraordinary person. His mastery of the phenomenon of capacitation took six years of unrelenting effort. His demonstration of-in vitro fertili- zation in a mammal took fourteen years. Such tenacity has become a rarity in biomedical research clue to the tenuous- ness of financial support. Chang's rise to prominence as a Chinese scholar (luring a perioc! of political unrest and so- cial upheaval at home was also out of the ordinary by a wide, nay very wicle, margin. Similarly, his contributions to human welfare are on a scale matched only by the few whose names are legencI.

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MIN CHUEH CHANG SELECTED BIBLIOGRAPHY 1940 59 With A. Walton. The effects of low temperature and acclimatization on the respiratory activity and survival of ram spermatozoa. Proc. R. Soc. Lond. (Series B) 857 ~ 129 ~ :517-27. 1943 Disintegration of epididymal spermatozoa by application of ice to the scrotal testis. i. Exp. Biol. 20~1~:16-22. 1947 Effects of testis hyaluronidase and seminal fluids on the fertiliza- tion capacity of rabbit spermatozoa. Proc. Soc. Exp. Biol. Med. 66:51- 54. 1950 Further study of the role of hyaluronidase in the fertilization of rabbit ova in vivo. Science 112 (2900) :118-19. 1951 Fertilizing capacity of sperm deposited in the fallopian tube. Nature 168:697. Fertilization in relation to the number of spermatozoa in the fallo- pian tubes of rabbits. Estratto das 2Fascicolo Speciale (7~:918-25. 1953 With G. Pincus. The effects of progesterone and related compounds on ovulation and early development in the rabbit. Acta Physiol. Latinoam. 3 (2-3) :177-83. 1955 Development of fertilizing capacity of rabbit spermatozoa in the uterus. Nature 175: 1036. 1956 With G. Pincus et al. Studies of the biological activity of certain 19- nor steroids in female animals. Endocrinology 59:695-707.

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60 BIOGRAPHICAL MEMOIRS 1957 A detrimental effect of seminal plasma on the fertilizing capacity of sperm. Nature 179:258-59. 1958 With T. Thorsteinsson. Effects of osmotic pressure arid hydrogen- ion concentration on the motility and fertilizing capacity of rab- bit spermatozoa. Fertil. Steril. 9~6~:510-20. Capacitation of rabbit spermatozoa in the uterus with special refer- ence to the reproductive phases of the female. Endocrinology 63~5~:619- 28. 1959 Fertilization of rabbit ova in vitro. Nature 184:466-67. 1960 With D. M. Hunt. Effects of in vitro radiocobalt irradiation of rabbit ova on subsequent development in viva with special reference to the irradiation of maternal organism. Anat. Rec. 137~4~:511-20. 1962 With }. M. Bedford. Fertilization of rabbit ova in vitro. Nature 193 (4818) :898-99. 1963 With R. Yanagimachi. Fertilization of hamster eggs in vitro. Nature 200 (4903) :281-82. 1964 With R. Yanagimachi. In vitro fertilization of golden hamster ova. {. Exp. Zool. 156~3~:361-76. 1966 With M. I. K. Harper. Effects of ethinyl estradiol on egg transport and development in the rabbit. Endocrinology 78~4~:860-72. 1968 In vitro fertilization of mammalian eggs. [. Anim. Sci. 27 (Suppl. 1 ) : 15- 22.

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MIN CHUEH CHANG 1969 61 With S. Pickworth. Fertilization of Chinese hamster eggs in vitro. I. Reprod . Fertil. 1 9: 371 -74. With T. Iwamatsu. In vitro fertilization of mouse eggs in the pres- ence of bovine follicular fluid. Nature 224:919-20. 1971 With T. Iwamatsu. Factors involved in the fertilization of mouse eggs in vitro. I. Reprod. Fertil. 26:197-208. 1973 With H. Miyamoto. In vitro fertilization of rat eggs. Nature 241:50- 52. 1974 With Y. Toyoda. Fertilization of rat eggs in vitro by epididymal sper- matozoa and the development of eggs following transfer. J. Reprod. Fertil. 36:9-22. With H. Miyamoto and Y. Toyoda. Effect of hydrogen-ion concen- tration on in vitro fertilization of mouse, golden hamster and rat eggs. Biol. Reprod. 10:487-93. 1984 The meaning of sperm capacitation. A historical perspective. [. Androl. 5 (it) :45-50.

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