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M. C. CHANG

The interruption of early pregnancy in the rabbit by oral administration of ethinyl oestradiol (ee) has been reported by Parkes, Dodds & Nobles (1938). Recent work by Chang & Harper (1966) has shown that rabbits fed with various oestrogens, especially ee, 1 to 3 days after insemination will induce the degeneration of blastocysts and that the effect is due to the rapid transport of eggs from the tube to the uterus and their expulsion from the uterus. Chang (1966) has further reported that administration of an orally effective progestin, medroxyprogesterone acetate (MPA, Upjohn Company), for 3 days before ovulation has an effect similar to that observed following the administration of oestrogen after ovulation in the rabbit. This communication describes an experiment concerning the fertilization and transport of ferret eggs following oral administration of MPA before ovulation, or of

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M. C. CHANG

Summary.

Intra-uterine insemination of ten Snowshoe hares with rabbit spermatozoa resulted in the fertilization of 10% of the eggs. Following the intravaginal insemination of Snowshoe hares with epididymal hare spermatozoa and intravenous injection of hcg about 90% of the eggs were fertilized and two living young were born in captivity. When eleven, two-cell hare eggs were transferred into the Fallopian tubes of four rabbits, development to the blastocyst stage was possible, but decidua formation and implantation failed.

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A. Hanada and M. C. Chang

Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545, U.S.A.

In a study of the penetration of zona-free eggs by foreign spermatozoa in the mouse, rat and hamster, it was concluded that the incorporation of eggs with foreign spermatozoa may depend on sperm capacitation and the physiological affinity between the spermatozoa and the vitellus of different species, and that the zona pellucida appears to be the major block for interspecific fertilization (Hanada & Chang, 1972). Yanagimachi (1972) has reported that hamster zona-free eggs can be penetrated readily by capacitated but not by uncapacitated guinea-pig spermatozoa. Based upon our recent studies of sperm capacitation and fertilization of rat eggs in vitro (Toyoda & Chang, 1974; Niwa & Chang, 1974, 1975), this paper describes the penetration of hamster and rabbit zona-free eggs by mouse and rat spermatozoa as affected by the capacitation of spermatozoa.

The medium used for the manipulation of gametes was

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A. Hanada and M. C. Chang

Summary.

Hamster eggs with follicular cells were fertilized by epididymal spermatozoa in two chemically defined media. The proportion of penetrated eggs was significantly higher in a medium for rabbit (16%) than in a medium for rat eggs (6%), but much lower than in Tyrode's solution containing follicular fluid or blood serum as reported by others. The optimal sperm concentration for sperm penetration ranged from 0·5 to 5 × 106/ml but penetration of denuded eggs failed in these media. When exposed to hamster spermatozoa in the rabbit medium containing living or dead spermatozoa of guinea-pig, rat, mouse or hamster, high proportions of denuded eggs (24-96 %) and eggs with follicular cells (93%) were penetrated. By exposure of denuded hamster eggs to hamster spermatozoa in supernatant fluid of frozen-thawed guinea-pig spermatozoa, 97% of eggs were penetrated in 8 hr compared to 0% in the control group. Sperm capacitation was also efficiently induced by preincubation of hamster spermatozoa in the supernatant fluid. The fertilizing capacity of hamster spermatozoa was maintained for 12 hr during incubation with frozen-thawed guinea-pig spermatozoa when the concentration of hamster spermatozoa ranged between 10 and 20 × 106/ml. The beneficial factor of guinea-pig spermatozoa appeared to be from spermatozoa themselves, not from the vasal or epididymal fluids. The presence of follicular cells, blood serum, bovine serum albumin, or even polyvinylpyrrolidone in the media is essential for the capacitation and acrosome reaction of hamster spermatozoa. The components of guinea-pig spermatozoa appear to maintain the fertilizing capacity of hamster spermatozoa and stimulate the process of capacitation.

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Y. TSUNODA and M. C. CHANG

Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545, U.S.A.

(Received 21st December 1974)

It is well known that a large number of the spermatozoa in the ejaculate reach the uterus but only a few are found in the oviducts of mammalian species after mating (see Bishop, 1961). This suggests that a small number of spermatozoa competent to fertilize may be selected during their passage through the female tract. In a study of the optimal sperm concentration and the minimal number of spermatozoa required to fertilize a rat egg in vitro, Niwa & Chang (1974a) have shown that the optimal sperm concentration was about 0·5 to 1·5 × 106 spermatozoa/ml and that the minimal number of spermatozoa required to fertilize a rat egg was about 3000 to 6000 spermatozoa. The present experiment was designed to determine the optimal sperm concentration, the minimal number of spermatozoa required to fertilize a mouse

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H. MIYAMOTO and M. C. CHANG

Summary.

The removal of follicular cells decreased the proportion of mouse eggs penetrated by epididymal spermatozoa, but did not affect the proportion of hamster eggs penetrated, whether the spermatozoa were taken from the epididymis or from the uterus.

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Y. TOYODA and M. C. CHANG

Summary.

Spermatozoa from the cauda epididymidis of adult male rats were preincubated in different media for various times and used for fertilization in vitro. When examined at different times after insemination, sperm penetration occurred 5 to 6 hr after insemination with epididymal spermatozoa preincubated in a standard medium as used for fertilization, and a marked decrease of penetration rates was observed if the preincubation period exceeded 3 hr. Addition of rat serum to the standard medium did not improve the time and rate of sperm penetration. By contrast, preincubation of spermatozoa in the presence of 2 mm-dibutyryl cyclic 3′,5′-adenosine monophosphate (dCAMP) caused earlier sperm penetration although the overall penetration rates decreased as the time of preincubation increased. Preincubation of spermatozoa in a medium with a high potassium/sodium ratio (0·32) increased the penetration rates and caused early sperm penetration, while in a medium containing high K/Na ratio and 2 mm-dCAMP the latency of sperm penetration was shortened distinctively and approximately 90% of eggs were penetrated within 3 hr after insemination with spermatozoa preincubated for 5 to 6 hr. It is considered that rat epididymal spermatozoa can be capacitated in a chemically defined medium and that addition of potassium and/or dCAMP accelerates the process of capacitation.

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K. NIWA and M. C. CHANG

Summary.

Ovulated eggs from mature rats and superovulated eggs from immature rats were incubated with various concentrations of epididymal spermatozoa from mature rats. Few eggs from mature rats and only 16 to 20% of the superovulated eggs from immature rats were fertilized when the sperm concentration was 1·8 to 3·7 × 106 spermatozoa/ml. At concentrations of 0·7 to 1·5 × 106 spermatozoa/ml, 2 to 8% of the eggs from mature rats and 96% of the eggs from immature rats were fertilized. It appears that the eggs from the immature rats are much easier to fertilize in vitro than those from the mature rats, but the optimal concentration of spermatozoa also plays an important rôle.

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H. MIYAMOTO and M. C. CHANG

Summary.

Hamster spermatozoa recovered from the epididymis or the uterus were deposited into the uterine horns at various times before ovulation. It was found that the fertilizing life of hamster spermatozoa in the female tract is about 13 hr.

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Y. Tsunoda and M. C. Chang

Summary.

The fertilizing ability of epididymal spermatozoa from rats and mice treated for 3 or 4 or 9 or 10 days with various doses of μ-chlorohydrin was tested in vitro, and in vivo by intrauterine insemination. The minimum doses (per kg/day) needed to affect fertilization significantly were: rat, in vitro, 8·8 mg for 3 or 4 days, 4·4 mg for 4 days and 2·7 mg for 9 or 10 days; in vivo, 4·4 mg for 3 or 4 days and 2·7 mg for 9 or 10 days: mouse, in vitro, 4·4 mg for 3 days and 13·3 mg for 9 days; in vivo, 44·2 mg for 3 days and 26·5 for 9 days. Rats were infertile for at least 18 days after receiving 44·2 mg μ-chlorohydrin/kg/day for 3 days, but fertilizing ability, tested in vivo and in vitro, was restored 10-11 days and 15-18 days, respectively, after daily treatment with 11·1 mg μ-chlorohydrin/kg for 3 days.