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Y. Tsunoda

It has been reported that heteroimmune anti-ovary serum can block sperm penetration of the zona pellucida in vitro in hamsters (Shivers, Dudkiewicz, Franklin & Fussell, 1972; Oikawa & Yanagimachi, 1975; Tsunoda & Chang, 1976a), mice (Jilek & Pavlok, 1975) and rats (Tsunoda & Chang, 1976b). Passive immunization with anti-ovary serum can also prevent fertilization in vivo in hamsters (Oikawa & Yanagimachi, 1975; Tsunoda & Chang, 1976d), mice (Jilek & Pavlok, 1975; Tsunoda & Chang, 1976d) and rats (Tsunoda & Chang, 1976d). Inhibition of pregnancy by isoimmunization with ovarian extracts has been observed in guinea-pigs (Porter, 1965) and mice (Tsunoda & Chang, 1976c). It therefore seems possible that inhibition of fertilization by anti-ovary serum is caused by a zona pellucida-specific antigen(s) which is derived from the ovary. I therefore examined the effect of an antiserum against mouse eggs on fertilization in mice.

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Y. Kato and Y. Tsunoda

Summary. Mouse two-cell embryos were cultured in a medium supplemented with nocodazole or colcemid for 12·5–14·5 h in vitro, and development after elimination of these drugs was examined. All embryos cultured with nocodazole stopped at the metaphase of the second cell cycle. When nocodazole was removed, almost all embryos divided to the normal four-cell stage within 1 h and then developed into blastocysts (98%). The proportion of embryos that developed into young after transfer to recipients was not significantly different from the control (35 versus 36%), but the developmental ability of the embryos treated with colcemid was reduced, especially after transfer to recipients.

Keywords: blastocysts; nocodazole; colcemid; mouse

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Y. Tsunoda and Y. Kato

The developmental ability of enucleated mouse eggs that had received embryonic stem cells was examined. In a preliminary study, none of the reconstituted eggs formed a nucleus using inactivated Sendai virus (HVJ) after activation with ethanol. DC pulses were applied at 100 or 140 V mm−1 in addition to this treatment. After electrofusion, 29–40% of reconstituted eggs formed nuclei and 41–60%, 25–44%, 12–24% and12–18% developed to two-cell and four-cell and morulae and blastocysts, respectively. The ability of reconstituted eggs to form blastocysts did not depend on the age of recipient eggs, except in cases of very young and very old eggs. Although implantation sites were observed, no live fetuses were obtained after the transfer of reconstituted eggs to the recipients.

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Y. Tsunoda and Y. Kato

The nuclei of mouse trophectoderm cells were found to have developmental totipotency like inner cell mass cells after serial nuclear transfer. Single inner cell mass or trophectoderm cells from expanded blastocysts synchronized with the cell cycle by treatment with nocodazole and aphidicolin to the G1 stage were injected into the perivitelline space of enucleated metaphase II oocytes together with Sendai virus. All oocytes were given three electrical pulses to induce fusion and activation (first nuclear transfer). Aphidicolin was present in all media used until fusion. When reconstituted oocytes developed to the two-cell stage, the nuclei of the reconstituted eggs were fused with the enucleated blastomeres of fertilized two-cell embryos by inactivated Sendai virus (second nuclear transfer). The reconstituted embryos were cultured in vitro and transferred to recipients. After the second nuclear transfer, 23–64% (for inner cell mass cells) and 32–62% (for trophectoderm cells) developed to morula or blastocyst stage. Better development of second nuclear transfer embryos was observed when oocytes fused with trophectoderm nuclei did not extrude a polar body after the first nuclear transfer. After transfer of morulae and blastocysts to recipients, four males were obtained, two from inner cell mass and two from trophectoderm nuclei. These findings indicate that the nucleus of inner cell mass and trophectoderm cells of mouse blastocysts can be reprogrammed within the cytoplasm of unfertilized oocytes and then in fertilized embryos.

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Y. Tsunoda and T. Sugie

The development of frozen–thawed rabbit eggs to near term or term young has been reported by Bank & Maurer (1974), Whittingham & Adams (1976) and Tsunoda & Sugie (1977). The proportions of frozen–thawed eggs surviving in the first two studies were similar (6–7%) and poor compared with that of unfrozen rabbit eggs. In our study (Tsunoda & Sugie, 1977) the proportion of frozen–thawed late morulae developing to term did not differ from that of unfrozen rabbit eggs (64 versus 63%), but the proportion of eggs frozen at the 8–16-cell (early morula) and 2-cell stages was still poor (37 and 13%). Since Whittingham (1971) reported that mouse eggs survived after freezing and thawing when the eggs were suspended in modified Dulbecco's buffered medium (PBS), this medium has been used in most studies, irrespective of species, except those of Utsumi & Yuhara (1975) and Bilton & Moore (1976) who used Ringer or PBS supplemented with rat or goat serum for freezing rat or goat eggs. The importance of serum in the suspending medium for low-temperature preservation of eggs has been pointed out by Hafez (1971). We therefore examined the effects of different freezing media, especially the presence of serum, on the survival of frozen–thawed rabbit eggs.

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Y. Tsunoda and T. Sugie

Since the first successful report on the deep-freeze preservation of mouse embryos by Whittingham (1971), the development of frozen embryos after transplantation has been reported for the cow (Wilmut & Rowson, 1973), rabbit (Whittingham & Adams, 1974), rat (Whittingham, 1975), and sheep (Willadsen, Polge, Rowson & Moor, 1976). For the rabbit, Bank & Maurer (1974) found that 7–15% of 8-cell eggs or morulae transplanted after freezing and thawing developed into fetuses, and Whittingham & Adams (1974) also obtained a 17% development of frozen 8-cell eggs to term. These results are considerably poorer than those obtained for the mouse (Whittingham, 1974).

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Y. Tsunoda and Anne McLaren

Summary. Half embryos produced from 8-cell or compacted stages were cultured in vitro for 1–2 days and transferred to oviducts or uteri of recipients at different stages of pseudopregnancy. The proportion of live fetuses was low (8–12%), except for one group (27%) in which half embryos were cultured in vitro for 1 day and transferred into oviducts on the 1st day of pregnancy. Monozygotic twin production rate, however, was low (1 out of 10) even in this group. Fetal weight on the 18th day of gestation was significantly lower after transfer of half embryos than after transfer of similarly treated but undivided embryos.

Half embryos produced from the 2-cell stage were inserted into empty zonae, embedded in agar, cultured in ligated mouse oviducts for 2–4 days and transferred to oviducts of recipient females on the 1st day of pregnancy or pseudopregnancy. When twin embryos cultured for 2–3 days were transferred to pseudopregnant recipients together with control embryos, 4 sets of monozygotic twins and 5 singletons out of 10 sets of twin embryos were obtained on Days 18–19 of gestation, giving a survival rate of 65%.

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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.

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Y. Tsunoda and M. C. Chang

Summary.

Female rats and mice were injected with homologous spermatozoa without adjuvant, or with homogenates of ovary or testis+epididymis in adjuvant. Ovulation was not inhibited and the numbers of eggs released were normal. Fertilization rates were significantly reduced in the mouse by all tissues injected and in the rat by injection of ovarian homogenate only. Fracture of and a weak precipitate on the zona pellucida of the eggs were observed in the mouse but not in the rat after injection of ovarian homogenates. The number of pregnancies and litter sizes were reduced following the first and second mating after treatment in the mouse but not in the rat. When eggs with or without follicular cells were examined in vitro, fertilizability was very low if the donor had been treated with ovarian homogenate, better with testis+epididymis homogenate and best after treatment with sperm suspension. Failure of fertilization appeared to be the major cause of infertility after such treatments.

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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