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K. Niwa and A. Iritani

Summary. Fertilization of rat eggs in vitro could not be achieved when epididymal spermatozoa were preincubated and eggs in clots incubated in a chemically defined medium without D-glucose. Very high proportions (84–100%) of eggs examined were undergoing fertilization when 2·78–8·34 mM-D-glucose were included in the medium. The substitution of D-fructose or D-galactose for D-glucose resulted in very poor penetration rates (0–4%), but D-mannose was effective for fertilization (59–99% penetration). Incubations for sperm capacitation and egg fertilization in different media containing the various hexoses showed that rat epididymal spermatozoa could be partly capacitated without hexose, that D-glucose, D-mannose or D-galactose but not D-fructose is effective for sperm capacitation and that only D-glucose and D-mannose supported penetration of eggs in vitro.

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A. Iritani and K. Niwa

The maturation of ovarian oocytes in vitro and fertilization after transfer to mated animals have been reported for rabbits (Chang, 1955), sheep (Crosby, Ryan & Gordon, 1971), pigs (Motlik & Fulka, 1974; Leman & Dziuk, 1971) and cattle (Sreenan, 1970; Hunter, Lawson & Rowson, 1972). In-vitro maturation of follicular oocytes followed by successful fertilization in vitro has been reported for the mouse (Cross & Brinster, 1970; Iwamatsu & Chang, 1972; Mukherjee, 1972), rat (Niwa & Chang, 1975), hamster (Barros & Munoz, 1974), guinea-pig (Yanagimachi, 1974), rabbit (Brackett, Mills & Jeites, 1972) and man (Edwards, Bavister & Steptoe, 1969). There does not, however, appear to be any report on the fertilization in vitro of follicular oocytes matured in vitro for large domestic animals such as cattle. The present experiment was therefore performed to examine the possibility of capacitation of bovine spermatozoa in reproductive tracts isolated from oestrous cows or in the uteri in situ of oestrous rabbits.

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K. NIWA and M. C. CHANG

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

(Received 23rd December 1974)

After dissolution of the zona pellucida by chymotrypsin, rat eggs can be penetrated by uterine (capacitated) and epididymal (uncapacitated) spermatozoa (Toyoda & Chang, 1968). Yanagimachi & Noda (1970) stated that "capacitated spermatozoa quickly fused and became incorporated with the [zonafree hamster] eggs, while uncapacitated spermatozoa were never fused with eggs." Uncapacitated guinea-pig spermatozoa also failed to enter the zona-free eggs (Yanagimachi, 1972). In a study of the role of cumulus cells and the zona pellucida in the fertilization of mouse eggs in vitro, Pavlok & McLaren (1972) concluded that "capacitation of mouse spermatozoa appears to be required for the penetration of the zona pellucida only, not for entry into the vitellus."

Recently, we have found that the capacitation of rat spermatozoa can be achieved by preincubation of epididymal spermatozoa in a diluted rather than a concentrated form

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K. NIWA and M. C. CHANG

Summary.

Oocytes recovered at various times from immature rats treated with PMSG and HCG were incubated with capacitated epididymal spermatozoa of mature rats. In the presence of follicular cells, sperm penetration was not observed 4 hr after incubation in the oocytes at stages from the intact germinal vesicle to the chromatin mass, but 7 to 55% of oocytes were penetrated at stages from the condensed germinal vesicle to metaphase II. After the removal of follicular cells, 15 to 72% of the oocytes at any stage were penetrated. After further incubation for 15 hr, the proportion of penetrated oocytes increased from 8 to 98% from early to late stages and that of penetrated oocytes with a male and female pronucleus increased from 9 to 100% as maturation progressed. Although the average number of spermatozoa/oocyte was not correlated with its maturation, transformation of the sperm head into a male pronucleus was retarded or failed, especially in the younger oocytes. Following incubation in a defined medium for 13 hr, 85% of oocytes at the intact germinal vesicle stage matured to the stage of the first polar body formation, but only 18 to 22% of these mature oocytes were penetrated by spermatozoa and only a few of the penetrated oocytes cleaved into normal two-cell eggs. When eggs recovered from oviducts 14 to 20 hr after ovulation were exposed to capacitated spermatozoa, the proportion of penetrated eggs (86 to 98%) and that of polyspermic eggs (11 to 27%) were not related to the ages of the eggs, but failure of transformation of the sperm head and the proportion of abnormal eggs increased 14 to 20 hr after ovulation.

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L. R. Abeydeera and K. Niwa

Summary. Bovine oocytes at the germinal vesicle stage were inseminated in Brackett & Oliphant's medium with bovine serum albumin, caffeine and heparin. Eight hours after insemination, oocytes were transferred into tissue culture medium-199 containing 10% fetal calf serum and cultured for 5–40 h at 39°C in 5% CO2 in air. The proportions of unpenetrated and penetrated oocytes reaching metaphase II increased as the time of examination increased, reaching 70 and 65% 40 h after transfer, respectively. When oocytes were penetrated by more than four spermatozoa, meiotic maturation was greatly retarded. Sperm nuclei were decondensed in most (81%) penetrated oocytes 5 h after transfer. The decondensed sperm nuclei were recondensed and then transformed to metaphase chromosomes which were morphologically compacted at first but became slightly dispersed later. The formation of the metaphase chromosomes was observed in 86% of penetrated oocytes examined 40 h after transfer, and occurred in all metaphase II oocytes at that time. In oocytes penetrated by more than nine spermatozoa, no such transformation of sperm nuclei was observed. Well-developed male and female pronuclei were observed in only three (6%) of 51 oocytes penetrated 40 h after transfer.

Keywords: cow; oocytes; sperm penetration; sperm metaphase chromosomes; in vitro

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K. NIWA and M. C. CHANG

It has been reported that rat eggs can be fertilized in vitro (Miyamoto & Chang, 1973; Toyoda & Chang, 1974). Further study has revealed that the eggs from immature rats are easier to fertilize in vitro with a lower concentration of spermatozoa (Niwa & Chang, 1973) and that the capacitation of rat spermatozoa can be better achieved in a lower concentration than in a higher concentration of spermatozoa (Niwa & Chang, 1974). The present experiment was designed to determine the optimal sperm concentration and the minimal number of spermatozoa for the fertilization of rat eggs in vitro.

The medium for fertilization was prepared according to the description of Toyoda & Chang (1974). The eggs recovered from naturally ovulating mature rats or from immature rats which had been induced to superovulate were inseminated with epididymal spermatozoa from mature rats, incubated and examined as described by Niwa & Chang (1973). In order

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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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K. Niwa, C.-K. Park and K. Okuda

Summary. Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen–thawed spermatozoa in Medium BO with caffeine (5 mm) and heparin (10 μg/ml). Very high penetration rates (95–100%) were obtained in all oocytes which had been cultured for 0–20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20–22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.

Keywords: in-vitro fertilization; immature oocytes; maturation; bovine; sperm nuclear decondensation

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L. R. Abeydeera, K. Niwa and K. Okuda

Bovine oocytes at the germinal vesicle stage were inseminated in Brackett and Oliphant's medium in the presence of BSA (10 mg ml−1), caffeine (5 mmol l−1) and heparin (10 μg ml−1). When oocytes were transferred into tissue culture medium (TCM)-199 containing 10% fetal calf serum (FCS) with or without 6-dimethylaminopurine (6-DMAP; 2 mmol l−1) 8 h after insemination and cultured for 15–40 h at 39°C in 5% CO2 in air, 74–83% of oocytes were penetrated and polyspermy (67–80%) was common. At 40 h after culture in 6-DMAP-free medium, 65% and 63% of unpenetrated and penetrated oocytes, respectively, reached metaphase II or beyond. A few (6%) oocytes were activated and contained both male and female pronuclei. Sperm metaphase chromosomes were observed in 90% of the penetrated oocytes. Penetration by more than four spermatozoa greatly retarded the meiotic maturation of the oocyte. However, sperm chromosomes were never observed in oocytes cultured in 6-DMAP supplemented medium and oocyte maturation did not proceed beyond the stage of prometaphase I. These results demonstrate the possible participation of maturation-promoting factor in metaphase chromosome formation in spermatozoa.

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C.-K. Park, O. Ohgoda and K. Niwa

Summary. Bovine follicular oocytes matured in culture were inseminated with frozen–thawed spermatozoa which were either preincubated for 5–5·5 h or not preincubated in a medium with caffeine (5 mM) and heparin (10 μg/ml). When the oocytes with cumulus and corona cells were inseminated, spermatozoa started to penetrate oocytes 3 h later regardless of whether spermatozoa were preincubated or not. However, a significantly higher proportion of oocytes was penetrated by preincubated than non-preincubated spermatozoa. When the oocytes were freed from cumulus and corona cells, penetration was observed to start 1 h after insemination and there were no differences in penetration rates 1–5 h after insemination between preincubated and non-preincubated spermatozoa. This study demonstrates that capacitation and the acrosome reaction of bovine spermatozoa can be induced within 1 h in a medium containing both caffeine and heparin when denuded oocytes are inseminated.

Keywords: in-vitro fertilization; follicular oocytes; bovine; caffeine; heparin