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J. Motlík and J. Fulka

Summary. Rabbit ovarian oocytes co-cultured with granulosa cells were transferred for fertilization to the oviducts of recipient does. Oocytes with cumulus cells and membrana granulosa cells were isolated before or 3 h after hCG injection. Granulosa cells did not prevent the resumption of meiosis but the time sequence of nuclear maturation was retarded by about 3 h. When oocytes were isolated from FSH-stimulated ovaries and cultured with only their cumulus cells or with cumulus and granulosa cells of the same origin, non-decondensed sperm heads were detected in about 30% oocytes after fertilization. Culture of FSH-stimulated oocytes with granulosa cells isolated 3 h after hCG injection substantially enhanced the development of male pronuclei to 88·2% and regular cleavage to 85·2% 10 h and 20 h after transfer, respectively. Further improvement was observed if the oocytes and their co-cultured granulosa cells had been exposed to hCG for 3 h.

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J. MOTLÍK and J. FULKA

Pincus & Enzmann cultivated rabbit ovarian oocytes in vitro as early as 1935. In a study of the development of cultivated oocytes transferred to the oviducts of mated females, Chang (1955a) obtained 81% cleavage. In further experiments, Chang (1955b) evaluated embryonic development after the transplantation of cultivated oocytes and observed positive results with the oocytes isolated for cultivation from the ovaries 6 hr or more after injecting sheep pituitary extract. Thibault & Gérard (1970) fertilized cultivated oocytes in vitro and described the formation of anomalies in the development of the male pronuclei. The aim of the present study was to determine whether oocytes collected shortly after mating followed by an injection of HCG and then cultivated in vitro would be capable, after fertilization in the oviduct, of normal development in the

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J. MOTLÍK and J. FULKA

Maturation of primary oocytes in vitro has been described in several species of mammals. Mature pig oocytes can also be obtained in vitro after cultivation in a suitable medium (Edwards, 1965; Foote & Thibault, 1969; McGaughey & Polge, 1971; Motlík, 1972). Nuclear changes characteristic of maturation occur in vitro at the same time intervals as they would in vivo after the administration of HCG in vivo (Hunter & Polge, 1966). A cytogenetic analysis (McGaughey & Polge, 1971) revealed some anomalies, but these ought not to preclude penetration by spermatozoa and their transformation to pronuclei. Edwards, Bavister & Steptoe (1969) detected spermatozoa in the cytoplasm of cultivated human oocytes. Mouse oocytes can also be cultivated and fertilized in vitro (Cross & Brinster, 1970; Mukherjee, 1972), although the fertilization rate is

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J. Fulka Jr, A. Pavlok, and J. Fulka

Summary. Bovine oocytes removed from 3–5 mm follicles were matured in vitro for 22–24 h. The zonae pellucidae were then dissolved by pronase and the eggs were transferred to droplets of preincubated bovine epididymal spermatozoa. Of 575 oocytes, 510 (88·7%) were fertilized and in 261 (45·4%) of them normal male and female pronuclei were present. In the rest male pronucleus formations were arrested or were undetermined (9 · 7%). Approximately half of the oocytes were fertilized by ≥2 spermatozoa. In some of the polyspermic cells male pronuclei of normal size were accompanied by less advanced stages, including sperm heads at the beginning of decondensation. We conclude that a cytoplasmic substance responsible for male pronucleus formation is present in about 50% of randomly selected bovine oocytes.

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J. Fulka Jr, J. Motlík, J. Fulka, and F. Jílek

Summary. Culture of mouse oocytes in medium with 1 or 100 μg cycloheximide/ml did not prevent germinal vesicle breakdown (GVBD). In contrast, GVBD in pig oocytes was absolutely blocked at concentrations of 1, 5, 10, 50 and 100 μg cycloheximide/ml, respectively. The inhibition of GVBD was not influenced by the presence or absence of cumulus cells and it was fully reversible. When cycloheximide treatment (5 μg/ml) was given after preincubation for 6, 12 and 16 h, GVBD occurred in 15, 46 and 75% of oocytes, respectively. It is concluded that proteins important for GVBD of pig oocytes were present in sufficient amounts at about 12 h of culture.

The fusion of pig oocytes in metaphase I to oocytes with an intact germinal vesicle revealed that cycloheximide did not inhibit GVBD induced by maturing ooplasm. Therefore, induction of prematurely condensed chromosomes by the maturing ooplasm did not require protein synthesis. However, continuous protein synthesis was necessary to maintain metaphase I and prematurely condensed chromosomes in a typical configuration.

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J. Fulka Jr and A. Okolski

Summary. Oocytes were removed from follicles 5–30 mm in diameter. The germinal vesicle was present in 69·6% (23/33) of the oocytes at the start of culture, but after 20–24 and 40 h 70·5% (12/17) and 68·2% (43/63) of the oocytes were in metaphase I and metaphase II with first polar body extruded, respectively.

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J. Motlik, J. Fulka, and J.-E. Fléchon

Summary. Cumulus expansion and cumulus cell-oocyte coupling during in-vivo and in-vitro maturation of pig oocytes were studied by measuring [3H]uridine uptake. In vivo, cumulus expansion started before germinal vesicle breakdown (GVBD) (16 h versus 20 h after hCG) but no significant change occurred in the coupling index until 32 h after hCG. Intercellular coupling was decreasing at 32 h after hCG in oocytes at anaphase I and telophase I. Complete uncoupling was closely correlated with corona radiata expansion. In vitro, partial uncoupling was observed in oocyte—cumulus cell complexes from prepubertal and PMSG-stimulated gilts cultured for 16 and 32 h, respectively. The addition of FSH caused cumulus expansion, and the functional coupling between the cumulus cells and the oocyte was maintained up to at least 16 h of culture in complexes from prepubertal gilts.

We conclude that, under our conditions, neither hormone-free nor FSH-supplemented medium ensured the same [3H]uridine uptake and uncoupling kinetics as during in-vivo maturation.

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J. Motlík, A. Pavlok, and J. Fulka

Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, Department of Genetics, Liběchov, Czechoslovakia

Prostaglandin F-2α (PGF-2α) and its analogues have been proposed as agents for control of the oestrous cycle by inducing regression of the functional CL in several mammals, but few data are available on fertility at the synchronized oestrus in cattle. The first successful pregnancy was reported by Rowson, Tervit & Brand (1972) and encouraging results with small numbers of animals have since been published (Inskeep, 1973; Philipsen & Rasbech, 1973; Roche, 1974). Experiments on a larger scale were evaluated by Lauderdale et al. (1974). Except for Rowson et al. (1972), who used intrauterine application, the above authors all administered PGF-2α intramuscularly.

Although Nakahara, Kaneda, Domeki & Yamauchi (1974) and Ohta, Umezu & Takeuchi (1974) reported satisfactory results after intrauterine administration of PGF-2α through the cervix, Henricks, Long, Hill & Dickey (1974) observed lower fertility.

The

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J. Motlik, Nicole Crozet, and J. Fulka

Summary. Pig oocytes were isolated from early antral follicles of different sizes and their abilities to resume and complete meiotic maturation in vitro were compared. After 24 h of culture, more than 80% of the oocytes from follicles 0·3–0·7 mm in diameter remained at the germinal vesicle stage, while 66, 94·3 and 100% oocytes from follicles 0·8–1·6, 1·7–2·2 and 3–5 mm in diameter, respectively, completed germinal vesicle breakdown. After 48 h of culture, 35% of the oocytes in the smallest follicle class progressed to prometaphase and only 4% to metaphase I. Of the oocytes from follicles 0·8–1·6 mm in diameter, 23% reached metaphase I and 17·3% metaphase II. About 50 and 76% of the oocytes from follicles 1·8–2·2 mm and 3–5 mm in diameter, respectively, extruded the first polar body.

The ability to resume meiosis (i.e. to undergo germinal vesicle breakdown) is reached by porcine oocytes when they approach their full size in antral follicles >0·8 mm in diameter and before they are capable of completing it (i.e. reaching metaphase II). The ability to complete meiotic maturation acquired in antral follicles of about 2 mm in diameter coincided with a significant decrease in the nucleolar transcriptional activity of the oocytes.

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J. Motlík, V. Kopečný, J. Pivko, and J. Fulka

Summary. The fate of proteins formed during meiotic maturation was examined after fertilization. Rabbit ovarian oocytes were labelled in vitro with [3H]lysine and fertilized after transfer to recipients. A significant accumulation of the label was detected autoradiographically only in fully grown male and female pronuclei.

Pig oocytes at the germinal vesicle and metaphase I stages were labelled with [3H]lysine, [3H]methionine or [3H]tryptophan and fertilized. Pronuclei were labelled by all 3 precursors. During cleavage, eggs labelled with [3H]lysine lost the nuclear label by the 4-cell stage. However the [3H]methionine label was present in the cytoplasm and marked in the nuclei at the 4-cell stage, while the [3H]tryptophan label was still clear in 8-cell embryos.