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
J. MOTLÍK and J. FULKA
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
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.
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.
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.
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.
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.
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.
R. Procházka, J. Kaňka, P. Šutovský, J. Fulka, and J. Motlík
Summary. Pig oocytes were matured in vitro in a modified M-199 medium for 44 h, subjected to electrical stimulation and scored for activation 6 h later. Sham pulsed oocytes, exposed to electroporation medium and an a.c. field, did not develop the female pronucleus any more frequently than occurs spontaneously (8·3% within 50 h of culture). However, a single d.c. pulse proved extremely efficient in activating pig oocytes. Pulses of 0·75–1·65 kV cm−1 lasting 30 or 100 μs activated at least 90% of matured oocytes. The developmental pathway taken by the activated oocytes depended on the parameters of the pulse. The lowest effective stimulation (0·45 and 0·60 kV cm−1 for 30 μs) frequently produced oocytes that remained in pre–pronuclear stages of activation (29·4 and 42·3%, respectively). Extrusion of the second polar body and creation of one pronucleus was the most frequent type of activation (in up to 88·2% among the activated oocytes). The strongest stimulations used (1·05–1·65 kV cm−1 for 100 μs) often yielded oocytes that failed to extrude the second polar body and formed two or more pronuclei (up to 56·3%). Under optimal stimulation (0·75 kV cm−1), the activated oocytes proceed synchronously to interphase of the first mitotic division. Anaphase II is reached within 30 min and telophase II at 1 h after application of the pulse. The second polar body is extruded about 2 h after activation. Well-defined swelling pronuclei were found in oocytes 5–6 h after activation. The relationship between the stage of oocyte maturation and susceptibility to activation was investigated. The period of culture in which the oocytes develop the activation competence (32–36 h of culture) overlapped with the period in which the oocytes complete meiosis (28–38 h). This suggests that ageing in meiotic arrest is not essential for pig oocytes to become activated by electric pulses. Activation of pig oocytes was accompanied by release of cortical granules. In sections of control (metaphase II) oocytes, an average of 7·3 intact cortical granules per 10 μm of overlying cytoplasmic membrane was found. This number dropped to 1·5 in 10 μm within 30 min after the pulse.
Keywords: pig; oocyte; in vitro maturation; pronucleus; cortical granules
R. Procházka, E. Nagyová, Z. Rimkevičová, T. Nagai, K. Kikuchi, and J. Motlík
Summary. Oocyte–cumulus cell complexes (OCC) and complexes with an attached piece of membrana granulosa (C + P), isolated from prepubertal or cyclic gilts stimulated with pregnant mares' serum gonadotrophin, were cultured in media supplemented with follicle-stimulating hormone (FSH; 0·01–1·0 μg/ml) or forskolin (50–100 μmol/l) for 24 and 32 h. FSH and forskolin each induced dose-dependent cumulus and membrana granulosa expansion. After 2 h of culture, FSH (0·1 μg/ml) or forskolin (100 μmol/l) increased the contents of intracellular adenosine 3′,5′-phosphate (cAMP) in OCC from prepubertal gilts to almost 10 times that in unstimulated complexes. After 24 h of culture in media supplemented with FSH (0·1 μg/ml) or forskolin (100 μmol/l), the oocytectomized OCC and C + P showed similar expansion to that of the control groups. The intracellular cAMP contents in intact and oocytectomized OCCs were similar in all groups except those treated with FSH, in which the intact OCCs had significantly higher contents than their oocytectomized counterparts (P < 0·01). After hyaluronidase treatment, cumulus and membrana granulosa cells of intact and oocytectomized OCC and C + P were suspended, except for those of the innermost layers of the corona radiata. The results suggest that increases in cAMP contents and synthesis of an extracellular, hyaluronidase-sensitive mucus by pig OCC and C + P induced by FSH or forskolin are not dependent on the oocyte.
Keywords: FSH; cumulus expansion; hyaluronidase; oocytectomy; pig