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A. Pavlok and J. E. Fléchon

Summary. Variability in the interaction of ram spermatozoa with zona-free hamster eggs was recorded not only amongst individual males but also between the first and second ejaculates of the same male collected 30 min apart. Fusion ability also differed according to the conditions of gamete mixing. This ability decreased after in-vitro storage of undiluted ejaculates at room temperature but lasted for 48–192 h. The kinetics of sperm—egg fusion during the time of gamete incubation varied not only with the time of sperm storage in vitro but also with the ejaculate. When the semen was frozen, the ability of the spermatozoa to fuse was markedly reduced.

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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.-E. Fléchon and J.-P. Renard

Summary. The blastocysts were recovered from cows 7–10 days after oestrus and cultured. The zona pellucida has a spongy fibrous structure. Hatching begins, about 24 h after culture, through a relatively small hole out of which the blastocyst appears to escape by its own activity. Later the zona becomes split and the two edges of the slit surround part of the blastocyst. The emerging cells were large or small blastomeres which were generally covered by a dense mass of microvilli.

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M. Guillomot, J.-E. Fléchon, and Suzanne Wintenberger-Torres

Summary. A glycoprotein coat was demonstrated on the outer surface of both the uterine and trophoblastic cells using ruthenium red, cationized ferritin, concanavalin A–peroxidase and phosphotungstic acid in HCl. No changes were observed on the uterine epithelial surface of cyclic or pregnant animals before or during blastocyst attachment (Day 15). However, the cytochemical reactions were different on the trophoblastic cells of blastocysts at Days 13 and 15, the ruthenium red and cationized ferritin sites of reaction and the concanavalin A receptors being more homogeneously distributed on the outer surface of Day-15 trophoblast. The phosphotungstic acid staining demonstrated a glycoprotein substance between the trophoblast and the uterine epithelium in adhesion areas by Day 18.

The results suggest that biochemical changes occur in the composition or distribution of the trophoblastic cell coat during the process of blastocyst attachment in the ewe.

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M. L. Bernardi, J-E. Fléchon, and C. Delouis

The development and quality of ovine zygotes matured and fertilized in vitro were compared after coculture with oviductal cells (CZB–199 system) and culture in synthetic oviduct fluid medium without cells (SOF system). The effect of two oxygen concentrations (5% and 20%) on the development of ovine zygotes in SOF medium was also studied. More ovine zygotes reached the blastocyst stage when culture in SOF medium was performed in 5% O2 rather than 20% O2. A greater number of blastocysts was obtained after culture in the SOF system than coculture in the CZB–199 system. Proportions of grade I (excellent), II (good), III (fair) and IV (poor) blastocysts did not differ significantly between the SOF and CZB–199 systems. Histological examination of hatched blastocysts revealed a superiority of the SOF system for the following: a greater number of total and trophoblastic cells in grade I and II blastocysts; more endodermic cells in grade I blastocysts, higher mitotic index in the inner cell mass of grade II blastocysts and in total and trophoblastic cells of grade I, II and III blastocysts; more grade III blastocysts with mitosis in the inner cell mass; and a lower pyknotic index in the inner cell mass of grade I, II and III blastocysts. Culture in the SOF system improved the rate and quality of blastocysts in comparison with the CZB–199 system. Furthermore, culture in SOF medium with 5% O2 provided more blastocysts than culture in the presence of 20% O2.

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R. A. P. Harrison, J.-E. Fléchon, and C. R. Brown

Summary. Acrosin and its zymogen form proacrosin were located in various sperm fractions by biochemical and immunocytochemical techniques, using an anti-acrosin serum that cross-reacts strongly with proacrosin.

If activation of proacrosin was prevented, the zymogen was associated almost entirely with the sperm heads, where it was confined to the anterior segment of the acrosome. Electron microscopy revealed that the acrosomal lumen of such heads remained full of matrix material, and the outer acrosomal membrane remained closely apposed to the other head structures. However, if activation had been allowed to take place, the resultant acrosin and the remaining proacrosin were associated with all sperm fractions and no specific location was observed. In these circumstances, the matrix material was almost entirely absent from the heads, and the outer acrosomal membrane, though usually still present, was only loosely attached.

It is concluded that (1) neither acrosin nor proacrosin are truly membrane-bound; they behave as 'diffusible' or partly soluble proteins, although they display a non-specific affinity for cell membrane and other surfaces; (2) proacrosin is part of the acrosomal matrix material, and is not located specifically on the inner or the outer acrosomal membrane; (3) the matrix material plays an important mechanical role in stabilizing the position of the outer acrosomal membrane relative to the inner acrosomal membrane; it disperses during proacrosin activation.

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S. D'Andrea, S. Chousterman, J. E. Flechon, and C. La Bonnardière

Interferon-γ IFN-γ and a type I IFN (spl IFN) are transiently coexpressed by trophoblastic cells of pig conceptuses at implantation between day 12 and day 20 of gestation. The local effects of these trophoblastic IFNs were examined on endometrial cells and on trophoblast by measuring antiviral activity and the induction of (2′,5′)-oligoadenylate synthetase activity. Trophoblastic vesicles were shown to be susceptible to infection by vesicular stomatitis virus and transmissible gastroenteritis virus. Vesicular stomatitis virus multiplied by about 1000 times in trophoblastic vesicles, and endogenous trophoblastic IFNs or exogenous recombinant IFN-γ or spl IFN had no effect on virus production. No (2′,5′)-oligoadenylate synthetase activity could be measured on the trophoblast, even after treatment with IFN-γ or spl IFN. These results clearly show that trophoblastic IFNs cannot induce antiviral resistance or (2′,5′)-oligoadenylate synthetase activity in the trophoblast, suggesting that these IFNs have no autocrine function. Endometrial epithelial and stromal cells in primary cultures displayed distinct sensitivity to the antiviral effect of IFN-γ and spl IFN. Stromal fibroblasts were highly sensitive to spl IFN but weakly sensitive to IFN-γ; epithelial cells were sensitive to both IFNs. The same sensitivity pattern was obtained when measuring the (2′,5′)-oligoadenylate synthetase activity. Flushing fluid, containing IFN-γ and type I IFN, was a potent inducer of antiviral effect and (2′,5′)-oligoadenylate synthetase activity. It is therefore postulated that the endometrial epithelium is the most likely target of trophoblastic IFNs. It is possible that these IFNs play a role in the viral protection of conceptuses.