Jacques E. Fléchon

Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells

Muscle and skin biopsies taken from bovine fetuses and young calves have been used as a source of donor nuclei for cloning experiments. After culture, cells were individually fused to enucleated matured oocytes and the resulting blastocysts obtained after 7 d of culture (3-8% depending on the cell type) were transferred to foster recipient heifers. Two calves, a female and a male, both originating from muscle cells were born, and four additional pregnancies have surpassed mild-term gestation. The pregnancies include one fetus established from a transgenic nucleus from a fetal skin cell, and another one resulting from a skin biopsy performed on a female calf. Our data demonstrate that nuclei from cultured bovine somatic cells obtained from differentiated tissues can be made multipotent.

Developmental regulation of effect of epidermal growth factor on porcine oocyte-cumulus cell complexes: nuclear maturation, expansion, and F-actin remodeling.

Epidermal growth factor (EGF) efficiently stimulates expansion of mouse and rat oocyte–cumulus complexes (OCC). Contradictory data have been published by several laboratories about the ability of EGF to stimulate expansion of porcine OCC. We assumed that these contradictions may have resulted from heterogeneous conditions used for isolation, culture, and assessment of OCC. The present experiments were designed to test the hypothesis that porcine OCC acquire the ability to synthesize hyaluronic acid (HA) and undergo expansion following EGF‐stimulation gradually during the growth of follicles. For this reason, we isolated OCC from follicles of different sizes and assessed quantity of produced HA and proportions of expanding OCC after stimulation by EGF. In addition, we assessed in those OCC changes in morphology of cumulus cells and assembly of F‐actin microfilaments, which are necessary for expansion to occur. Finally, nuclear maturation of EGF‐stimulated OCC was assessed and its relationship with occurrence of expansion was evaluated. In all experiments, OCC stimulated with FSH were used as positive controls. The results showed that EGF did not stimulate production of HA, rearrangement of F‐actin and expansion in OCC isolated from small follicles (<4 mm in diameter). OCC isolated from large preovulatory follicles (6–7 mm in diameter and PMSG‐stimulated follicles) underwent efficient expansion when stimulated by EGF (93% and 100%, respectively). EGF dramatically stimulated total production of HA in these OCC and its retention in extracellular matrix of the expanding cumulus. Cumulus cells of the large OCC underwent essential changes of their morphology and extensive rearrangement of F‐actin microfilaments following stimulation with EGF. Interestingly, EGF enhanced nuclear maturation of OCC isolated from both small and large follicles, which suggest diversity of signaling pathways controlling maturation and expansion. FSH caused cumulus expansion, F‐actin remodeling, and enhancement of oocyte nuclear maturation in OCC originated from both small and large follicles. We conclude that EGF can stimulate expansion of porcine OCC in vitro; however, only of those isolated from large follicles. This indicates that EGF may have a physiological role in regulation of porcine cumulus expansion in preovulatory follicles, presumably as a mediator of signals elicited by the LH surge. Mol. Reprod. Dev. 56:63–73, 2000.

Comparative immunohistochemical distribution of connexin 37 and connexin 43 throughout folliculogenesis in the bovine ovary

Among gap junctional proteins previously identified in the mouse ovary, connexins (Cx) Cx37 and Cx43 appeared to be essential for normal follicular growth. The aim of this work was to detect Cx37 expression in the bovine ovary, then to quantify and compare its follicular distribution pattern with that of Cx43 using quantitative analysis of immunofluorescently labeled ovary sections viewed with a confocal laser scanning microscope. Cx37 immunoreactivity was detected in bovine ovarian follicles and was predominantly localized at preantral stages. Unlike follicular Cx43 expression which was restricted to granulosa cells, Cx37 staining was observed in both oocyte and granulosa cell compartments. While no changes were seen during early follicular growth, the level of Cx37 expression decreased significantly at the onset of antral cavity formation (P ≤ 0.01). On the contrary to what was found for Cx37, Cx43 was weakly expressed in preantral follicles. Concomitant with antrum formation, the level of Cx43 expression increased significantly (P ≤ 0.01). A further increase was correlated with antral follicular size (P ≤ 0.01). Cx43 immunoreactivity declined significantly in morphologically atretic follicles (P ≤ 0.01). A comparative analysis showed that Cx37 and Cx43 expression patterns were differentially regulated and could reflect specific physiological roles for each gap junction protein throughout folliculogenesis in cow. Mol. Reprod. Dev. 57:60–66, 2000.

The extracellular matrix of porcine mature oocytes: Origin, composition and presumptive roles

The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine – precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans – for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.

Expression of fetal-type intermediate filaments by 17-day-old rat Sertoli cells cultured on reconstituted basement membrane

SummaryThe expression of cytokeratin- and vimentin-type intermediate filaments was studied by means of immunohistochemistry in Sertoli cells cultured on two types of reconstituted basement membrane in two-compartment culture chambers. In situ, the Sertoli cells of 17-day-old rats contained only vimentin intermediate filaments. During culture, a gradual reorganization of intermediate filaments accompanied by an increased cytokeratin immunoreactivity was observed. After 6 days, Sertoli cells contained both cytokeratin and vimentin, and the same cytokeratin type as in fetal and newborn testis was revealed by electrophoresis and immunoblotting. The present study shows that the isolation and culture of Sertoli cells causes, even in an improved culture system qualitative changes in the expression of intermediate filament proteins.

RGD-mediated adhesion of porcine granulosa cells modulates their differentiation response to FSH in vitro

Summary— Porcine granulosa cells cultured in serum‐free medium undergo metabolic and morphologic changes after follicle stimulating hormone (FSH) stimulation. Under these conditions, granulosa cells differentiate and tend to round up and their links with the plastic support are reduced. Coating of culture substratum with PepTite‐2000, an integrin‐binding synthetic peptide containing RGD (Arg‐Gly‐Asp) sequences enhanced the plating of granulosa cells. Whether the peptide be present or not, cells cultivated in basal synthetic medium (without FSH) were flattened and attached to the substratum by stress fibers at focal contacts where integrin β1, extracellular fibronectin, and urokinase plasminogen activator colocalized. After FSH stimulation, part of the cells rounded up and F‐actin took a more uniform, cortical localization. Correlatively, extracellular fibronectin aggregated in a clump, while integrin β1 and urokinase plasminogen activator spread over rounded cells. These morphological changes elicited by FSH were little affected by the presence of PepTite‐2000, yet a larger number of cells remained flattened. However, concerning steroidogenesis, increasing concentrations of peptide seemed to favor progesterone rather than estrogen production, and to restrain luteinizing hormone (LH) receptor expression, suggesting a premature committment of cells towards luteinization rather than completion of follicular preovulatory differentiation.

Characterization of embryonic germ cells derived from primordial germ cells in the mouse

TCFP MEDIATES THE ACTION OF RETINOIDS AND VITAMIN D3 ON U937 CELLS DIFFERENTIATION. COMMES T&&e, PIQUEMAL David, DEFACQIJE H&ne , SEVILLA Claude et MARTI Jacques. INSERM u431. Univ-A4ontpII 34 095 Montpellier FRANCE Retinoids and vitamin D (VD) cooperate to inhibit the proliferation and induce the differentiation of human myelomonocytic leukemia cells. in order to investigate the role of TGFP as a possible mediator in this process, we used antibodies neutralizing the cytokine activity (aTGFb-Ab) r,nd studied their effects on the differentiation of U937 cells induced by various combinations of VD and synthetic retinoids. Our data demonstrate that aTGFP-Ab partially inhibit the expression of the differentiated phenotype, as assessed by measurement of phagocytic activity, response to chemobctic peptides, secretion of lysozyme and CD1 1 b expression. We also used a& sense oligonucleotides (TGF-AS) to inhibit the expression of TGFP in U937 cells, as monitored by immunofluorescence labelling. We found hat combinations of TGF-AS and aTGF-Ab had cumulative effects. Cell growth inhibition induced by VD and retinoids was nearly completely revened ad cell differentiation was largely reduced. Tie-course experiments, based on the delayed additions of aTGFb-Ab and differentiation inducers, showed that neutralizing antibodies have an effect only if added tit& he first 24h of treatment, suggesting that TGFP is involved in an early step of the differentiation process. Studies on the expression of TGFP receptor n&NAs by RT-PCR revealed that, while typ-e II receptor r&NA level remained stable, the amount of Type I TGFP receptor mRNA decreased within the first 12 h of treatment by differentiation inducers, suggesting further desensitization of cells to TGFP. Northern blot analyses showed detectable levels of TGFP mRNA in proliferating U937 cells. No variations were observed at this level upon differentiation, but the secretion of the latent TGFP protein precursor, as measured by ELBA, was increased from <SO0 pg/ml up to 2.5 &ml after 96 h in cultures supematants (normalized for 106 cells/ml) indicating a positive regulation at the post-transcriptional level. The amount of TGFP protein precursor remained low during the first 24 hours and the biologically active form of thi cytokine was not d&e&d in cell supematants. However, independent experiments showed that active recombinant TGFP actually potentiates the action of VD on cell growth inhibition and differentiation, at concenlra;ltions as low as 10 pg/ml. All these results support the notion that an autocrine TGFP pathway, activated in U937 cells treated by VD and retinoids, is involved in the early steps of the process leading to cell growth arrest and differentiation.