In vivo gene expression in granulosa cells during pig terminal follicular development.

Ovarian antral follicular development is clearly dependent on pituitary gonadotrophins FSH and LH. Although the endocrine mechanism that controls ovarian folliculogenesis leading to ovulation is quite well understood, the detailed mechanisms and molecular determinants in the different follicular compartments remain to be clarified. The aim of this study was to identify the genes differentially expressed in pig granulosa cells along the terminal ovarian follicle growth, to gain a comprehensive view of these molecular mechanisms. First, we developed a specific micro-array using cDNAs from suppression subtractive hybridization libraries (345 contigs) obtained by comparison of three follicle size classes: small, medium and large antral healthy follicles. In a second step, a transcriptomic analysis using cDNA probes from these three follicle classes identified 79 differentially expressed transcripts along the terminal follicular growth and 26 predictive genes of size classes. The differential expression of 18 genes has been controlled using real-time PCR experiments validating the micro-array analysis. Finally, the integration of the data using Ingenuity Pathways Analysis identified five gene networks providing descriptive elements of the terminal follicular development. Specifically, we observed: (1) the down-expression of ribosomal protein genes, (2) the genes involved in lipid metabolism and (3) the down-expression of cell morphology and ion-binding genes. In conclusion, this study gives new insight into the gene expression during pig terminal follicular growth in vivo and suggested, in particular, a morphological change in pig granulosa cells accompanying terminal follicular growth.

A first catalog of genes involved in pig ovarian follicular differentiation.

As a first step toward the characterization of genetic expression in pig ovaries, we have selected 238 clones by differential hybridization from a pig granulosa cell cDNA library, using probes prepared from RNA extracted from either untreated or FSH-treated cells and, in order to generate expressed sequence tags (ESTs), we have performed 3' and 5' single-pass sequencing of these clones. Sequences of the 3' end of the 167 clones that produced informative sequence data were first compared with each other, revealing a redundancy level of 21%. Sequences from the 136 unique clones were analyzed for similarities with sequence data included in Genbank and EMBL databases. Among these unique clones, 54 (40%) matched significantly with sequences from either Genbank of EMBL: 4 with known genes in pig, 35 matched with previously reported human genes, and 15 with other mammalian genes. Eighty-two clones (60%) showed no significant match with any gene or DNA sequence in the Genbank and EMBL databases and thus may represent new pig transcripts.

Expression of messenger ribonucleic acids of insulin-like growth factor binding protein-2, -4, and -5 in the ovine ovary: localization and changes during growth and atresia of antral follicles.

In the sheep as in many mammalian species, growth and atresia of antral follicles are characterized, respectively, by a decrease and a high increase in the intrafollicular levels of insulin-like growth factor binding proteins of less than 40 kDa (IGFBPs < 40 kDa), mainly IGFBP-2, -4, and -5. The objective of this study was to investigate whether such changes are associated with changes in follicular expression of the corresponding mRNA. For this purpose, ovaries were recovered from ewes slaughtered at the end of follicular phase (i.e., 30 h after progestagen sponge removal; control ewes) or at 24 h, 36 h or 72 h after hypophysectomy (hypox) performed 30 h after sponge removal. The expression of mRNA of IGFBPs of less than 40 kDa (IGFBPs < 40 kDa mRNA) was studied in ovine antral follicles from control and hypox ewes by in situ hybridization using [35S]-labeled human IGFBP-2, -4, and -5 cRNA as probes. In control ewes, IGFBP-2 mRNA was mainly expressed in granulosa as a gradient in healthy follicles, the expression being higher in granulosa cells close to the basal membrane than in granulosa cells bordering the antrum and within the cumulus. The level of IGFBP-2 mRNA was lower both in granulosa cells close to the basal membrane and in those bordering the antrum from small follicles than in the corresponding compartments of granulosa cells from large healthy follicles (p < 0.05). In healthy follicles, IGFBP-4 and -5 mRNA were mainly expressed in thecal cells. No change in level of IGFBP-4 mRNA was observed between small and large follicles, whereas the level of IGFBP-5 mRNA tended to be lower in thecal cells from large compared to small follicles (p = 0.055). In atretic follicles, expression of IGFBPs < 40 kDa mRNA strongly increased in granulosa (IGFBP-2 and -5, p < 0.01) and in thecal cells (IGFBP-2 and -4, p < 0.01). In hypox ewes, the chronology of changes in expression of follicular IGFBPs < 40 kDa mRNA and in intrafollicular levels of the corresponding proteins was studied during atresia of large antral follicles. Early atresia of large follicles was associated with a strong decrease in intrafollicular estradiol levels (p < 0.001); an increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001) an increase in both IGFBP-2 (p < 0.001) and -5 (p < 0.01) mRNA expression in granulosa and thecal cells; but no changed in IGFBP-4 mRNA expression. Late atresia of large follicles was associated with a further decrease in intrafollicular estradiol levels (p < 0.01); a further increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001); an increase in IGFBP-4 (p < 0.01) and -5 (p < 0.05) mRNA expression in theca and granulosa, respectively; a decrease in IGFBP-5 mRNA expression in theca (p < 0.05); but no further increase in IGFBP-2 mRNA expression. Overall, these data suggest that the decrease and the increase in expression of mRNA of follicular IGFBPs < 40 kDa during follicular growth and atresia, respectively, are involved in the decrease and the increase in intrafollicular levels of the corresponding proteins. Moreover, the increases in expression of follicular IGFBPs < 40 kDa during atresia of large follicles in hypophysectomized ewes followed a specific time course, the increase in IGFBP-2 and -5 mRNA expression being early than the increase in IGFBP-4 mRNA expression.

Protein kinase C inhibition of in vitro FSH-induced differentiation in pig granulosa cells.

In granulosa cells, growth factor IGF I plays a major role in both growth and differentiation, acting through an autocrine/paracrine mechanism, and its production is regulated by FSH, via cyclic AMP (cAMP). As protein kinase C is also involved in granulosa cell function, we investigated the possibility that its activation could balance the positive effects of FSH. Using pig granulosa cells cultured in vitro, we studied the effects of protein kinase C activation by tetradecanoylphorbol acetate (TPA) on IGF I mRNA level. We also checked morphological modifications, cAMP production and steroidogenesis at the P450 side chain cleavage mRNA and progesterone levels. Our data demonstrate that protein kinase C activation antagonizes the in vitro FSH-induced differentiation, particularly morphological modifications and accumulation of IGF I mRNA. These inhibitory effects on FSH responses suggest that there could be a balance between protein kinase A and protein kinase C pathways in regulating differentiation in pig granulosa cells.

Gonadotropins induce accumulation of insulin-like growth factor I mRNA in pig granulosa cells in vitro.

Pig granulosa cells have been shown to synthesize insulin-like growth factor (IGF) I peptide in vitro, and this expression is regulated by gonadotropins via the cAMP pathway. By hybridizing an IGF I cDNA probe with total RNA isolated from pig granulosa cells cultured in vitro, we show that these cells contain two IGF I transcripts of about 0.9 kb and 9 kb in size. Treatment of the cells with gonadotropins (follicle-stimulating hormone, luteinizing hormone) or cAMP agonists (dibutyryl-cAMP, forskolin) induces an accumulation of the transcripts which can be abolished by transcriptional inhibitors, but not by translational inhibitors. We thus provide new evidence that pig granulosa cells are a site of IGF I synthesis, and we conclude that (1) gonadotropins increase IGF I mRNA levels; (2) the accumulation of IGF I mRNA results from an increased transcription; (3) the stimulation of IGF I gene transcription does not require ongoing protein synthesis; (4) these effects of follicle-stimulating hormone can be mimicked by cAMP agonists.

[Gene mapping in the pig (Sus scrofa 1.). I. Study of two syntenic groups G6PD, PGK, HPRT and PKM2, MPI (author's transl)]. / Carte génique du porc (Sus scrofa .1). I. Etude de deux groupes synténiques G6PD, PGK, HPRT et PKM2, MPI.

Thirteen pig-hamster and fifteen pig-mouse hybrid ceil lines were developed. These lines eliminate the pig chromosomes preferentially. The following syntenies were demonstrated: G6PD, PGK, HPRT, and PKM2, MPI.