The effects of insulin, and insulin-like growth factors I and II on human ovarian follicles in long-term culture.

The aim of this study was to investigate the effects of insulin and insulin-like growth factors I and II (IGF-I and IGF-II) on human ovarian follicles in vitro. Ovarian cortical tissue slices (0.1-0.3 cm) were cultured for 7 or 14 days on an artificial extracellular matrix and with FSH. The ovarian tissue cultures were stimulated by insulin (33 ng/ml), IGF-I (20 or 50 ng/ml) or IGF-II (20 ng/ml). Combined effects of IGF-I (20 ng/ml) or IGF-II (20 ng/ml) and insulin (33 ng/ml) were also studied. Proliferating cell nuclear antigen (PCNA) was selected for immunohistochemical examination activation of the mitotic cell cycle in granulosa cells. After 1 week of culture the number of follicles had decreased in all cases. After 2 weeks of culture the number of healthy follicles had decreased dramatically in control cultures. However, the loss of follicles could be prevented with insulin and IGFs. The number of atretic follicles was significantly lower in insulin cultures compared with control cultures after 2 weeks. The proportion of primary follicles was significantly increased in cultures treated with insulin, IGF-I (50 ng/ml) or IGF-II (20 ng/ml) compared with control cultures after 2 weeks. A similar effect was seen after co-treatment with IGF-II and insulin. There were significantly more PCNA-positive follicles in IGF-I cultures than in control cultures. These results suggest that insulin, IGF-I and IGF-II may act as survival factors for early stage human follicles. IGFs may also be involved in activation of the mitotic cell cycle of granulosa cells.

Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B.

Although targeted gene disruption of GDF-9, an oocyte derived growth factor, leads to an arrest of folliculogenesis and causes infertility in female mice, little is known on the expression of GDF-9 protein in the ovary. We show that GDF-9 protein is expressed in rat oocytes during folliculogenesis from the early primary follicle stage onwards but the most intensive immunostaining was seen in primary and preantral follicles. Northern blot analyses of the ontogeny of GDF-9 gene expression in postnatal rat ovaries showed that the GDF-9 transcript levels are clearly increased on the second postnatal day concomitant with the appearance of primary follicles. Interestingly, Northern blot and in situ hybridization analyses indicate a similar expression pattern for GDF-9B, the rat ortholog of a mouse GDF-9 like factor for which we recently reported the partial amino acid sequence. The polypeptide sequences deduced from isolated ovarian cDNAs indicate that the rat GDF-9 prepropeptide is 440 amino acids (aa) in length and the putative mature peptide is 135 aa whereas rat GDF-9B is 391 aa long and the mature region is 125 aa. We conclude that (1) the GDF-9 protein is highly expressed in the oocytes of primary follicles of rat ovaries suggesting that it plays a role mainly in early folliculogenesis and that (2) the full-length polypeptide sequence of GDF-9B suggests that this novel TGF-beta family member is likely to be a secreted growth factor that may regulate folliculogenesis at similar developmental stages as GDF-9.

Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis.

Growth differentiation factor 9 (GDF-9) is a transforming growth factor-beta family member that is required for normal folliculogenesis in female mice, but its role as a regulator of human fertility is still unclear. We determined here by in situ hybridization and immunohistochemical analyses the localization of the GDF-9 messenger ribonucleic acid (mRNA) and protein during human folliculogenesis. The GDF-9 transcripts were not detected in primordial follicles, but they are abundantly expressed in primary follicles in frozen sections of ovarian cortical tissue material obtained at laparoscopic surgery. We raised antipeptide antibodies against GDF-9 and showed by immunohistochemical studies on paraffin sections of whole human ovaries that the GDF-9 protein is most abundantly expressed in primary follicles. We recently demonstrated that a novel GDF-9-related factor, GDF-9B, is coexpressed with GDF-9 during murine folliculogenesis. We now isolated human GDF-9B complementary DNA and genomic clones and report the unusually restricted expression pattern of human GDF-9B. The human GDF-9B transcript can be detected only in the gonads by RT-PCR analysis, and in situ hybridization studies indicate that it is not expressed in small primary follicles but, rather, in the oocytes of late primary follicles. Functional studies using the Xenopus laeuis embryo model indicate that unlike the transforming growth factor-beta family members activin and bone morphogenetic protein-4, neither GDF-9 nor GDF-9B affects mesoderm induction, suggesting that they may use signaling pathways distinct from those well defined for activin and bone morphogenetic protein-4. We conclude that 1) both GDF-9 mRNA and protein are abundantly expressed in oocytes of primary follicles in human ovary, suggesting that the GDF-9 transcript is translated at this early stage of folliculogenesis; 2) human GDF-9B is specifically expressed in gonads at low levels; and 3) the expression of GDF-9 mRNA begins slightly earlier than that of GDF-9B in the human oocytes during follicular development. Our results are consistent with the suggestion that GDF-9 and GDF-9B may regulate human folliculogenesis in a manner specific to the ovary.