Oocyte-derived growth factors and ovulation rate in sheep.

The physiological mechanisms controlling ovulation rate in mammals involve a complex exchange of endocrine signals between the pituitary gland and the ovary, and a localized exchange of intraovarian hormones between the oocyte and its adjacent somatic cells. The discoveries in sheep of mutations in bone morphogenetic protein 15 (BMP15) and bone morphogenetic protein receptor type IB (BMPR-IB) together with recent findings on the physiological effects of growth differentiation factor 9 (GDF9) and BMP15 on follicular development and ovulation rate highlight some important differences in the way in which the oocyte may function in mammals with different ovulation rate phenotypes. In sheep, BMP15 and GDF9 have each been shown to be essential for the early and later stages of follicular development. In addition, ovulation rate is sensitive to changes in the dose of either of these two oocyte-derived growth factors. These findings are in contrast to those reported for mice in which GDF9, but not BMP15, is essential for follicular development. The evidence to date is consistent with the hypothesis that the oocyte plays a central role in regulating key events in the process of follicular development and hence, is important in determining ovulation rate. Moreover, it appears that the mechanisms that the oocyte uses to control these processes differ between species with low and high ovulation rate phenotypes.

Comparison of serous and mucinous ovarian carcinomas: distinct pattern of allelic loss at distal 8p and expression of transcription factor GATA-4.

Using comparative genomic hybridization (CGH), we have previously demonstrated frequent loss of 8p, especially its distal part, in ovarian carcinoma. To compare the deletion map of distal 8p in serous and mucinous ovarian carcinomas, we performed allelic analysis with 18 polymorphic microsatellite markers at 8p21-p23. In serous carcinoma, loss of heterozygosity (LOH) was detected in 67% of the samples, and the majority of the carcinomas showed loss of all or most of the informative markers. In contrast, only 21% of mucinous carcinomas showed allelic loss, with only one or two loci showing LOH in each sample. In serous carcinomas, LOH was associated with higher grade tumors. Three distinct minimal common regions of loss could be defined in serous carcinomas (at 8p21.1, 8p22-p23.1, and 8p23.1). Expression of a transcription factor gene, GATA4, located at one of these regions (8p23.1) was studied in serous and mucinous ovarian carcinomas by Northern blotting and immunohistochemical staining of tumor microarray. Expression was found to be lost in most serous carcinomas but retained in the majority of mucinous carcinomas. Our results suggest distinct pathogenetic pathways in serous and mucinous ovarian carcinomas and the presence of more than one tumor suppressor gene at 8p involved in the tumorigenesis of serous carcinoma.

Transcription factors GATA-4 and GATA-6 and a GATA family cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors.

Previous studies have implicated transcription factors GATA-4 and GATA-6 in the regulation of murine ovarian development and function. In rodents, GATA-4 is expressed in granulosa cells of primary and early antral follicles, whereas GATA-6 is expressed in granulosa cells of late antral follicles and luteal glands. Both transcription factors can be detected in lesser amounts in theca cells and interstitial cells. We have now examined the expression of GATA-4 and GATA-6 in human ovaries, human granulosa-luteal (GL) cells and sex cord-derived tumors. We show by in situ hybridization and immunohistochemistry that GATA-4 and GATA-6 messenger RNA (mRNA) and GATA-4 protein are present in granulosa and theca cells in both preantral and antral follicles. Both human ovarian tissue samples and freshly isolated GL cells derived from preovulatory follicles of gonadotropin-treated women express GATA-4, GATA-6, and FOG-2 transcripts, and GATA-6 mRNA expression in GL cell cultures is stimulated by human CG and 8-bromo-cAMP. The vast majority of granulosa and theca cell tumors examined expressed GATA-4 and GATA-6. We also found that mRNA for FOG-2, a recently discovered regulator of GATA-4, is coexpressed with GATA-4 in human ovary samples, normal granulosa cells, and in sex cord-derived tumors. Our results demonstrate that GATA-4, GATA-6, and FOG-2 are expressed in human ovary and in granulosa and theca cell tumors. Our findings support a role for GATA-binding proteins in human ovarian folliculogenesis. Moreover, these data suggest that GATA factors may contribute to the phenotypes of sex cord-derived ovarian tumors.

Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner.

Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrophins and as yet unidentified ovarian factors precisely regulate follicular development so that, normally, only one follicle is selected to ovulate. The Inverdale (FecXI) sheep, however, carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+; ref. 1), but primary ovarian failure in homozygotes (FecXI/FecXI; ref. 2). Germ-cell development, formation of the follicle and the earliest stages of follicular growth are normal in FecXI/FecXI sheep, but follicular development beyond the primary stage is impaired. A second family unrelated to the Inverdale sheep also has the same X-linked phenotype (Hanna, FecXH). Crossing FecXI with FecXH animals produces FecXI/FecXH infertile females phenotypically indistinguishable from FecXI/FecXI females. We report here that the FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2-11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B (GDF9B)). Whereas BMP15 is a member of the transforming growth factor beta (TGFbeta) superfamily and is specifically expressed in oocytes, its function is unknown. We show that independent germline point mutations exist in FecXI and FecXH carriers. These findings establish that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.

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.

Immunochromatographic assay for quantitation of milk progesterone.

We describe a rapid immunochromatographic method for the quantitation of progesterone in bovine milk. The method is based on a 'competitive' assay format using the monoclonal antibody to progesterone and a progesterone-protein conjugate labelled with colloidal gold particles. The monoclonal antibody to progesterone is immobilized as a narrow detection zone on a porous membrane. The sample is mixed with colloidal gold particles coated with progesterone-protein conjugate, and the mixture is allowed to migrate past the detection zone. Migration is facilitated by capillary forces. The amount of labelled progesterone-protein conjugate bound to the detection zone, as detected by photometric scanning, is inversely proportional to the amount of progesterone present in the sample. Analysis is complete in less than 10 min. The method has a practical detection limit of 5 ng of progesterone per ml of bovine milk.

Affinity immunosensor for milk progesterone: identification of critical parameters.

The effects of several experimental parameters on the performance characteristics of a competitive-type immunochromatographic assay of milk progesterone were studied. Increasing the size of the colloidal gold particles used as a label increased both maximal signal obtained and sensitivity of the assay measured as slope of the progesterone standard curve. The concentration of the antibody used to prepare the detection zone was found to be critical factor, in that low concentrations of antibody resulted in a poor sensitivity. The compatibilities of various buffer systems with the assay were studied. The assay worked well with buffers having a broad pH range of 4.5-8.5.