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We demonstrated previously a negative association of granulosa cell cocaine- and amphetamine-regulated transcript (CARTPT) expression with follicle health status and inhibitory effects of the mature CARTPT peptide (CART) on follicle-stimulating hormone (FSH) signal transduction in vitro, resulting in reduced bovine granulosa cell CYP19A1mRNA and estradiol production. The objectives of this study were to investigate temporal regulation of granulosa cell CARTPT expression (granulosa cell mRNA and follicular fluid CART peptide concentrations) during follicular waves, CART regulation of androstenedione production (precursor for estradiol biosynthesis) by thecal tissue collected at specific stages of a follicular wave, FSH regulation of granulosa cell CARTPTmRNA expression, and the ability of CART to inhibit granulosa cell estradiol production and CYP19A1mRNA expression when administered in vivo. CART concentrations in healthy, estrogen-active follicles (estradiol greater than progesterone in follicular fluid) decreased after dominant follicle selection, and CARTPTmRNA was lower in healthy, estrogen-active versus estrogen-inactive atretic follicles (progesterone greater than estradiol) collected at the predeviation and early dominance stages. CART treatment reduced luteinizing hormone-induced androstenedione production by thecal tissue collected at predeviation and early dominance stages but not at later stages of a follicular wave. The FSH or insulin-like growth factor 1 treatment in vitro reduced granulosa cell CARTPTmRNA in a dose-dependent fashion. Administration of CART in vivo into follicles at the early dominance stage reduced follicular fluid estradiol concentrations and granulosa cell CYP19A1mRNA. Collectively, results support a potential stage-specific regulatory role for CART in negative regulation of estradiol production associated with selection of the dominant follicle.

The X-linked Rhoxgene cluster encodes homeobox transcription factors selectively expressed in reproductive tissues. The founding member of the mouse Rhoxgene cluster, Rhox5, is highly expressed in Sertoli cells, where it promotes the survival of the adjacent germ cells and modestly promotes fertility. The in vivo functions of the other 32 genes in the mouse Rhoxcluster have not been elucidated. Here, we report that a set of highly related Rhoxgenes (encoding proteins with >96% sequence identity) that have been duplicated eight times in mice - Rhox3ato Rhox3h- are essential for spermatogenesis. Because these eight Rhox3paralogs are interspersed with other Rhoxgenes, a knockout strategy to determine their in vivo function was not feasible, and so we employed a conditional RNA interference (RNAi) approach instead. Towards this end, we first identified a short interfering RNA (siRNA) displaying 100% sequence complementary with all eight Rhox3paralogs that efficiently downregulated RHOX3 level in cell lines (by 90%). This siRNA sequence was inserted (in the form of a short hairpin [sh]) downstream of a ubiquitously expressed Pol III promoter rendered non-functional by a DNA insertion flanked by loxP sites. Several transgenic lines harboring this Rhox3shRNA construct were obtained and mated with transgenic mice lines expressing Cre recombinase to remove the insert and activate Rhox3shRNA expression. Double-transgenic mice lines expressing Cre from a male germ cell promoter (Stra8) exhibited reduced RHOX3 protein levels in the testes (up to 90%) and had a profound block in post-meiotic germ cell progression and >1% of normal sperm count. These Rhox3-knockdown mice also had reduced expression of some meiotic genes, suggesting that the Rhox3paralogs also have a role in meiosis. Given that the Rhox3paralogs encode transcription factors, we propose that they regulate key genes essential for spermatogenesis, which we are currently in the process of identifying. The conditional RNAi approach that we used to identify the in vivo function of the Rhox3paralogs has the potential to be a generally applicable and efficient means to elucidate the cell type-specific functions of genes.(platform)

A growing body of evidence suggests oocyte developmental competence is a limiting factor in pregnancy success in multiple species, including humans. However, the inherent phenotypic characteristics of competent oocytes are not well understood. We have conducted fundamental studies using the bovine model to elucidate differences in oocyte and cumulus cell transcriptome composition associated with poor oocyte competence and the potential diagnostic and therapeutic significance of such results. The bovine model was chosen because ovarian physiology and several aspects of embryo development are similar between the two single-ovulating species and because of existence of multiple, well established bovine models of poor oocyte competence. Of particular interest from microarray studies is the increased transcript abundance for members of the cathepsin family of lysosomal cysteine proteinases (cathepsin Z, S and B) in cumulus cells surrounding oocytes collected from prepubertal (model of poor oocyte competence) versus adult animals. Variation in cumulus cell expression of mRNA for these genes for oocytes collected from adults is also associated with success of blastocyst development. Treatment of bovine oocytes with a cathepsin inhibitor during meiotic maturation enhances rates of blastocyst development following in vitro fertilization (IVF) demonstrating therapeutic potential of inhibition of cathepsin activity. Stimulatory effects of cathepsin inhibitor treatment on subsequent blastocyst development following IVF are both dependent on presence of cumulus cells during meiotic maturation and associated with reduced rates of cumulus cell apoptosis. We have also observed a positive relationship between oocyte expression of follistatin and oocyte quality in two distinct models of poor oocyte competence. Follistatin supplementation and ablation experiments in early embryos were performed to determine the functional significance of such findings. Treatment of presumptive zygotes with follistatin during embryo culture in vitro (prior to embryonic genome activation) enhances blastocyst development and blastocyst cell allocation in favor of trophectoderm. In contrast, follistatin ablation via microinjection of follistatin siRNA into presumptive zygotes results in reduced rates of blastocyst development and decreased trophectoderm cell numbers. These effects of follistatin ablation can be rescued by treatment of siRNA injected embryos with exogenous follistatin. To begin to address the potential translational relevance of our results, expression of above markers of oocyte competence in the rhesus monkey model is under investigation using the Primate Embryo Gene Expression Resource (PREGER). Results to date indicate that cathepsin expression is increased in rhesus monkey blastocysts in response to in vitro culture, suggesting that cathepsin expression may also be negatively associated with embryo competence in primates. Collectively, results indicate that cathepsins and follistatin are potential markers to identify oocytes with enhanced developmental competence and that manipulation of cathepsin activity and follistatin levels during in vitro culture has therapeutic potential to improve success of embryo development.