Females with Reduced Fertility Have Excess Androstenedione in Follicular Fluid, Altered Theca Gene Expression and Increased VEGFA164b, Maternal Effect, and microRNA Processing mRNA Levels in Cumulus-Oocyte Complexes.

Abstract

Ovarian dysfunction contributes significantly to female infertility. However, the intrinsic and exogenous factors that result in abnormal ovarian function are poorly defined. Thus, we have established a cow model of fertility to identify mechanisms regulating follicular growth, steroidogenesis and oocyte growth and maturation. Culling age due to failure to establish pregnancy was used to classify animals with low (>/= 2 years-of-age), moderate (> 2 - < 6 years-of-age), and high (>/= 6 years-of-age) fertility. Previous experiments using this model identified class-dependent changes in mural granulosa cell gene expression. The objective of this study was to identify the effect of ovarian and follicular environment on the oocyte molecular phenotype. To attain this objective, females from each fertility group were synchronized using a modified Co-Sync (CIDR) protocol and ovariectomies were performed 36 h after PGF2-alpha injection and CIDR removal. Surface antral follicle counts (AFC) for each ovary pair and the follicular fluid, theca cells, mural granulosa cells and cumulus-oocyte complex (COC) from each dominant follicle were collected. Dominant follicles were confirmed based on size (i.e. largest follicle on the ovaries) and an estrogen- to-progesterone ratio greater than 1.0 in the follicular fluid. Antral follicle counts were lower in low fertility (n = 14) than moderate (n = 17) or high fertility (n = 17) cows. Furthermore, androstenedione (A4) concentrations were 4 fold (P < 0.002) greater in follicular fluid from low compared to high fertility group cows. Given that increased androgens is a hallmark characteristic of the PCOS phenotype, gene expression in theca cells from the different fertility groups was compared. The affect of increased intrafollicular androgen levels on cumulus-oocyte gene expression in the dominant follicle was also examined. Quantitative, real-time RT-PCR demonstrated that mRNA abundance of maternal effect genes NLRP5 (8-fold P < 0.0001), Zar1 (2-fold; P < 0.0001) and Dnmt1 (P < 0.006) and miRNA processing genes DGCR8 (P < 0.005) and RNAsen (P < 0.005) were increased in low compared to high fertility cows. Increased mRNA abundance in low fertility animals is consistent with previous findings in PCOS oocytes and suggests that abnormal endocrine profiles of follicular fluid has a global effect on transcriptional and post-transcriptional regulation of mRNAs in the oocyte. Previous studies identified differences in the expression profile of vascular endothelial growth factor A (VEGFA) isoforms in mural granulosa cells between low, moderate, and high fertility group cows. In the current study, VEGFA_164B mRNA abundance was also increased in cumulus granulosa cells of low fertility cows (P < 0.005). Taken together, these data indicate that females with low fertility have intrinsic differences in ovarian morphology and steroidogenesis. Furthermore, the data suggest that increased androgen production in low fertility animals alters gene expression and/or mRNA stability during oocyte growth and maturation. These changes in oocyte gene expression may reduce the developmental competence of the oocyte and contribute to the reduced pregnancy rates in low fertility cows. USDA is an equal opportunity employer. (poster)