172 RELATIONSHIPS AMONG GRANULOSA CELL FGF9 mRNA, FOLLICLE SIZE, AND APOPTOSIS IN CATTLE

Abstract

Fibroblast growth factor 9 (FGF9) has been suggested to act as a dedifferentiation factor during bovine folliculogenesis, reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells, but whether endogenous GC production of FGF9 change during bovine folliculogenesis and atresia/apoptosis is unknown. The objective of these studies was to investigate the relationship between FGF9 mRNA, follicle size, and health status of follicles. Ovaries (n = 10 cows) from a local abattoir classified visually as in midcycle phase (i.e. presence of corpus luteum and large follicles) were collected and categorized as small (1–5 mm), medium (5.1–8 mm) or large (8.1–22 mm) in size (Experiment 1). Follicular fluid (FFL) was aspirated for measurement of oestradiol (E2) and progesterone (P4) via radioimmunoassay and GC collected for RNA extraction. Abundance of mRNA for FGF9 and Caspase-3 (CASP3), an effector of apoptosis, were measured by real-time PCR (qPCR). Data were analysed via factorial ANOVA with main factors: follicle size, follicle estrogenic status, and their interaction. The abundance of GC FGF9 mRNA was greater (P < 0.05) in large E2-inactive (E2P4 concentrations) follicles (10.5 ± 22). The abundance of GC CASP3 mRNA was greater (P < 0.01) in small E2-inactive follicles than in large and medium E2-active and E2-inactive follicles. FGF9 mRNA abundance was not correlated with E2/P4 ratio in FFL, but it was positively correlated with CASP3 mRNA abundance (r = 0.35; P < 0.05). GC CASP3 mRNA abundance was negatively correlated with E2/P4 ratio (r = –0.48; P < 0.01). To investigate the relationship between FGF9 and CASP3 mRNA abundance during experimentally-induced apoptosis, GC from large and small follicles were collected (Experiment 2) and GC were plated in medium containing 10% FCS. GC (n = 3 independent pools for small and large follicles) were then treated with or without 10% FCS for an additional 24 h or 48 h followed by RNA extraction and qPCR for measurement of abundance of FGF9 and CASP-3 mRNA. Statistical analyses with ANOVA included main factors: treatment, duration of treatment, and their interaction. In small-follicle GC, FGF9 and CASP3 mRNA abundance were not correlated and were not affected by treatments. In large follicles, FGF9 mRNA abundance was greater in GC treated without FCS (27.5 ± 2.7) than in GC treated with 10% FCS (6.6 ± 2.7) and tended to differ (P < 0.08) between 24 h (22.5 ± 2.7) and 48 h (11.6 ± 2.7). CASP3 mRNA abundance was greater in GC treated without FCS (310 ± 36) than in GC treated with 10% FCS (140 ± 36) but did not differ (P > 0.10) between 24 h and 48 h. In Experiment 2, there was no significant correlation between FGF9 and CASP3 mRNA (r = 0.28; P = 0.2). These results indicate that FGF9 mRNA abundance is greater in GC from large E2-inactive than from E2-active follicles and its production may be increased in large follicles undergoing apoptosis.