Small noncoding RNA molecules (miRNA) regulate protein levels in a post-transcriptional manner by partial base pairing to the 3'-UTR of target genes thus mediating degradation or translational repression. Previous studies indicate that numerous miRNA regulate the biosynthesis of intraovarian hormones, and emerging evidence indicates that one of these, miRNA-221 (MIR221), may be a modulator of ovarian function. However, the hormonal control of ovarian MIR221 is not known. The objectives of this study were to investigate the developmental and hormonal regulation of MIR221 expression in granulosa (GC) and theca cell (TC) and its possible role in regulating follicular function. Bovine ovaries were collected from a local abattoir and GC and TC were obtained from small (<6 mm) and large (≥8 mm) follicles. In Exp. 1, GCs of small follicles had 9.7-fold greater (P < 0.001) levels of MIR221 than those of large follicles, and TCs of large follicles had 3.7-fold greater (P < 0.001) levels of MIR221 than those of small follicles. In large follicles, abundance of MIR221 was 66.6-fold greater (P < 0.001) in TCs than in GCs. In small follicles, MIR221 abundance did not differ (P = 0.14) between GC and TCs. In vitro Exp. 2, 3, and 4 revealed that treatment of bovine TCs with various steroids, phytoestrogens, IGF1, forskolin, and dibutyryl cyclic adenosine monophosphate had no effect (P > 0.35) on MIR221 expression, whereas treatment with fibroblast growth factor 9 (FGF9) and FGF2 increased (P < 0.001) TC MIR221 abundance 1.7- to 2.5-fold. In Exp. 5, FGF9 increased (P < 0.05) GC MIR221 abundance by 1.7- and 2.0-fold in small and large follicles, respectively. The role of MIR221 in GC steroidogenesis was investigated in Exp. 6 and it was found that transfection with a MIR221 mimic reduced (P < 0.01) GC estradiol and progesterone production induced by FSH and IGF1, whereas transfection with MIR221 inhibitor had little or no effect. We conclude that thecal MIR221 expression is increased by FGF9 and increased MIR221 may act to inhibit GC steroidogenesis in cattle.

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The E2F family of transcription factors plays an important role in the control of the cell cycle, cell proliferation, and differentiation, and their role in ovarian function is just emerging. Although some evidence suggests a possible role of E2F1 in ovarian follicular development, what regulates its production in ovarian cells is unknown. Objectives of this study were to determine whether: (i) E2F1 gene expression in granulosa cells (GCs) and theca cells (TCs) change with follicular development and (ii) E2F1 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F1 mRNA abundance in GC was 5.5-fold greater (P &lt; 0.05) in small (SM; 1 to 5 mm) than large (LG; &gt;8 mm) follicles, but in TC, E2F1 expression did not differ among follicle sizes. SM-follicle GC had 2.1-fold greater (P &lt; 0.05) E2F1 mRNA than TC. In SM-follicle GC, FGF9 induced a 7.6-fold increase in E2F1 mRNA abundance; however, FGF9 did not affect (P &gt; 0.10) abundance of E2F1 mRNA in LG-follicle TC or GC. Follicle-stimulating hormone (FSH) had no effect (P &gt; 0.10) on E2F1 gene expression in SM- or LG-follicle GC. SM-follicle GC were concomitantly treated with insulin-like growth factor 1 (30 ng/mL), FSH (30 ng/mL), and either 0 or 30 ng/mL of FGF9 with or without 50 µM of an E2F inhibitor (E2Fi; HLM0064741); FGF9 alone increased (P &lt; 0.05) GC numbers, whereas E2Fi alone decreased (P &lt; 0.05) GC numbers, and concomitant treatment of E2Fi with FGF9 blocked (P &lt; 0.05) this stimulatory effect of FGF9. Estradiol production was inhibited (P &lt; 0.05) by FGF9 alone and concomitant treatment of E2Fi with FGF9 attenuated (P &lt; 0.05) this inhibitory effect of FGF9. SM-follicle GC treated with E2Fi decreased (P &lt; 0.05) E2F1 mRNA abundance by 70%. Collectively, our studies show that GC E2F1 mRNA is developmentally and hormonally regulated in cattle. Inhibition of E2F1 reduced FGF9-induced GC proliferation and attenuated FGF9-inhibited estradiol production, indicating that E2F1 may be involved in follicular development in cattle.

Recently, we discovered using microarray analysis that expression of cingulin (CGN), a cytoplasmic tight junction protein, is significantly down‐regulated by fibroblast growth factor 9 (FGF9) in cultured ovarian theca cells (TC). FGF9 is known to increase cell proliferation and decrease steroidogenesis in bovine TC. The objective of the present studies was to investigate the hormonal regulation of CGN gene expression in bovine TC. Ovaries of cattle were collected at a local slaughterhouse, and TC isolated from large follicles (8‐22 mm). TC were cultured for 2 days in 10% fetal bovine serum followed by 24 h in serum‐free medium containingvarious treatments in three experiments, and TC RNA was isolated to measure abundance of CGN mRNA by real‐time PCR. Exp. 1: TC were treated with 30 ng/ml of FGF9 for either 0, 4, 12, or 24 h, and CGN mRNA measured; data were analyzed via 3×2 factorial ANOVA. CGN mRNA abundance averaged 60% less (P < 0.05) in FGF9‐treated than in control TC at both 12 h and 24 h. Exp. 2: TC were treated with 30 ng/ml of either FGF9, insulin‐like growth factor 1 (IGF1), tumor necrosis factor (TNFα), IGF1 plus FGF9, or dexamethasone (300 ng/ml) for 24 h, and CGN mRNA measured; data were analyzed via one‐way ANOVA. CGN mRNA abundance was 64% less (P < 0.01) in TNFα‐treated TC and 49% greater (P < 0.05) in dexamethasone‐treated TC than in controls. IGF1 tended (P < 0.10) to increase CGN mRNA abundance while FGF9 attenuated (P < 0.01) the IGF1 effect. Exp. 3: Estrogenic and androgenic compounds had no significant effect on CGN mRNA expression. These results indicate that FGF9 and TNFα inhibit whereas glucocorticoids stimulate CGN mRNA expression in TC, and suggests a role of inflammatory and stress hormones in regulating the tight junction protein CGN.

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