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Ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) is a multi-domain nuclear protein that plays an important role in epigenetics and tumorigenesis, but its role in normal ovarian follicle development remains unknown. Thus, the present study evaluated if UHRF1 mRNA abundance in bovine follicular cells is developmentally and hormonally regulated, and if changes in UHRF1 are associated with changes in DNA methylation in follicular cells. Abundance of UHRF1 mRNA was greater in granulosa cells (GC) and theca cells (TC) from small (

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 8 mm) follicles, but in TC, E2F1 expression did not differ among follicle sizes. SM-follicle GC had 2.1-fold greater (P 0.10) abundance of E2F1 mRNA in LG-follicle TC or GC. Follicle-stimulating hormone (FSH) had no effect (P > 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

Feeding N-carbamylglutamate (NCG) and arginine (ARG) improves reproductive measures in pigs and reduces systemic steroid levels in pregnant ewes. We hypothesized that the effects of NCG and ARG on reproduction were due to direct effects on the ovary. Thus, the objectives of this study were to investigate the effects of NCG and ARG on granulosa cell (GC) steroidogenesis, gene expression, and cell proliferation in vitro. GC were collected from small (1-5mm) bovine follicles and treated in vitro with NCG or ARG in serum-free medium for 24h to 48h. Both NCG and ARG inhibited (P0.05) IGF1- and FSH-induced GC estradiol production but only NCG inhibited (P0.05) progesterone production. In contrast, NCG and ARG increased (P0.05) GC numbers induced by IGF1 and FSH. NCG inhibited (P0.05) StAR, CYP11A1 and CYP19A1 mRNA abundance in small-follicle GC, whereas ARG had no effect (P0.10) on StAR, CYP11A1 or CYP19A1 mRNA abundance. We conclude that NCG and ARG may act directly on GC and therefore may regulate ovarian function by slowing follicular differentiation via inhibiting IGF1 action, and steroid synthesis while stimulating GC proliferation in cattle.

Melatonin affects granulosa cell function in several species but its function in theca cells is less clear, particularly in monotocous animals. Thus, the objectives of this study were to determine the effects of melatonin on theca cell steroidogenesis, gene expression and cell proliferation in a monotocous species, namely cattle. Ovaries were collected from a local bovine abattoir, from which theca cells were isolated from large (8–22 mm) follicles and treated with various hormones in serum-free medium for 24 h or 48 h. Melatonin caused a dose-dependent inhibition (P 0.10) on cytochrome P450 11A1 (CYP11A1) and cytochrome P450 17A1 (CYP17A1) mRNA abundance. In LH+IGF1-treated theca cells, melatonin decreased caspase 3 (CASP3) mRNA to levels similar to those observed in LH-treated theca cells. In contrast, melatonin increased (P

Overexpression of the transcription factor, E2F8, has been associated with ovarian cancer. Objectives of this study were to determine: 1) if E2F8 gene expression in granulosa cells (GC) and theca cells (TC) change with follicular development, and 2) if E2F8 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F8 mRNA abundance in GC and TC was greater (P 0.05) in small than large follicles. FGF9 induced an increase (P 0.05) in E2F8 mRNA abundance by 1.6- to 7-fold in large-follicle (8-20 mm) TC and GC as well as in small-follicle (1-5 mm) GC. Abundance of E2F8 mRNA in TC was increased (P 0.05) with FGF2, FGF9 or VEGFA treatments alone in vitro, and concomitant treatment of VEGFA with FGF9 increased (P 0.05) abundance of E2F8 mRNA above any of the singular treatments; BMP4, WNT3A and LH were without effect. IGF1 amplified the stimulatory effect of FGF9 on E2F8 mRNA abundance by 2.7-fold. Collectively, our studies show for the first time that follicular E2F8 is developmentally and hormonally regulated indicating that E2F8 may be involved in follicular development.

Results of in vivo studies indicate dietary N-carbamylglutamate (NCG) and arginine (ARG) can enhance reproductive performance in gilts. It was hypothesized that both NCG and ARG will alter hormone-induced estradiol (E2) production by granulosa cells (GC), explaining why these compounds could improve reproductive performance in pigs. The objective of these studies, therefore, was to evaluate the direct effects of NCG and ARG on porcine GC proliferation and steroidogenesis, using an in vitro cell culture system. The GC from small (SM; 1-5 mm) and large (LG;5 mm) pig follicles were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing 500 ng/mL of testosterone (as an E2 precursor), and NCG or ARG at various doses in the presence of either follicle-stimulating hormone (FSH; 30 ng/mL), insulin-like growth factor-1 (IGF1; 30 ng/mL), or both. Numbers of GC were determined at the end of the experiment and concentrations of progesterone (P4) and E2 in culture medium were determined. Results indicated that LG-follicle GC were more responsive to NCG and ARG than SM-follicle GC. Specifically, in LG-follicle GC, NCG inhibited (P 0.05) basal and FSH-induced P4 and E2 production but stimulated cell numbers; whereas ARG inhibited FSH-induced E2 production and cell numbers. In SM-follicle GC, treatment with NCG and ARG decreased IGF1 plus FSH induced P4 production, but E2 production and cell proliferation were not affected. These studies indicate that NCG and ARG may directly affect follicular function in pigs.