Developmental and hormonal regulation of ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 gene expression in ovarian granulosa and theca cells of cattle

Abstract

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 (<6 mm) than large (≥8 mm) follicles and was greater in small-follicle GC than TC. In GC and TC, fibroblast growth factor 9 (FGF9) treatment increased (P < 0.05) UHRF1 expression by 2-fold. Also, luteinizing hormone (LH) and insulin-like growth factor 1 (IGF1) increased (P < 0.05) UHRF1 expression in TC by 2-fold, and forskolin (an adenylate cyclase inducer) alone or combined with IGF1 increased (P < 0.05) UHRF1 expression by 3-fold. An E2F transcription factor inhibitor (E2Fi) decreased (P < 0.05) UHRF1 expression by 44% in TC and by 99% in GC. Estradiol, progesterone, and dibutyryl-cAMP decreased (P < 0.05) UHRF1 mRNA abundance in GC. Treatment of GC with follicle-stimulating hormone (FSH) alone had no effect but when combined with IGF1 enhanced the UHRF1 mRNA abundance by 2.7-fold. Beauvericin (a mycotoxin) completely inhibited the FSH plus IGF1-induced UHRF1 expression in small-follicle GC. Treatments that increased UHRF1 mRNA (i.e., FGF9) in GC tended to decrease (by 63%; P < 0.10) global DNA methylation, and those that decreased UHRF1 mRNA (i.e., E2Fi) in GC tended to increase (by 2.4-fold; P < 0.10) global DNA methylation. Collectively, these results suggest that UHRF1 expression in both GC and TC is developmentally and hormonally regulated, and that UHRF1 may play a role in follicular growth and development as well as be involved in ovarian epigenetic processes.

Keywords: cattle; follicle; gene expression; granulosa cell; theca cell; ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1.

Figure 1.

Levels of UHRF1 mRNA in freshly collected GC and TC from small and large follicles (exp. 1). Real-time PCR quantified steady-state mRNA levels were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of large follicle TC value. ^a–cMeans (± SEM; n = 7) without a common letter differ (P < 0.05).

Figure 2.

In vitro effect of FGF9 (0, 30, or 100 ng/mL) on UHRF1 mRNA abundance in bovine TC from large follicles (exp. 2). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. ^a,bMeans without a common letter differ (P < 0.05).

Figure 3.

In vitro effects of LH (30 ng/mL) and IGF1 (I; 30 ng/mL), alone or combined, and Fsk (4.1 μg/mL), alone or combined with IGF1, and E2Fi (50 μM) on UHRF1 mRNA abundance in bovine TC from large follicles (exp. 3). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. ^a,bMeans without a common letter differ (P < 0.05).

Figure 4.

In vitro effects of steroids, EGF, and E2Fi on UHRF1 mRNA abundance in bovine TC from large follicles (exp. 4). Treatments were applied for 24 h and were 300 ng/mL of P4, E2, or DHT, and 10 ng/mL of EGF and 50 µM of E2Fi. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and are expressed as a ratio of control values. ^a,bMeans without a common letter differ (P < 0.05).

Figure 5.

In vitro effects of A4 (300 ng/mL), P4 (300 ng/mL), dbcAMP (0.25 mM), FGF9 (30 ng/mL), and EGF (30 ng/mL) on UHRF1 mRNA abundance in bovine GC from large follicles (exp. 5). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. ^a–dMeans without a common letter differ (P < 0.05).

Figure 6.

In vitro effects of E2 (300 ng/mL), A4 (300 ng/mL), IGF1 (30 ng/mL), E2Fi (50 μM), and FGF9 (30 ng/mL) on UHRF1 mRNA abundance in bovine GC from small follicles (exp. 7). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. ^a–dMeans without a common letter differ (P < 0.05).

Figure 7.

In vitro effects of GLYP (30 μM), BEA (30 μM), and FB1 (30 μM), in the presence of FSH (30 ng/mL) plus IGF1 (30 ng/mL), on UHRF1 mRNA abundance in bovine GC from small follicles (exp. 8). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. ^a,bMeans without a common letter differ (P < 0.05).

Figure 8.

In vitro effect of FGF9 (30 ng/mL) or E2Fi (50 µM) on global cellular methylation in bovine GC from small follicles (exp. 9). Treatments were applied in serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. ^a–cMeans without a common letter tended to differ (P < 0.10).

Figure 9.

Schematic model summarizing the hormonal regulation of UHRF1 production by GC and TC in small and large follicles. The hormones IGF1, FSH, and LH are stimulatory to E2 and A4 production. Increased free IGF1 in large follicles increases E2 production and decreases FGF9 mRNA in GC, and these changes cause a reduction in the UHRF1 production by GC and TC, which induces an overall increase in global DNA methylation by the dominant follicle.