Effects of asprosin on estradiol and progesterone secretion and proliferation of bovine granulosa cells.

Asprosin is an adipokine synthesized by the white adipose tissue that regulates glucose homeostasis and that has been reported to affect bovine theca cell function and follicular growth, but its role on granulosa cell functions remains to be unveiled. Hence, the objective of this study was to investigate asprosin impacts on granulosa cell steroidogenesis. Bovine granulosa cells from small ovarian follicles were cultured in vitro to investigate the effects of asprosin on cell proliferation, production of steroids, mRNA abundance of genes that encode steroidogenic enzymes and cell cycle regulators, and protein relative abundance of steroidogenic signaling pathways. Asprosin was shown to affect granulosa cell functions in a dose-dependent manner. In the presence of FSH, asprosin enhanced estradiol production and stimulated an increase in mRNA expression of FSHR and CYP19A1 in a dose-dependent manner. In the presence of IGF1, asprosin decreased estradiol production, increased progesterone production, altered PKA relative protein expression, and tended to alter the ratio of p-ERK1/2/total ERK1/2 protein expression in a dose-dependent manner. Furthermore, asprosin increased p-53 gene expression in basal culture conditions and with or without FSH and IGF1. Taken together, findings of this study show that asprosin is a regulator of granulosa cell functions and the effects of asprosin depend on dose and cell culture conditions.

In Vitro Effects of Enniatin A on Steroidogenesis and Proliferation of Bovine Granulosa Cells.

The emerging Fusarium mycotoxins enniatins (ENNs) have been the focus of new research because of their well-documented existence in various cereal and grain products. Research findings indicate that reproductive disorders may be caused by exposure to Fusarium mycotoxins, but little work has evaluated ENNs on reproductive function. Therefore, to determine the effects of ENNA on the proliferation and steroidogenesis of granulosa cells (GC), experiments were conducted using bovine GC cultures. In vitro, ENNA (1−5 µM) inhibited (p < 0.05) hormone-induced GC progesterone and estradiol production. The inhibitory effect of ENNA on estradiol production was more pronounced in small- than large-follicle GC. In large-follicle GC, 0.3 µM ENNA had no effect (p > 0.10) whereas 1 and 3 µM ENNA inhibited GC proliferation. In small-follicle GC, ENNA (1−5 µM) dramatically decreased (p < 0.05) GC proliferation. Using cell number data, the IC50 of ENNA was estimated at 2 µM for both follicle sizes. We conclude that ENNA can directly inhibit ovarian function in cattle, decreasing the proliferation and steroid production of GC.

A potential role of fibrillin-1 (FBN1) mRNA and asprosin in follicular development in water buffalo.

Fibrillin-1 (FBN1) functions as a structural protein in the ovary, while the role of its protein product asprosin remains unknown. Both proteins are encoded by the FBN1 gene and when it is cleaved at the C-terminal end, asprosin is produced. Asprosin is associated with various metabolic parameters and sex-related hormones in women. One goal of this research was to quantify FBN1 and the presumed asprosin receptor, olfactory receptor family 4 subfamily M member 1 (OR4M1) mRNA in water buffalo granulosa cells and correlate them to aromatase (CYP19A1) gene expression. A second goal was to determine the effect of asprosin on follicular growth in vivo. In Exp. 1, ovaries were collected from a local slaughterhouse, follicular fluid and granulosa cells from small (<6 mm) and large (6-13 mm) follicles were aspirated, cellular RNA extracted for gene expression analysis, data analyzed using ANOVA, and Pearson correlation coefficients were calculated among FBN1, OR4M1, and CYP19A1 gene expression. In Exp. 2, an intra-follicular injection of asprosin (600 ng of asprosin/194 µL of PBS) or vehicle (200 µL of PBS; Controls) was given via the theca layer of the dominant follicle of synchronized cows (n = 5/group) 1 day after injection of PGF2α, follicle sizes were measured daily via transrectal ultrasonography for 3 days, a two-way repeated measures ANOVA was used to determine the effect of asprosin on growth rate of follicles from day 0-2, and Chi-square analysis for the percentage of cows ovulated 2 days following asprosin injections. In Exp. 1, FBN1 mRNA abundance was 1.9-fold greater in cells of follicular aspirates from small than large follicles (P < 0.05), but abundance of OR4M1 and CYP19A1 mRNA did not differ (P > 0.10) between the two sizes of follicles. Abundance of FBN1 mRNA was positively correlated with CYP19A1 (r = 0.55, P < 0.05) and OR4M1 mRNA (r = 0.50, P < 0.06) across follicle sizes. In Exp. 2, cows treated with asprosin revealed a greater follicle growth rate from day 0-2 (63.4% increase in diameter) than placebo cows (36.8% increase in diameter) post-injection, and more follicles from asprosin treatment vs. control group (100% vs. 20%; P < 0.05) ovulated within 2 days. These findings suggest that FBN1 may be developmentally regulated in follicular cells, and that asprosin may induce follicular growth in buffaloes, but further studies will be required to determine if asprosin directly regulates estradiol production during follicle development.

Effects of selected hormones and their combination on progesterone and estradiol production and proliferation of feline granulosa cells cultured in vitro.

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. The present study aimed to develop a hormone responsive granulosa cell culture system to measure steroidogenic and cell proliferation responses to help identify factors that might regulate ovarian function in queens. Five experiments were conducted each with 75 or more ovaries, three in spring and two in fall seasons. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48 h after an initial 48 h plating in 10% fetal calf serum. In granulosa cells isolated from spring and fall collected feline ovaries, IGF1 alone and combined with FSH stimulated (P < 0.05) cell proliferation, whereas FSH alone had no effect (P > 0.10) on cell proliferation. Also, in granulosa cells collected in spring and fall, IGF1 alone and FSH alone increased (P < 0.05) estradiol production by severalfold, and a combination of FSH and IGF1 increased (P < 0.05) estradiol production above either FSH or IGF1 treatment alone. The FSH plus IGF1 treatment increased (P < 0.05) CYP19A1 mRNA abundance by 27-fold. In contrast, EGF decreased (P < 0.05) FSH plus IGF1-induced estradiol production by over 80% in granulosa cells of both spring and fall collected ovaries. In granulosa cells isolated from spring and fall collected ovaries, IGF1 plus FSH inhibited (P < 0.05) progesterone production. Melatonin increased (P < 0.05) FSH plus IGF1-induced cell proliferation and amplified (P < 0.05) the FSH plus IGF1-induced inhibition of progesterone production. However, melatonin and GH had no effect (P > 0.10) on estradiol production either alone or in combination with FSH plus IGF1 in both spring and fall. Prolactin, FGF9 and activin had no effect (P > 0.10) on cell proliferation or steroidogenesis. FGF2 decreased (P < 0.05) estradiol production without affecting progesterone production or cell numbers. Growth differentiation factor 9 (GDF9) increased (P < 0.05) progesterone production but had no effect (P > 0.10) on granulosa cell proliferation or estradiol production. In conclusion, the in vitro system described herewithin may be useful to assess and evaluate ovarian function in feline species and has identified EGF, FSH and IGF1 as major regulators of feline ovarian follicular function.

Discovery of a possible role of asprosin in ovarian follicular function.

Asprosin is a novel fasting-induced protein encoded by fibrillin-1 (FBN1) gene, produced when FBN1 is cleaved by the enzyme furin, and is associated with insulin resistance and polycystic ovarian syndrome in humans. To characterize mRNA abundance of FBN1, FURIN, and the presumed asprosin receptor, olfactory receptor family 4 subfamily M member 1 (OR4M1) in granulosa (GC) and theca cells (TC), and identify hormones regulating FBN1 mRNA expression, GC and TC from small (1-5 mm; SM) and large (>8 mm; LG) follicles were collected from ovaries of heifers obtained at an abattoir and used for real-time PCR gene expression analysis or in vitro evaluation of hormone regulation and asprosin effects. SMTC had 151-fold greater (P < 0.05) FBN1 mRNA abundance than SMGC, and LGTC had 50-fold greater FBN1 mRNA than LGGC. In contrast, OR4M1 mRNA was 81-fold greater in SMGC than LGGC and did not differ from SMTC, but LGTC had 9-fold greater OR4M1 mRNA than LGGC. FURIN mRNA was 2.6-fold greater in SMTC than SMGC, but did not differ among follicular sizes. In cultured TC, leptin, insulin, LH, IGF1 and steroids did not affect FBN1 mRNA, but TGFB1 increased (P < 0.05) FBN1 mRNA by 2.2-fold; EGF and FGFs increased FBN1 mRNA by 1.3- to 1.5-fold. Asprosin enhanced LH-induced TC androstenedione production, reduced IGF1-induced TC proliferation, and had no effect on progesterone production. Developmental regulation of FBN1, FURIN and OR4M1 along with direct effects of asprosin on TC suggests that asprosin may be a novel regulator of ovarian follicular function.

Regulation of the transcription factor E2F1 mRNA in ovarian granulosa cells of cattle.

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 < 0.05) in small (SM; 1 to 5 mm) than large (LG; >8 mm) follicles, but in TC, E2F1 expression did not differ among follicle sizes. SM-follicle GC had 2.1-fold greater (P < 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 > 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 < 0.05) GC numbers, whereas E2Fi alone decreased (P < 0.05) GC numbers, and concomitant treatment of E2Fi with FGF9 blocked (P < 0.05) this stimulatory effect of FGF9. Estradiol production was inhibited (P < 0.05) by FGF9 alone and concomitant treatment of E2Fi with FGF9 attenuated (P < 0.05) this inhibitory effect of FGF9. SM-follicle GC treated with E2Fi decreased (P < 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.

Regulation of the transcription factor E2F8 gene expression in bovine ovarian cells.

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.

Effects of follicle-stimulating hormone followed by gonadotropin-releasing hormone on embryo production by ovum pick-up and in vitro fertilization in the river buffalo (Bubalus bubalis).

In this study, we examined the effects of superstimulation using follicle-stimulating hormone (FSH) followed by gonadotropin-releasing hormone (GnRH) on buffalo embryo production by ultrasound-guided ovum pick-up (OPU) and in vitro fertilization (IVF). Nine Murrah buffaloes were subjected to OPU-IVF without superstimulation (control). The morphologies of the oocytes collected were evaluated, and oocytes were then submitted to in vitro maturation (IVM). Two days after OPU, same nine buffaloes were treated with twice-daily injections of FSH for 3 days for superstimulation followed by a GnRH injection. Oocytes were collected by OPU 23-24 hr after the GnRH injection and submitted to IVM (the superstimulated group). The total number of follicles, number of follicles with a diameter > 8 mm, and number of oocytes surrounded by multi-layered cumulus cells were higher in the superstimulated group than in the control group (p ≤ 0.05). After IVF, the percentages of cleavage and development to blastocysts were higher in the superstimulated group than in the control group (p < 0.05). In conclusion, superstimulation improved the quality of oocytes and the embryo productivity of OPU-IVF in river buffaloes.