No abstract

Brain ribonuclease (BRB) is a member of the ribonuclease A superfamily that is constitutively expressed in a range of tissues, and is the functional homolog of human ribonuclease 1. This study was designed to characterize BRB gene expression in granulosa cells (GC) during development of bovine dominant ovarian follicles, and to determine the hormonal regulation of BRB in GC. Estrous cycles of Holstein cows (n = 18) were synchronized and cows were ovariectomized on either day 3 to 4 or day 5 to 6 post-ovulation during dominant follicle growth and selection. Ovaries were collected, follicular fluid (FFL) was aspirated, and GC were collected for RNA isolation and quantitative PCR. Follicles were categorized as small (1 to 5 mm; pooled per ovary), medium (5 to 8 mm; individually collected) or large (8.1 to 17 mm; individually collected) based on surface diameter. Estradiol (E2) and progesterone (P4) levels were measured by RIA in FFL. Abundance of BRB mRNA in GC was 8.6- to 11.8-fold greater (P < 0.05) in small (n = 31), medium (n = 66) and large (n = 33) subordinate E2-inactive (FFL E2 < P4) follicles than in large (n = 16) dominant E2-active (FFL E2 > P4) follicles. In the largest 4 follicles, GC BRB mRNA abundance was negatively correlated (P < 0.01) with FFL E2 (r = −0.65) and E2/P4 ratio (r = −0.46). In Exp. 2, GC from large (8 to 22 mm diameter) and small (1 to 5 mm diameter) follicles were treated with IGF1 (0 or 30 ng/mL), and/or tumor necrosis factor α (TNFα) (0 or 30 ng/mL); IGF1 increased (P < 0.05) BRB mRNA abundance and TNFα decreased (P < 0.001) the IGF1-induced BRB mRNA abundance in large-follicle GC. In Exp. 3 to 6, E2, FSH, fibroblast growth factor 9 (FGF9), cortisol, wingless 3A (WNT3A), or Sonic hedgehog (SHH) did not affect (P > 0.10) abundance of BRB mRNA in GC; thyroxine and LH increased (P < 0.05) whereas prostaglandin E2 (PGE2) decreased (P < 0.05) BRB mRNA abundance in small-follicle GC. Treatment of small-follicle GC with recombinant human RNase1 increased (P < 0.05) GC numbers and estradiol production. In conclusion, BRB is a hormonally and developmentally regulated gene in bovine GC and may regulate estradiol production during follicular growth in cattle.

No abstract

No abstract

Glyphosate (GLY) is a common herbicide used worldwide but its effect on ovarian function in mammals is unknown. The aim of this study was to determine the potential endocrine disruptor effects of GLY on ovarian function evaluating cell proliferation, steroidogenesis and gene expression using bovine granulosa cells (GC) and theca cells as in vitro models. GC proliferation was impaired (P < 0.05) after exposure to GLY at 0.5, 1.7 and 5 μg ml . GC progesterone production was not affected (P ≥ 0.05) at all doses tested while estradiol production was inhibited (P < 0.05) by GLY at 5 μg ml . At the same concentration GLY showed no effect (P ≥ 0.05) on theca cell proliferation and steroidogenesis. At higher concentrations (0.01 and 0.3 mg ml ), GLY had no significant effect (P ≥ 0.05) on GC proliferation and steroidogenesis. These studies, for the first time, suggest that GLY may affect the reproductive system in cattle via direct action on ovarian function; however, further studies will be required to understand better the mechanism of action and to determine the in vivo reproductive effects of GLY. Copyright © 2016 John Wiley & Sons, Ltd.

Tight junctions (TJ) are common paracellular sealing structures that control the transport of water, ions, and macromolecules across cell layers. Because the role of TJ in bovine follicular development is unknown, we investigated the developmental and hormonal regulation of the transmembrane TJ protein, occludin (OCLN), and the cytoplasmic TJ proteins, TJ protein 1 (TJP1) and cingulin (CGN) in bovine granulosa cells (GC) and theca cells (TC). For this purpose, bovine GC and TC were isolated from large (>8 mm) and/or small (1 to 5 mm) follicles and either extracted for real-time PCR (qPCR) or cultured in vitro. The abundances of both and mRNA were greater ( < 0.05) in TC than GC, whereas the mRNA abundance was greater ( < 0.05) in GC than TC. The abundance of mRNA in both GC and TC was greater ( < 0.05) in small follicles compared with large follicles, whereas the GC of large follicles had less ( < 0.05) mRNA abundance than the GC of small follicles. The abundance of mRNA in GC or TC did not differ ( > 0.10) among follicle sizes. In vitro treatment with various growth factors known to affect ovarian folliculogenesis indicated that , , and were hormonally regulated. Fibroblast growth factor 9 (FGF9) decreased ( < 0.05) the and mRNA abundances. Tumor necrosis factor α (TNFα) and vascular endothelial growth factor A (VEGFA) increased ( < 0.05) the mRNA abundance but decreased ( < 0.05) the mRNA abundance. Dexamethasone (DEX) increased ( < 0.05) and mRNA abundances. Epidermal growth factor (EGF) decreased ( < 0.05) and dihydrotestosterone (DHT) increased ( < 0.05) the abundances of , , and mRNA. We propose that the downregulation of OCLN and other TJ proteins during follicular development could reduce barrier function, thereby participating in increasing follicle size by allowing for an increase in the volume of follicular fluid as well as by allowing additional serum factors into the follicular fluid that potentially may directly impact GC functions. The results of the current study indicate the following in cattle: 1) gene expression of TJ proteins (i.e., , , and ) differs between GC and TC and changes with follicle size, and 2) autocrine, paracrine, and endocrine regulators, such as FGF9, EGF, DHT, TNFα, and glucocorticoids, modulate , , and mRNA abundance in TC in vitro.

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 TGFβ1 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.

No abstract