Changes in brain ribonuclease (BRB) messenger RNA in granulosa cells (GCs) of dominant vs subordinate ovarian follicles of cattle and the regulation of BRB gene expression in bovine GCs

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 (GCs) during development of bovine dominant ovarian follicles and to determine the hormonal regulation of BRB in GCs. Estrous cycles of Holstein cows (n = 18) were synchronized, and cows were ovariectomized on either day 3 to 4 or day 5 to 6 after ovulation during dominant follicle growth and selection. Ovaries were collected, follicular fluid (FFL) was aspirated, and GCs were collected for RNA isolation and quantitative polymerase chain reaction. Follicles were categorized as small (1–5 mm; pooled per ovary), medium (5–8 mm; individually collected), or large (8.1–17 mm; individually collected) based on surface diameter. Estradiol (E2) and progesterone (P4) levels were measured by radioimmunoassay (RIA) in FFL. Abundance of BRB messenger RNA (mRNA) in GCs 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, GCs BRB mRNA abundance was negatively correlated (P < 0.01) with FFL E2 (r = −0.65) and E2:P4 ratio (r = −0.46). In experiment 2, GCs from large (8–22 mm diameter) and small (1–5 mm diameter) follicles were treated with insulin-like growth factor 1 (IGF1; 0 or 30 ng/mL) and/or tumor necrosis factor alpha (0 or 30 ng/mL); IGF1 increased (P < 0.05) BRB mRNA abundance, and tumor necrosis factor alpha decreased (P < 0.001) the IGF1-induced BRB mRNA abundance in large-follicle GCs. In experiment 3 to 6, E2, follicle-stimulating hormone, fibroblast growth factor 9, cortisol, wingless 3A, or sonic hedgehog did not affect (P > 0.10) abundance of BRB mRNA in GCs; thyroxine and luteinizing hormone increased (P < 0.05), whereas prostaglandin E2 (PGE2) decreased (P < 0.05) BRB mRNA abundance in small-follicle GCs. Treatment of small-follicle GCs with recombinant human RNase1 increased (P < 0.05) GCs numbers and E2 production. In conclusion, BRB is a hormonally and developmentally regulated gene in bovine GCs and may regulate E2 production during follicular growth in cattle.