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 (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.

Changes in fibroblast growth factor 9 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle

“…Nonlactating Holstein cows (n = 18) were used for this experiment as previously described (Dentis et al, 2016). Briefly, estrous cycles were synchronized using 2 injections (im) of PGF 2α (Lutalyse, 25 mg, Pharmacia & Upjohn Co., Kalamazoo, MI) with an interval of 11 d. From the first injection of PGF 2α to the occurrence of ovulation after the second injection, follicle development was monitored daily via ultrasonography using an Aloka 500V (Aloka Co., Tokyo, Japan) with a 7.5-MHz probe.…”

“…From the 18 cows used in the synchronization program, 2 failed (1 from d 3 and 1 from d 6 groups) to ovulate and were excluded from this experiment. After each ovariectomy, ovaries were put on ice and transported to the laboratory where diameters of all follicles ≥5 mm (surface diameter) in diameter were recorded, the numbers of all follicles ≥1 mm in diameter on the ovarian surface determined, and ovarian tissue and fluid collected (Dentis et al, 2016). The animal experimentation described in this report was approved by the Oklahoma State University Institutional Animal Care and Use Committee.…”

“…Follicular fluid ( FFL ) from medium and large follicles was aspirated individually and centrifuged (200 × g at 4°C for 8 min) to obtain GC; FFL from small follicles was pooled within each ovary and then centrifuged to obtain GC as previously described (Stewart et al, 1996; Dentis et al, 2016). After centrifugation, FFL was aspirated and stored at −20°C for subsequent RIA.…”

“…After centrifugation, FFL was aspirated and stored at −20°C for subsequent RIA. After collection of FFL, each medium and large follicle was bisected in situ, the inner wall was scraped, rinsed with Ham’s F-12 (Sigma-Aldrich, St. Louis, MO) to remove any remaining GC, and these GC were combined with GC collected from FFL as previously described (Schreiber and Spicer, 2012; Dentis et al, 2016). The GC collected from small follicles were kept separate for each ovary; GC were lysed in 0.5 mL of TRIzol reagent (Life Technologies Inc., Grand Island, NY) and stored frozen at −80°C until RNA extraction.…”

“…The RNA was extracted from GC and TC as described previously (Lagaly et al, 2008; Dentis et al, 2016). Samples of RNA were solubilized in diethylpyrocarbonate-treated water (Life Technologies Inc.), quantified at 260 nm using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE), and stored at −80°C.…”