(308) Exploring Neuregulin-4 as a Regulator of Granulosa Cells

Isadora M. Batalha¹; Tezozomoc A. Martinez²; Neva Bennet¹; Nicco Mayor²; Luis F. Schutz¹

1. Department of Agriculture, Veterinary & Rangeland Sciences, University of Nevada-Reno, Reno, NV, United States

2. Department of Biochemistry & Molecular Biology, University of Nevada-Reno, Reno, NV, United States

Abstract Text:

Neuregulin-4 (NRG4) is an adipokine produced by both brown and adipose tissue that influence several physiological mechanisms, including energy metabolism and inflammation. NRG4 activates the epidermal growth factor receptor tyrosine kinase-4 (ErBb-4). ERBB4 knock-out mice displayed impairment of ovulation. Despite NRG4 and ErBb-4 being expressed in granulosa cells (GC) and correlated with clinical ovulatory disorders and polycystic ovarian syndrome (PCOS), effects of NRG4 on GC function remain to be explored. As a plethora of adipokines are known to regulate mammalian ovarian physiology, we hypothesized that NRG4 plays a role in GC function. To test this hypothesis, in vivo and in vitro experiments were employed using cattle as a model of mono-ovulatory species. To investigate whether mRNA expression of NRG4 and ERBB4 changes in bovine GC during ovarian folliculogenesis, Angus heifers' (n=6) estrous cycle was synchronized using a progesterone (P4) implant for 5 days, an injection of 86mcg gonadorelin on day 0, and a single intramuscular injection of 25 mg of prostaglandin F2α (PGF2α) at the time of implant removal. Ovaries were collected 27.21 ± 1.9h after the PGF2α injection and follicles were categorized based on surface diameter and the ratio of estradiol (E2):P4 as subordinate (1-5mm; E2:P4< 1; n=9) and dominant ( >10mm; E2:P4 >1; n= 4). GC was isolated and mRNA abundance of NRG4 and ERBB4 was investigated via qPCR. Results from the in vivo experiment showed that NRG4 and ERBB4 mRNAs are more abundant (p < 0.001) in GC from subordinate follicles than those from dominant. Due to the in vivo results, primary in vitro culture of bovine GC from subordinate follicles (1-5 mm; n=11pools) was performed to investigate the effects of NRG4 on GC E2 synthesis and cell proliferation. Using ovaries collected from USDA-inspected slaughterhouses, GC were isolated and allowed to grow for 48h in the presence of 10% fetal bovine serum (FBS) with medium replacement every 24h until reaching 60% confluence. Then, cells were treated with NRG4 (0, 5, 10, 20, 40 ng/mL) in combination with follicle-stimulating hormone (FSH; 30 ng/ml) for 24 h in FBS-free medium for assessment of E2 levels via ELISA and cell numbers via Muse Cell Analyzer. Doses of NRG4 were based on the maximum levels detected in serum of women with PCOS. Results from the in vitro study show that NRG4 10ng/mL decreased (p≤0.05) by 0.6-fold E2 production by GC from subordinate follicles in comparison to the control group (NRG4 0 ng/mL). Furthermore, NRG4 20 ng/mL significantly stimulated (p< 0.05) the proliferation of GC by 1.52-fold compared to the control group (NRG4 0 ng/mL). Additionally, a trend (p≤0.06) to increase cell numbers was observed with NRG4 10ng/mL. Hence, although cell numbers tended to increase under NRG4 treatments, E2 production was impaired. Through this study, we showed for the first time the presence of NRG4 and its receptor ErBb-4 in the ovary of cattle. Collectively, the results here establish that NRG4 directly influences GC physiology and suggest that the role of NRG4 and its receptor may change through the different stages of ovarian folliculogenesis.