Optimisation of hormonal treatment to improve follicular development in one-day-old mice ovaries cultured under in vitro condition

Abstract

Context Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles. Aims The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries. Methods One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight. Key results In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k), KIT ligand (Kitl), growth differentiation factor 9 (Gdf9) and follicle stimulating hormone receptor (Fshr) (P < 0.05). Combination of testosterone and FSH, moreover, boosted gene expression of bone morphogenetic protein 15 (Bmp15), Connexin-43 (Cx43) and luteinising hormone and choriogonadotropin receptor (Lhcgr) (P < 0.05). In experiment II, the effect of various gonadotropins including FSH, equine chorionic gonadotropin (eCG) and human menopausal gonadotropin (hMG) on cultured ovaries was assessed and hMG supported development of primary follicles greater than eCG and FSH did (P < 0.05). Additionally, eCG and hMG increased gene expression of Lhcgr (P < 0.05), but FSH did not (P > 0.05). Conclusions Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development. Implications This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.