Synergistic Interplay of Hyperandrogenism and Hyperinsulinism on Primary Culture of Luteinized Granulosa Cells – an “ in-vitro †Model Mimicking Ovarian Microenvironment of Poly-Cystic Ovary Syndrome (PCOS)

Abstract

Poly-Cystic Ovary Syndrome (PCOS) is the most prevalent endocrine disorder, characterized by hyperandrogenism and hyperinsulinemia, both at systemic and ovarian levels. This study investigated the synergistic effect of hyperinsulinemia and hyperandrogenism on the regulatory mechanism of ovarian steroidogenesis using Luteinized Granulosa Cells (LGCs). LGCs were isolated from 40 weaning female Charles Foster rats by superovulation by PMSG and characterized for purity and stability in modified DMEM: F12 media. The isolated cells were divided into following groups- control, hyperinsulinic group (0.1-2 mIU/ mL of insulin), excess androgen (10-100 ng/mL of DHT) and combination of both. One-way ANOVA was performed with a Bonferroni post-hoc test. Results demonstrate that the LGCs exhibit reduced expression of FSHR and CYP19A and increased expression of LHR, StAR and CYP17A1 at 72 hours. There was reduction in cell viability of LGCs when induced with hyperinsulin and hyperandrogen doses individually or in combination.0.1 mIU/mL of insulin and 50 ng/mL of DHT in combination were the minimum effective dose in inducing PCO like ovarian microenvironment in the primary culture of LGCs. There was exaggerated androgen biosynthesis, reduced progesterone secretion and non-significant change is estradiol levels in the LGCs. The abnormal steroidogenesis can be attributed to upregulation of key genes such as StAR, CYP17A1, AMH and SREBP1-c and down-regulation of genes like CYP19A1, HSD3B2, IGFBP1 and SHBG. This model can be used to study downstream signaling pathways involved with dysregulated ovarian microenvironment as observed in PCOS at cellular level and for screening of drug targets for such pathological conditions.