Dual effect of insulin resistance and cadmium on human granulosa cells - In vitro study.

Combined exposure of cadmium (Cd) and insulin resistance (IR) might be responsible for subfertility. In the present study, we investigated the effects of Cd in vitro in IR human granulosa cells. Isolated human granulosa cells from control and polycystic ovary syndrome (PCOS) follicular fluid samples were confirmed for IR by decrease in protein expression of insulin receptor-ß. Control and IR human granulosa cells were then incubated with or without 32µM Cd. The combined effect of IR with 32µM Cd in granulosa cells demonstrated significant decrease in expression of StAR, CYP11A1, CYP19A1, 17ß-HSD, 3ß-HSD, FSH-R and LH-R. Decrease was also observed in progesterone and estradiol concentrations as compared to control. Additionally, increase in protein expression of cleaved PARP-F2, active caspase-3 and a positive staining for Annexin V and PI indicated apoptosis as the mode of increased cell death ultimately leading to decreased steroidogenesis, as observed through the combined exposure. Taken together the results suggest decrease in steroidogenesis ultimately leading to abnormal development of the follicle thus compromising fertility at the level of preconception.

Investigating the potential role of swertiamarin on insulin resistant and non-insulin resistant granulosa cells of poly cystic ovarian syndrome patients.

BACKGROUND AND AIM: Conventional drugs have limitations due to prevalence of contraindications in PCOS patients. To explore the potential effects of swertiamarin, on abrupted insulin and steroidogenic signaling in human luteinized granulosa cells from PCOS patients with or without insulin resistance. EXPERIMENTAL PROCEDURE: hLGCs from 8 controls and 16 PCOS patients were classified for insulin resistance based on down regulation of protein expression of insulin receptor-ß (INSR- ß) as shown in our previous paper. Cells were grouped as control, PCOS-IR and PCOS-NIR, treated with swertiamarin (66 µM) and metformin (1 mM). Expression of key molecules involved in insulin signaling, fat metabolism, IGF system and steroidogenesis were compared between groups. RESULTS: Swertiamarin significantly (P < 0.05) reversed the expression of INSR-ß, PI(3)K, p-Akt, PKC-ζ, PPARγ, (P < 0.01) IRS (Ser 307) and IGF system in PCOS-IR group and was equally potent to metformin. In the same group, candidate genes viz SREBP1c, FAS, ACC-1 and CPT-1 were down regulated by swertiamarin (P < 0.001) and metformin (P < 0.001). Significant upregulation was demonstrated in expression of StAR, CYP19A1, 17ß-HSD and 3ß-HSD when treated with swertiamarin (P < 0.01) and metformin (P < 0.01) in PCOS-IR followed by increase in 17ß-HSD and 3ß-HSD enzyme activity along with estradiol and progesterone secretions. However, swertiamarin did not reveal any effect on PCOS-NIR group as compared to metformin that significantly (P < 0.01) reversed all the parameters related to steroidogenesis and down regulated basal expression of insulin signaling genes. CONCLUSION: Swertiamarin, presents itself as a potential fertility drug in hLGCs from PCOS-IR patients.

Differential insulin and steroidogenic signaling in insulin resistant and non-insulin resistant human luteinized granulosa cells-A study in PCOS patients.

Insulin resistance (IR) is one of the significant aberrations in polycystic ovarian syndrome (PCOS), however is only observed in 70%-80% of obese PCOS and 20%-25% of lean PCOS. Hyperinsulinemia accompanies PCOS-IR along with hyperandrogenemia against normal insulin and androgen levels in PCOS-non insulin resistance (NIR). This could possibly be due to defects in the downstream signaling pathways. The study thus aims to unravel insulin and steroidogenic signaling pathways in luteinized granulosa cells isolated from PCOS-IR and NIR vs matched controls. Luteinized granulosa cells from 30 controls and 39 PCOS were classified for IR based on a novel method of down regulation of protein expression of insulin receptor-ß (INSR- ß) as shown in our previous paper. We evaluated expression of molecules involved in insulin, steroidogenic signaling and lipid metabolism in luteinized granulosa cells followed by analysis of estradiol, progesterone and testosterone in follicular fluid. Protein expression of INSR- ß, pIRS (ser 307), PI(3)K, PKC-ζ, pAkt, ERK1/2, pP38MAPK and gene expression of IGF showed differential expression in the two groups. Increased protein expression of PPAR-γ was accompanied by up regulation in SREBP1c, FAS, CPT-1 and ACC-1 genes in PCOS-IR group. Expression of StAR, CYP19A1, 17 ß- HSD and 3 ß- HSD demonstrated significant decrease along with increase in CYP11A1, FSH-R and LH-R in both the groups. Follicular fluid testosterone increased and progesterone decreased in PCOS-IR group. This study shows how candidate molecules that were differentially expressed, aid in designing targeted therapy against the two phenotypes of PCOS.

Modulation of steroidogenic pathway in rat granulosa cells with subclinical Cd exposure and insulin resistance: an impact on female fertility.

Changes in lifestyle lead to insulin resistance (IR) in females ultimately predisposing them towards infertility. In addition, cadmium (Cd), an environmental endocrine disruptor, is reported for detrimental effects on granulosa cells, thus leading to ovarian dysfunction. A combination of these factors, lifestyle and environment, seems to play a role in etiology of idiopathic infertility that accounts for 50% amongst the total infertility cases. To address this issue, we made an attempt to investigate the extent of Cd impact on insulin-resistant (IR) granulosa cells. We exposed adult female Charles Foster rats to dexamethasone and confirmed IR condition by fasting insulin resistance index (FIRI). On treatment of IR rats with Cd, the preliminary studies demonstrated prolonged estrous cyclicity, decrease in serum estradiol concentrations, abnormal histology of ovary, and increased granulosa cell death. Further gene and protein expression studies of steroidogenic acute regulatory (StAR) protein, 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and cytochrome P450 aromatase (CYP19A1) were performed. Protein expression studies demonstrated significant decrease in treated groups when compared with control. Study revealed that, in spite of the molecular parameters being affected at varied level, overall ovarian physiology is maximally affected in IR and Cd coexposed group, thus mimicking the condition similar to those prevailing in infertile females.

Basal expression of pluripotency-associated genes can contribute to stemness property and differentiation potential.

Pluripotency and stemness is believed to be associated with high Oct-3/4, Nanog, and Sox-2 (ONS) expression. Similar to embryonic stem cells (ESCs), high ONS expression eventually became the measure of pluripotency in any cell. The threshold expression of ONS genes that underscores pluripotency, stemness, and differentiation potential is still unclear. Therefore, we raised a question as to whether pluripotency and stemness is a function of basal ONS gene expression. To prove this, we carried out a comparative study between basal ONS expressing NIH3T3 cells with pluripotent mouse bone marrow mesenchymal stem cells (mBMSC) and mouse ESC. Our studies on cellular, molecular, and immunological biomarkers between NIH3T3 and mBMSC demonstrated stemness property of undifferentiated NIH3T3 cells that was similar to mBMSC and somewhat close to ESC as well. In vivo teratoma formation with all three germ layer derivatives strengthen the fact that these cells in spite of basal ONS gene expression can differentiate into cells of multiple lineages without any genetic modification. Conclusively, our novel findings suggested that the phenomenon of pluripotency which imparts ability for multilineage cell differentiation is not necessarily a function of high ONS gene expression.