Estrogen Modulates Specific Life and Death Signals Induced by LH and hCG in Human Primary Granulosa Cells In Vitro

Abstract

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are glycoprotein hormones used for assisted reproduction acting on the same receptor (LHCGR) and mediating different intracellular signaling. We evaluated the pro- and anti-apoptotic effect of 100 pM LH or hCG, in the presence or in the absence of 200 pg/mL 17β-estradiol, in long-term, serum-starved human primary granulosa cells (hGLC) and a transfected granulosa cell line overexpressing LHCGR (hGL5/LHCGR). To this purpose, phospho-extracellular-regulated kinase 1/2 (pERK1/2), protein kinase B (pAKT), cAMP-responsive element binding protein (pCREB) activation and procaspase 3 cleavage were evaluated over three days by Western blotting, along with the expression of target genes by real-time PCR and cell viability by colorimetric assay. We found that LH induced predominant pERK1/2 and pAKT activation STARD1, CCND2 and anti-apoptotic XIAP gene expression, while hCG mediated more potent CREB phosphorylation, expression of CYP19A1 and procaspase 3 cleavage than LH. Cell treatment by LH is accompanied by increased (serum-starved) cell viability, while hCG decreased the number of viable cells. The hCG-specific, pro-apoptotic effect was blocked by a physiological dose of 17β-estradiol, resulting in pAKT activation, lack of procaspase 3 cleavage and increased cell viability. These results confirm that relatively high levels of steroidogenic pathway activation are linked to pro-apoptotic signals in vitro, which may be counteracted by other factors, i.e., estrogens.

Human Chorionic Gonadotropin Mediated Generation of Reactive Oxygen Species Is Sufficient to Induce Meiotic Exit but Not Apoptosis in Rat Oocytes

“… 48 Pituitary gonadotropin surge is the primary requirement for the induction of meiotic maturation in oocytes. 28 , 39 hCG has been frequently used as a surrogate for LH surge to induce ovulation in several mammalian species, 26 , 46 but its role in meiotic maturation remains ill understood. Data of the present study suggest that most of the oocytes (more than 90%) collected after 48 h PMSG surge were arrested at GV stage.…”

“…A moderate increase of ROS level has been reported to induce spontaneous meiotic resumption in rat oocytes 16 , 21 and beneficial for fertilization, as well as reproductive outcome, after ovarian stimulation protocol. 4 , 28–30 Studies from our laboratory using NO donor and iNOS inhibitor suggest that the reduction in NO level results in spontaneous EDA in rat COCs cultured in vitro. 49 Our results suggest that the increase of total ROS level (almost two fold increase) was associated with meiotic exit from diplotene, as well as M-II arrest.…”

“… 3 , 4 The increased level of ROS has been observed during luteinizing hormone (LH)-induced ovarian stimulation protocol. 4 , 28 The increase in level of ROS associates with meiotic resumption, fertilization, and improved reproductive outcome in human. 29 , 30 In contrast, excess amount of ROS could induce susceptibility towards oocyte apoptosis.…”

“…Human chorionic gonadotropin (hCG) has commonly been used in ovarian stimulation protocol to induce final maturation and ovulation in several mammalian species during assisted reproduction. 28 , 39 Due to structural and biological similarities with LH, hCG binds to LH receptors. 39 It is proposed that hCG surge generates ROS due to increased cell proliferation during final stages of folliculogenesis and mediate meiotic resumption.…”

Membrane estrogen receptor (GPER) and follicle-stimulating hormone receptor heteromeric complexes promote human ovarian follicle survival

“…In that respect, as FSH is thought to bind and activate FSHR trimers 45 , our data could support a role for these asymmetric lower order heterooligomers in regulating cell viability. This inhibitory role of GPER on cAMP is specific to FSHR, since GPER had no effect on LHCGR-mediated cAMP, reflecting structural differences between FSHR and LHCGR, in line with the different and specific physiological role of LH 46,47 .…”