Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells

Abstract

Context Human granulosa cells (hGCs) produce and respond to insulin-like growth factor 2 (IGF2) but whether the oocyte participates in IGF2 regulation in humans is unknown. Objective To determine the role of oocyte-secreted factors (OSFs) such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in IGF2 production by hGCs. Design Primary human cumulus GCs in culture. Setting University infertility center. Patients or Other Participants GCs of women undergoing in vitro fertilization. Intervention(s) Cells treated with GDF9 and BMP15 in the presence of vehicle, follicle-stimulating hormone (FSH), dibutyryl cyclic-AMP (dbcAMP), or mothers against decapentaplegic homolog (SMAD) inhibitors. Main Outcome Measure(s) Quantification of mRNA, protein, promoter activity, and DNA methylation. Results FSH stimulation of IGF2 (protein and mRNA) was significantly potentiated by the GDF9 and BMP15 (G+B) combination (P < 0.0001) in a concentration-dependent manner showing a maximal effect at 5 ng/mL each. However, GDF9 or BMP15 alone or in combination (G+B) have no effect on IGF2 in the absence of FSH. FSH stimulated IGF2 promoter 3 activity, but G+B had no effect on promoter activity. G+B potentiated IGF2 stimulation by cAMP. SMAD3 inhibitors inhibited G+B enhancement of IGF2 stimulation by FSH (P < 0.05) but had no effect on FSH induction. Moreover, inhibition of insulin-like growth factor receptor partially blocked G+B potentiation of FSH actions (P < 0.009). Conclusions For the first time, we show that the oocyte actively participates in the regulation of IGF2 expression in hGCs, an effect that is mediated by the specific combination of G+B via SMAD2/3, which in turn target mechanisms downstream of the FSH receptor.

Effects of Growth and Differentiation Factor 9 and Bone Morphogenetic Protein 15 overexpression on the steroidogenic metabolism in bovine granulosa cells in vitro

“…An oocyte producing high levels of GDF9 and BMP15 enhance the survival of its surrounding GCs in the presence of low levels of FSH (Hobeika et al, 2020), but in the present study, GCs survived without FSH in the media for more than 3 weeks, showing that the addition of FSH may be not essential for GC survival in vitro. The absence of FSH in our in vitro system also shows the evidence that expression of BMPR1B, BMPR2 and TGFβR1 receptors by GCs can be sustained independent of FSH, in accordance with the results of Chen et al (2009).…”

Molecular crosstalk between insulin‐like growth factors and follicle‐stimulating hormone in the regulation of granulosa cell function

“…Oocyte‐secreted factors (OSFs), such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), potentiate the stimulatory effect of FSH on IGF2 but have no effect by themselves. 120 , 121 These findings suggest an intricate interplay between OSFs, IGF2, and FSH is established in human follicles during GC differentiation. This interplay involves FSH stimulation of IGF2 that is strongly enhanced by OSFs.…”

Downregulation of LHCGR Attenuates COX-2 Expression and Induces Luteinized Unruptured Follicle Syndrome in Endometriosis

“…The cells were then seeded at a density of 2 × 10 5 cells/ml in a 6-well plate and incubated for 3 days at 37°C in humidified atmosphere with 5% CO 2 . The media was replenished every 24 h. To mimic the effect of LH and cAMP in vivo , we used hCG (10 IU/ml; LIVZON, China) and dibutyryl-cAMP (db-cAMP, 1 mM; HY-B0764A, MedChemExpress, USA) respectively to stimulate cells and further cultured for stated hours in vitro according to previous studies ( 21 ). KGN cell line was cultured in DMEM/F12 medium as mentioned above.…”