Effects of MiR-375-BMPR2 as a Key Factor Downstream of BMP15/GDF9 on the Smad1/5/8 and Smad2/3 Signaling Pathways

Abstract

Background/Aims: Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are secreted by oocytes, are important regulators of follicular growth and development and ovarian function. These two factors can regulate the proliferation and apoptosis of cumulus cells via modulation of the Smad signaling pathway. Studies have shown that BMP15 and GDF9 can affect the level of miR-375, whereas the target gene of miR-375 is BMPR2, the type II receptor of BMP15 and GDF9. However, whether or how the BMP15/ GDF9-miR-375-BMPR2 pathway affects the proliferation and apoptosis of bovine cumulus cells through regulation of the Smad signaling pathway remains unclear. Methods: In this study, cumulus cells were first obtained from cumulus-oocyte complexes (COCs). Appropriate concentrations of BMP15 and GDF9 were added during the in vitro culture process. Cell Counting Kit-8 (CCK-8) analyses and flow cytometry were used to determine the effects of BMP15/GDF9 on bovine cumulus cells proliferation and apoptosis. Subsequently, miR-375 mimics, miR-375 inhibitor and BMPR2 siRNA were synthesized and used for transfection experiments. Western Blot analysis was used to detect changes before and after transfection in the expression levels of the BMP15/GDF9 type I receptors ALK4, ALK5 and ALK6; the phosphorylation levels of Smad2/3 and Smad1/5/8, which are key signaling pathway proteins downstream of BMP15/GDF9; the expression levels of PTX3, HAS2 and PTGS2, which are key genes involved in cumulus cells proliferation; and Bcl2/Bax, which are genes involved in apoptosis. Results: The addition of 100 ng/mL BMP15 or 200 ng/mL GDF9 or the combined addition of 50 ng/mL BMP15 and 100 ng/mL GDF9 effectively inhibited bovine cumulus cell apoptosis and promoted cell proliferation. BMP15/GDF9 negatively regulated miR-375 expression and positively regulated BMPR2 expression. High levels of miR-375 and inhibition of BMPR2 resulted in increased expression of ALK4 and decreased expression of PTX3, HAS2 and PTGS2, whereas miR-375 inhibition resulted in the opposite results. BMP15 and GDF9 significantly activated the levels of p-Smad2/3 and p-Smad1/5/8, whereas miR-375 inhibited the levels of p-Smad2/3 and p-Smad1/5/8 by negatively regulating BMPR2 and also led to apoptosis. Conclusion: BMP15 and GDF9 have synergistic effects and can act through miR-375 to affect the expression levels of type I receptor ALK4 and type II receptor BMPR2 and the activation of Smad signaling pathway, which subsequently affected the proliferation, spread and apoptosis of cumulus cells.

GDF-9 and BMP-15 direct the follicle symphony

“…These findings are consistent with the fact that in humans, mutations in GDF-9 and BMP-15 have been associated with subfertility, high incidence of dizygotic twins, ovulation defects, and with premature ovarian failure [76][77][78][79][80]. Altered expression of these factors may cause damage to ovarian function and fertility in several species [35,37,81,82].…”

“…The BMP-15/GDF-9 heterodimer cumulin needs to bind to the receptor BMPRII-ALK4/5/7-ALK6 complex to activate SMAD 2 and 3 signaling [52,[78][79][80]. BMP-15 and GDF-9 have synergistic effects and can act through miR-375 to affect the expression levels of type I receptor ALK4 and type II receptor BMPRII and the activation of SMAD signaling pathway, which subsequently affected the proliferation, spread, and apoptosis of cumulus cells [81]. The activation of SMAD signaling by the oocytes and the requirement of SMAD2/3 signaling for cumulus cell expansion can also vary between species.…”

Transforming growth factor-β is involved in maintaining oocyte meiotic arrest by promoting natriuretic peptide type C expression in mouse granulosa cells

“…TGFBR2 was mainly expressed in MGCs. In contrast, bone morphogenetic protein type II receptor (BMPR2), the oocyte-derived paracrine factors (BMP15, growth differentiation factor 9) binding receptor 49 , was mostly expressed in cumulus cells (Fig. S2b).…”

Antioxidant hesperetin improves the quality of porcine oocytes during aging in vitro

“…BMP15 prevents apoptosis of cumulus cells (Hussein, Froiland, Amato, Thompson, & Gilchrist, 2005). BMP15 and GDF9 interact and thereby elicit synergistic effects (Liu et al, 2018). CCNB1 forms a complex with cyclin-dependent kinase 1 that permits transition from G2 to M phase of the cell (Robert, Hue, McGraw, Gagne, & Sirard, 2002;Zhang et al, 2011).…”