Signalling pathways involved in the cooperative effects of ovine and murine GDF9+BMP15-stimulated thymidine uptake by rat granulosa cells

Abstract

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted factors known to be involved in regulating the proliferation and differentiation of granulosa cells during follicular growth. The aims of this study were to determine the signalling pathways used by recombinant forms of murine and ovine GDF9 and BMP15 in combination (GDF9CBMP15) and the molecular complexes formed by combinations of these factors. Differences in the molecular forms of combinations of murine and ovine GDF9CBMP15 were observed by western blot analysis. Ovine GDF9CBMP15-stimulated 3 H-thymidine uptake was completely blocked by SMAD2/3 and nuclear factor-kB pathway inhibitors and partially blocked by a p38-mitogen-activated protein kinase (MAPK) inhibitor. Thymidine uptake by murine GDF9CBMP15 was reduced by the SMAD2/3 and extracellular signal-regulated kinase-MAPK pathway inhibitors and increased after addition of a c-Jun N-terminal kinase inhibitor. Stimulation of 3 H-thymidine uptake by GDF9CBMP15 from either species was not affected by the SMAD1/5/8 pathway inhibitor. In conclusion, both murine and ovine GDF9CBMP15-stimulated thymidine incorporation in rat granulosa cells was dependent on the SMAD2/3 signalling pathway but not the SMAD1/5/8 pathway. Divergence in the non-SMAD signalling pathways used by murine and ovine GDF9CBMP15 was also evident and may be due to the differences observed in the molecular complexes formed by these factors. These results are consistent with the hypothesis that the disparate cooperative functions of GDF9 and BMP15 in different species are mediated by divergent non-SMAD signalling pathways.

Smad7 is a transforming growth factor-beta–inducible mediator of apoptosis in granulosa cells

“…It is also expressed in granulosa cells and is able to inhibit the functions of Smad2 and Smad3. Moreover, Smad7 could also inhibit granulosa cell proliferation through inhibition of the GDF9/BMP15 pathway (34). If Smad7 promotes apoptosis over survival, the expression level of Smad7 present in a follicle could be a determinant of follicle fate.…”

Growth differentiation factor 9:bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions

“…7B). In contrast, ALK6 is expressed in sheep granulosa cells beginning at the primordial (type 2) follicle stage (17), and mutations in both GDF9 and BMP15 cause a similar block at the primary follicle stage (10,11). Thus, in sheep, we predict that the activity of the homodimers is somewhat irrelevant, because ALK6 is expressed and GDF9: BMP15 heterodimers are expected to be potent, as in humans (Fig.…”

“…Although an in vitro study has detected the GDF9:BMP15 heterodimer by immunoprecipitation (15), and cooperative effects of the two homodimers were studied by other groups (16)(17)(18), the functions of GDF9:BMP15 heterodimers in any species remain largely unknown.…”

The local regulation of folliculogenesis by members of the transforming growth factor superfamily and its relevance for advanced breeding programmes

“…However, FSHR mRNA was increased in preantral bovine follicles cultured with hBMP15 or mGDF9 alone, but this effect was lost when follicles were also exposed to FSH (Passos et al, 2013;Vasconcelos et al, 2013). It is also important to note that BMP15 and GDF9 from different species appear to activate different second messenger systems, and thus the source of the BMP15 or GDF9 could affect outcomes (Reader et al, 2011(Reader et al, , 2016. Granulosa cells collected from small follicles of homozygous Booroola ewes were more responsive to FSH when assessed by stimulation of oestradiol production (Campbell et al, 2006).…”

“…Additionally, reduction of SMAD2 expression, through either chimiRNA-4110 mimics or SMAD2 interference, reduced SMAD2 mRNA and protein in caprine granulosa cells and increased apoptosis . The SMAD2/3 pathway is important for the synergistic actions of ovine BMP15 and GDF9 (Reader et al, 2011). In contrast to the suppressive effects of some BMP family members on apoptosis, members of the TGFB subfamily may actually induce apoptosis in ruminants.…”