Differences in the participation of TGFB superfamily signalling pathways mediating porcine and murine cumulus cell expansion

Abstract

It is widely held that mammalian cumulus cell (CC) expansion requires oocyte-paracrine signalling, however in three of the four species studied to date, CC expansion occurs in the absence of the oocyte. This study was conducted to examine the paracrine and SMAD/MAPK intracellular signalling mechanism mediating porcine CC expansion, and to compare these to the mouse. Cumulus-oocyte complexes (COCs) and oocyte-free complexes (OOXs) from pigs and eCG-primed mice were treated in vitro with FSH and a broad range of TGFB superfamily antagonists. Expansion of porcine COCs and OOXs was unaffected by neutralisation of growth differentiation factor 9, TGFB, activin A, activin B and a broad spectrum bone morphogenetic protein antagonist. A SMAD-responsive luciferase reporter assay confirmed that porcine oocytes secreted factors that activate SMAD3 and SMAD1/5/8 in granulosa cells, but murine oocytes activated SMAD3 only. Treatment of COCs with a SMAD2/3 phosphorylation inhibitor (SB431542) partially inhibited porcine CC expansion and expression of TNFAIP6, but ablated murine CC expansion. SB431542 was equally effective at attenuating porcine CC expansion in the presence or absence of the oocyte. By contrast, a SMAD1/5/8 phosphorylation inhibitor (dorsomorphin) had no effect on porcine or murine CC function. Inhibition of ERK1/2 and p38 MAPK signalling pathways prevented porcine COC expansion and expression of most matrix genes examined. The activation of CC SMAD signalling by oocytes, and the requirement of SMAD2/3 signalling for expansion, is notably contrasted in pigs and mice. Nonetheless, porcine CC SMAD2/3 signalling is likely to be needed for optimal matrix formation, possibly by facilitating essential MAPK signals.

Signaling pathways regulating FSH- and amphiregulin-induced meiotic resumption and cumulus cell expansion in the pig

“…Therefore, through this mechanism, the MAPK14 is involved in regulation of mouse cumulus expansion. Such a MAPK14-dependent regulatory loop seems to also work in the pig as indicated by results presented in this study and also in other studies reporting inhibition of pig cumulus expansion by SB203580 (Yamashita et al 2009, Gilchrist & Ritter 2011. In the mouse, FSH-but not EGF-induced cumulus expansion and elevation of expansion-related transcripts (Has2 and Ptgs2) required MAPK14 activity (Diaz et al 2006).…”

Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family, Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality

“…In brief, cumulus-oocyte complexes were aspirated from small antral follicles (2-4 mm) and matured in protein-free porcine oocyte maturation basic IVM medium (35), supplemented for the first 22 h of IVM with 1 mM dibutyryl-cAMP and 100 ng/ml amphiregulin (R&D Systems) and treated with either vehicle (control), 20 or 100 ng/ml pro-cumulin, 20 or 100 ng/ml mature cumulin, 100 ng/ml pro-GDF9, 100 ng/ml pro-BMP15, or 100 ng/ml of each of pro-GDF9 ϩ pro-BMP15. Cumulus expansion was assessed at 22 h of IVM and scored as per standard criteria (36,37). At 22 h, cumulus-oocyte complexes were washed and matured for a further 22 h in porcine oocyte maturation without supplements or treatments (13,33,34).…”

The role of TGF superfamily gene expression in the regulation of folliculogenesis and oogenesis in mammals: a review

“…However, in three out of the four species studied to date, CCE did not require the presence of oocytes (Nagyova 2012;Watson et al 2012;Gharibi et al 2013). To study this process, Gilchrist and Ritter (2011) analysed the paracrine mechanisms of the Smad/MAPK signalling cascade comparing porcine and murine cumulus-oocytecomplexes (COCs). Treating COCs and oocyte-free complexes (OOXs) with FSH and TGFB superfamily antagonists, they found that the inhibition of TGFB superfamily genes, such as GDF9, TGFB, activin A, and activin B, and several BMP superfamily genes did not affect the CCE of porcine COCs.…”

“…Since Derynck et al (1985), first identified a partial amino acid sequence from transforming growth factor beta 1 (TGFB1) purified from blood platelets, numerous studies have highlighted the role of this peptide as a modulator of cell growth, proliferation and differentiation in many types of cells and tissues (Gilchrist and Ritter 2011;Nagamatsu et al 2012). It is also thought that TGF proteins are involved in the regulation of oocyte and embryonic growth and development.…”