Mouse Oocytes Promote the Mechanistic Target of Rapamycin (Serine/Threonine Kinase, MTOR) Pathway in Cumulus Cells by Suppressing the Expression of DNA-damage-inducible Transcript 4-like (Ddit4l).

Mechanistic (also known as mammalian) target of rapamycin (MTOR) is an evolutionally conserved kinase that plays an essential role in the control of cell growth and development by integrating and transmitting multiple extracellular cues, such as nutrient supply and signals of growth factor and stress. We reported previously that mouse oocytes promote several key metabolic processes including glycolysis, cholesterol biosynthesis and uptake of some amino acids in cumulus cells. This suggests that mouse oocytes promote MTOR activity in cumulus cells, but this implication had not been tested previously. Here, we show that mouse oocytes promote the MTOR pathway in cumulus cells by suppressing expression of DNA-damage-inducible transcript 4-like (Ddit4l) mRNA, a negative regulator of MTOR pathway. In situ hybridization of Ddit4l mRNA revealed its robust expression in mural granulosa cells, whereas expression in cumulus was significantly lower. Removal of oocytes from cumulus-oocyte complexes (COCs) by oocytectomy (OOX) caused dramatic up-regulation of Ddit4l mRNA in OOX cumulus cells, suggesting that the lower level of expression of Ddit4l in cumulus cells is due to suppression by oocytes. Coincident with up-regulation of Ddit4l expression, levels of phosphorylated MTOR (p-MTOR) in OOX cumulus cells was reduced. Up-regulation of Ddit4l mRNA and down-regulation of p-MTOR in wild type (WT) OOX cumulus cells was prevented by co-culture of these OOX cumulus cells with WT, but not Bmp15^-/- or Gdf9^+/-Bmp15^-/-, fully-grown, germinal vesicle stage oocytes. This suggests that the regulatory effect of oocytes on MTOR pathway in cumulus cells is mediated by oocyte-secreted factors, presumably, GDF9 and/or BMP15. Indeed, Ddit4l mRNA was significantly up-regulated in Bmp15^-/- and Gdf9^+/-Bmp15^-/- cumulus cells; and treatment with recombinant GDF9 and/or BMP15 suppressed expression of Ddit4l mRNA in WT OOX cumulus cells. Recombinant GDF9 and/or BMP15 were also found to promote the elevated level of p-MTOR in OOX cumulus cells. Treatment with inhibitors of SMAD2/3 but not of SMAD3 dramatically up-regulated the level of Ddit4l mRNA in WT COCs, and blocked GDF9-induced suppression of Ddit4l mRNA expression in WT OOX cumulus cells, indicating that a SMAD2-dependent pathway is involved in the process of oocyte and GDF9 regulation of MTOR pathway in cumulus. In conclusion, mouse oocytes, via GDF9 and BMP15, promote the MTOR pathway in cumulus cells, at least in part, by suppressing expression of Ddit4l, a negative regulator of this pathway. (Supported by NIH grant HD42137 and HD23839).

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