Divalent cation influx and calcium homeostasis in germinal vesicle mouse oocytes.

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca) influx and spontaneous Ca oscillations. The oscillations cease during maturation but Ca influx continues, as the oocytes' internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca influx has not been completely determined. GV and matured oocytes are known to express three Ca channels, Ca3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca homeostasis, suggesting a complex regulation of Ca influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. Ca3.2 and TRPM7 channels contributed the majority of Ca influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca entry. Sr influx was promoted by Ca3.2 channels, as Sr oscillations were negligible in Ca3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn entry relied on expression of Ca3.2 and TRPM7 channels, but Ni entry depended on the latter. Ca3.2 and TRPV3 channels combined to fill the Ca stores, although Ca3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies.