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

Cell calcium (2020-02-26)
Goli Ardestani, Aujan Mehregan, Andrea Fleig, F David Horgen, Ingrid Carvacho, Rafael A Fissore
ABSTRACT

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca2+) influx and spontaneous Ca2+ oscillations. The oscillations cease during maturation but Ca2+ 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 Ca2+ influx has not been completely determined. GV and matured oocytes are known to express three Ca2+ channels, CaV3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca2+ homeostasis, suggesting a complex regulation of Ca2+ 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. CaV3.2 and TRPM7 channels contributed the majority of Ca2+ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca2+ entry. Sr2+ influx was promoted by CaV3.2 channels, as Sr2+ oscillations were negligible in CaV3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn2+ entry relied on expression of CaV3.2 and TRPM7 channels, but Ni2+ entry depended on the latter. CaV3.2 and TRPV3 channels combined to fill the Ca2+ stores, although CaV3.2 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 Ca2+ 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Magnesium chloride hexahydrate, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Nickel(II) chloride hexahydrate, BioReagent, suitable for cell culture
Sigma-Aldrich
2-APB - CAS 524-95-8 - Calbiochem, A cell-permeable modulator of Ins(1,4,5)P3-induced Ca2+ release.
Sigma-Aldrich
NS8593 hydrochloride, ≥98% (HPLC), powder