Ca2+]i rise at in vitro maturation in bovine cumulus–oocyte complexes

Abstract

An intracellular calcium ([Ca(2+)]i) rise has been described in cumulus-oocyte complexes (COCs) following luteinizing hormone (LH) exposure. Together with cAMP, Ca(2+) is a candidate signal for resumption of meiosis. Here, we analyzed if the most common hormones involved in oocyte maturation can induce the same Ca(2+) signal. In addition, we characterized the source of this signal. Immature, in vitro-matured, and roscovitine-meiotically arrested COCs were loaded with Fluo-4 AM, stimulated with hormones/growth factors, and tested for [Ca(2+)](i) variations in cumulus cells. Reagents known to inhibit or stimulate [Ca(2+)](i) rises were used to characterize these [Ca(2+)](i) dynamics. Finally, expression of LH receptors (LHRs) in COCs was analyzed by immunofluorescence. In immature COCs, follicle-stimulating hormone (FSH) elicited a single [Ca(2+)](i) rise that was higher than those induced by LH and growth hormone (GH), whereas epithelial growth factor failed to induce any changes in [Ca(2+)](i). The [Ca(2+)](i) rise induced by FSH was higher in immature COCs; was reduced in roscovitine-arrested, immature COCs; and was negligible in gonadotropin-induced, in vitro-matured COCs. In the case of spontaneous- and GH-matured COCs, however, FSH stimulation caused a lower [Ca(2+)](i) rise. The hormone-induced [Ca(2+)](i) rise was due to: (i) external Ca(2+) entry; (ii) intercellular communication; and (iii) intracellular Ca(2+) stores. Immunofluorescence revealed that LHRs were expressed throughout the cumulus cells. The above results show that: (i) gonadotropins and GH cause a [Ca(2+)](i) rise in cumulus cells; (ii) this [Ca(2+)](i) rise results from extra-, inter-, and intra-cellular cumulative Ca(2+) fluxes; and (iii) LHRs are distributed on either outer or inner cumulus cells.