280 EFFECTS OF β-ENDORPHIN AND NALOXONE ON INTRACELLULAR CALCIUM LEVELS IN CUMULUS CELLS OF EQUINE OOCYTES

Abstract

Changes in intracellular calcium levels in the cumulus oocyte complex (COC) have a crucial role in oocyte maturation. In previous studies we demonstrated that the μ-opioid receptor is expressed in the bovine COC and participates in the signaling associated with oocyte maturation, by inducing an intracellular calcium increase (Dell'Aquila ME et al. 2002 Mol. Reprod. Dev. 63, 210–222). In this work we evaluated modifications of intracellular calcium induced by β-endorphin (β-end) or Naloxone (Nx) in cumulus cells of equine oocytes in relation to the time of the year and cumulus morphology at retrieval. Cumulus cells, isolated by mechanical treatment from compact (Cp, n = 120) or expanded (Exp, n = 120) COCs, recovered from the ovaries of slaughtered mares (follicles <20 mm in diameter) during anestrus, breeding season, spring transition, and autumnal transition, were cultured for 24 h and loaded with 5 μM Fura2-AM for microspectrofluorometric measurements of cytoplasmic ionized calcium (Dell'Aquila et al., 2002). The changes in β-end (30 μM)- or Nx (1mM and 10 μM)-induced calcium concentration were calculated in single cells (n = 194) and are expressed as Δ fluorescence (F_{maximal effect} – F_baseline) before and after 1-min perfusion with the drugs. The use of 1 mM Nx induced a significant increase of intracellular calcium levels in cumulus cells of oocytes recovered in all periods of the year in both Cp and Exp (P < 0.01). The addition of 10 μM Nx or 30 μM β-end significantly increased intracellular calcium only in cumulus cells from oocytes recovered in anestrus (P < 0.05). These results confirm previous observations, carried out on bovine oocytes, in which Nx behaved as a μ-receptor agonist when used at high concentration (Dell'Aquila et al. 2002). The effects of β-end and Nx may be explained in terms of a binding of the two subtances at the μ-receptor with consequent intracellular calcium increases due to extracellular calcium entry or depletion of intracellular stores. These findings could be related to differential espression and/or activation status of the μ-opioid receptor in COCs retrieved in different seasons. These substances can be used to modulate intracellular calcium in the equine COCs, and consequent effects on the stimulation/inhibition of oocyte maturation in this species need to be further investigated.

This work was supported by Grant MIUR COFIN PRIN 2003.