279 PLASMA MEMBRANE ELECTRICAL PROPERTIES AND INTRACELLULAR CALCIUM STORES IN IMMATURE AND IN VITRO-MATURED ADULT AND JUVENILE SHEEP OOCYTES

Abstract

The low developmental efficiency recorded in juvenile oocytes represents, besides its technological relevance, an opportunity for increasing the knowledge of mechanisms regulating developmental competence in the oocytes. To analyze the biological reasons that make an adult oocyte different from a juvenile one, we monitored membrane electrical properties, i.e. resting potential, steady-state conductance and calcium currents, and calcium stores in these two oocyte types both at immature (GV) stage and after in vitro maturation (MII). Ovaries of cycling ewes and 40-day-old lambs were collected at abattoir and transported at 30°C. Cumulus-oocyte complexes (COC) were recovered by mincing. In vitro maturation was carried out in TCM199 supplemented with 10% fetal calf serum, 10 IU/mL of LH, 0.1 IU/mL of FSH, and 1 mg/mN of 17β-estradiol at 39.0°C in 5% CO₂ for 24 h. Zona pellucida of immature and in vitro-matured oocytes was removed after incubation for 1–1.5 min in 0.5% (w/v) protease solution. Zona-free oocytes were placed in Ham F10 at 38.5°C and voltage clamped by standard techniques (Tosti et al. 2002 Reproduction 124, 835–846). After obtaining a giga-seal, the patch was ruptured. The permeability of the plasma membrane was verified by applying depolarizing and hyperpolarizing voltage steps of 10 mV and 500 ms before and at the peak current to generate the voltage-dependent currents. The voltage clamp was set at −80 and −30 mV to differentiate the Ca²⁺ current components, i.e. L-type Ca²⁺ channels. For intracellular calcium determinations, oocytes were placed in Ham F10 and injected with the 0.5 mM calcium green dextran (Mr 10,000). Ca²⁺ stores were evoked by the addition of 5 μM Ca²⁺ ionophore, monitored using a computer-controlled photo-multiplier system, and measured as relative fluorescence units (RFU) by normalizing fluorescence against baseline fluorescence. In lamb and ewe, differences in electrical features and calcium dynamics between GV (n = 36 and 17) and MII (n = 42 and 32) oocytes were tested by ANOVA and expressed as mean ± SEM. Resting potential was higher at MII than GV stages (−15.2 ± 0.9 vs. −12.1 ± 1.1 mV, respectively; P < 0.02) but it did not differ between animal age. GV stage and ewe showed either a higher steady-state conductance (25.4 ± 0.2 vs. 11.7 ± 0.2 nS and 21.7 ± 0.2 vs. 15.4 ± 0.2 nS, respectively; P < 0.01) or L-type Ca²⁺ channels (9.7 ± 1.4 vs. 2.7 ± 1.3 pA and 9.2 ± 1.5 vs. 3.2 ± 1.1 pA, respectively; P < 0.01). No differences were found between resting potential peaks yielded after Ca²⁺ ionophore exposure but a higher ion activation current was found in lamb oocytes (489 ± 56 vs. 300 ± 73 pA; P < 0.05). Ca²⁺ stores did not differ between animal age but they were larger at MII than at GV stage (0.70 ± 0.07 vs. 0.44 ± 0.07 RFU; P < 0.01). These results supply further information on both reproductive biology in ovine species and the physiology of oocytes collected from juvenile and adult individuals.

This work was supported by Italian Ministry of University and Research (MIUR) COFIN 2002 Project.