Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth

Oocytes grow within ovarian follicles in which the oocyte is coupled to the surrounding granulosa cells by gap junctions. It was previously found that small growing oocytes isolated from juvenile mice and freed of their surrounding granulosa cells (denuded) lacked the ability to regulate their intracellular pH (pH_i), did not exhibit the pH_i-regulatory HCO₃^-/Cl^- and Na⁺/H⁺ exchange activities found in fully-grown oocytes, and had low pH_i. However, both exchangers became active as oocytes grew near to full size, and, simultaneously, oocyte pH_i increased by approximately 0.25 pH units. Here, we show that, in the more physiological setting of the intact follicle, oocyte pH_i is instead maintained at 7.2 throughout oocyte development, and the growing oocyte exhibits HCO₃^-/Cl^- exchange, which it lacks when denuded. This activity in the oocyte requires functional gap junctions, as gap junction inhibitors eliminated HCO₃^-/Cl^- exchange activity from follicle-enclosed growing oocytes and substantially impeded the recovery of the oocyte from an induced alkalosis, implying that oocyte pH_i may be regulated by pH-regulatory exchangers in granulosa cells via gap junctions. This would require robust HCO₃^-/Cl^- exchange activity in the granulosa cells, which was confirmed using oocytectomized (OOX) cumulus-oocyte complexes. Moreover, in cumulus-oocyte complexes with granulosa cells coupled to fully-grown oocytes, HCO₃^-/Cl^- exchange activity was identical in both compartments and faster than in denuded oocytes. Taken together, these results indicate that growing oocyte pH_i is controlled by pH-regulatory mechanisms residing in the granulosa cells until the oocyte reaches a developmental stage where it becomes capable of carrying out its own homeostasis.