In this study, PNa/PK and Em, were evaluated in early and late oocyte development. These data indicate a significant change in PNa/PK and Em between oocytes collected at the metaphase II stage and those from preantral follicles, and the effect from removal of cumulus cells. First, cumulus intact oocytes were compared to denuded oocytes to determine the amount of regulation from cumulus cells in a stage dependant manner. Secondly, comparisons made between two chosen stages of oocyte development (preantral and metaphase II) determined the amount of change in both variables tested over a broad maturational time. These stages were chosen to look at the early stages of oocyte maturation compared to late in an effort to bridge the gap present in the current literature, in which the changes occurring at transitions from GV to MII have been studied.
These data indicate that while Em
of denuded oocytes at the MII stage were no different than cumulus enclosed MII oocytes, denuded oocytes from preantral follicles versus cumulus enclosed of the same stage did vary. Furthermore, if cumulus was removed from preantral stage oocytes the membrane potential became more positive and no longer significantly different than metaphase II oocytes, regardless of cumulus cell attachment. While present, heterologous gap junctions between the oocyte and cumulus cells may play a key role in the maintenance of oocyte membrane potential. Previous studies in several species have shown functional gap junctions in the cumulus-oocyte complex present during many stages and required for meiotic resumption [[11
] for review]. When the cumulus cells begin to undergo cumulus expansion, cytoplasmic extensions protruding through the zona pellucida contacting the oolema are retracted, thus breaking heterologous gap junction communication. The loss of gap junctions during cumulus expansion could account for the shift in oocyte membrane potential observed between early and late maturation stages. The mechanism could be either direct reduction in ion diffusion through gap junctions, or indirectly by causing the oocyte to adjust oolemma properties in response to loss of a cumulus derived signal.
The data presented here support previous studies (11) and could explain the change in membrane potential seen when cumulus cells are chemically removed, as in the case of the preantral oocyte experiments or physiologically removed as in the case of the metaphase II oocyte. Work done by Gilula, Epstein and Beers, 1978 describes a similar conclusion, ionic coupling of cumulus cells and oocytes decrease to zero from preovulatory to postovulatory specimens [12
]. Other work from the pig model shows a loss of gap junctions in the same maturational time, progression from metaphase I to metaphase II [13
The presence of gap junctions is well documented but the regulation of these connexin pores is not well defined in any model. Thus it is interesting to note the difference in the membrane potential of the cumulus cells and oocyte at the preantral stage. This indicates that while the two cells are highly electrically coupled before ovulation [12
], they maintain a different membrane potential.
When the Em
of denuded and cumulus-intact oocytes were compared within their maturational stage, either preantral or MII, there was a significant difference during the preantral stage (p < 0.0001, 0.001 respectively) but not at MII. This substantiates the above importance of cumulus regulation. Grenfield, Hackett, and Linden investigated Xenopus oocyte K+
currents in 1990 to reveal that an outward K+
current in response to cAMP is abolished by inhibition of gap junctions or removal of cumulus cells [14
], effecting the permeation of the cell to K+
and substantiating these data. The loss of gap junctions in Grenfield's paper caused the outward potassium current to decrease, just as the PNa
value became closer to one in our studies.
Results for the membrane potential of MII oocytes is similar to values obtained by Racowsky and Saterlie [9
]. This value (-19.7 ± 0.8 mV) from the cumulus-enclosed oocyte is less negative than the potential we report from oocytes removed from preantral oocytes (-38.4 ± 4.5 mV, p < 0.001). Racowsky and Saterlie have also investigated the importance of changes in oocyte and cumulus cell membrane potential during the resumption of meiosis and progression to metaphase II [10
]. The aforementioned authors indicate that a shift in Em
to a more positive value does not seem to be requisite for this progression. Our data suggest there is a requisite change in membrane potential from the early stage to late stage oocytes. The change in oocyte membrane potential and PNa
we describe here suggest there is a requisite change in oocyte development between the preantral and metaphase II stage. This alteration in membrane potential may not be necessary for progression from germinal vesicle to MII, but it is likely that it is required to produce an oocyte competent for fertilization. This may be a more gradual change or a multi-step rise in the Em
and decrease in PNa
from preantral to GVBD.