PARENT SESSION

(T395) OOCYTE-DIRECTED DEPLETION OF CONNEXIN43 USING THE Cre-LoxP SYSTEM.

Gershon, Eran¹, Plaks, Vicki¹, Aharon, Idan¹, Galiani, Dalia¹, Petrovich, Katia¹, Kalma, Yael¹, Reizel, Yitzhak¹, Sela-Abramovitch, Sagit¹, Granot, Irit², Winterhager, Elke³, Dekel, Nava¹, ¹ The Weizmann Institute of Science, Rehovot, Israel² Kaplan Medical Center, Rehovot, Israel³ University Hospital Duisburg-Essen, Duisburg, Essen, Germany

ABSTRACT- Gap junctions are transmembrane channels that consist of protein subunits referred to as connexins (Cxs). Several Cxs have been detected in ovarian follicles of different species, among which the necessity of connexin37 (Cx37) and connexin43 (Cx43) for normal ovarian function has been demonstrated. Mice lacking Cx43 die soon after birth due to cardiac malfunction. To avoid postnatal lethality allowing the examination of the role of Cx43 in oogenesis, we established a model of mice lacking Cx43 in the oocyte by mating Cre transgenic males, expressing the enzyme under the control of ZP3 promoter with loxP-Cx43 females. Superovulation experiments and histological evaluations detected no abnormalities in the ovaries of the Cx43 depleted mice. Furthermore, dye microinjection into Cx43^del/del oocytes revealed the presence of established communication with the surrounding cumulus cells. In addition, depletion of Cx43 does not affect the expression levels of other connexins such as Cx26, Cx32 and Cx37. Despite the above mentioned findings, mating of the oocyte Cx43^del/del females with wild type males resulted in a 50% reduction in the rate of parturition and a similar decrease in litter size. Examining the tissue-specificity of the Cx43 gene ablation revealed in addition to the complete deletion of Cx43 in the oocytes a substantial reduction in the amount of Cx43 mRNA and protein in the ovarian somatic cells. Along this line, the granolusa cells expressed the Cre-recombinase mRNA and the deletion of the Cx43 gene in their genome was observed. Our data suggest that, unexpectedly, the ZP3 Cre mice did not restrict the ablation of Cx43 to the oocyte, rather directing partial deletion of this gene to the granolusa cells as well. Nevertheless, full depletion of Cx43 in the oocyte combined with its partial deletion in the somatic follicle cells neither affect ovarian development nor ovulation but severely impair the rate of fertility. Further investigations will clarify whether subfertility in our model is attributable to corpus luteum insufficiency that is subsequent to reduced Cx43 expression in granolusa cells.

KEY WORDS: oocyte, cre-lox, connexin43, gap-junction