PARENT SESSION
GRANULOSA CELL FUNCTION AND REGULATION
Laurel
7:30 AM-10:00 AM

(274) EXPRESSION AND QUANTIFICATION OF GAP JUNCTIONAL PROTIEN CONNEXIN 43 IN CUMULUS OOCYTE COMPLEX OF EWES TREATED IN VIVO WITH FSH.

Pant, Disha¹, Reynolds, Lawrence^1,2, Lopes, Flavia¹, Petry, Kimberley¹, Bilski, Jerzy¹, Redmer, Dale¹, Grazul-Bilska, Anna^1,2, ¹ Department of Animal and Range Sciences, Fargo, ND² Cell Biology Centre, Fargo, ND

ABSTRACT- Gap junctions play an important role in oocyte growth and maturation, and Cx43 seems to be a major gap junctional protein in ovarian follicles. Non-pregnant ewes were grouped into control (n=13) and FSH treated (n=14) groups. The latter were treated with FSH for two to three days beginning day 13 of the estrous cycle and ewes were slaughtered on days 15 or 16. COC were collected from all visible small (<3 mm) and large (>3 mm) antral follicles. Immediately after collection on day 16, COC were embedded in agarose gel and fixed in Carnoys solution. However, after collection on day 15, a portion of COC was embedded and fixed as for day 16, but the other portion of COC were incubated in maturation medium (TCM-199 containing 10% FBS, FSH, LH, and estradiol for 24 h before embedding and fixing. Cx43 was immunolocalized in histological sections (6 m) of each COC, followed by nuclear fast red to stain cumulus cell nuclei. The area of positive staining for Cx43 and the number of nuclei were determined for each COC using computerized image analysis. Cx43 data are expressed as area of positive staining per cumulus cell (m²/cell). Before maturation, cumulus cells were compacted, but after maturation cumulus cells were expanded. Cx43 was localized on cellular borders in COC. The expression of Cx43 in COC was greater (P<0.01) on day 16 (0.60±0.09) than on day 15 (0.22±0.02) of the estrous cycle. In vivo FSH treatment decreased (P<0.02) Cx43 expression on day 16 (from 0.71±0.09 for control to 0.25±0.05 for FSH treated), but not on day 15 of the estrous cycle. Cx43 expression tended (P<0.1) to be greater for matured (0.36±0.06) than for nonmature (0.31±0.03) COC. Expression of Cx43 was similar for COC from small and large follicles. These results indicate that day of estrous cycle, stage of maturation and duration of FSH treatment affect expression of Cx43 in ovine COC. (Supported by ND EPSCoR grant to ATGB and ND SBARE grant to ATGB, DAR & LPR)

KEY WORDS: Gap junctions, cumulus oocyte complex, FSH, sheep