269 Connexins in Sheep Ovaries/Grazul-Bilska et al. Vol. 8, No. 3 Received January 6, 1998; Revised February 18, 1998; Accepted March 5, 1998. Author to whom all correspondence and reprint requests should be addressed: Dr. Anna T. Grazul-Bilska, Cell Biology Center, 119b Hultz Hall, North Dakota State University, Fargo, ND 58105. E-mail: grazul@plains.nodak.edu Current Address: Dr. Albina Jablonka-Shariff, Dept. Obstetrics/Gynecology, Washington University School of Medicine, St. Louis, MO 63110. Endocrine, vol. 8, no. 3, 269–279, June 1998 0969–711X/98/8:269–279/$10.75 © 1998 by Humana Press Inc. All rights of any nature whatsoever reserved. 269 Gap Junctional Proteins, Connexin 26, 32, and 43 in Sheep Ovaries Throughout the Estrous Cycle Anna T. Grazul-Bilska, 1,2 Dale A. Redmer, 2 Jerzy J. Bilski, 1,2 Albina Jablonka-Shariff, 2 Vinayak Doraiswamy, 2 and Lawrence P. Reynolds 1,2 1 Cell Biology Center, Biotechnology Institute and 2 Department of Animal and Range Sciences, North Dakota State University, Fargo, ND Introduction Ovarian follicles and corpora lutea (CL) are the most rapidly growing normal tissues in adults that grow, differ- entiate, and regress during each estrous cycle and/or preg- nancy (1–4). Successful development of follicles to the preovulatory stage and subsequent development of the CL require cellular interactions among ovarian cells to coordi- nate the functions of these diverse cell types that are prolif- erating and differentiating extremely rapidly (1–7). Such cellular interactions may be mediated by several mecha- nisms, including humoral pathways as well as the contact- dependent gap junctional pathway (8). Gap junction-mediated intercellular communication has been shown to be important for coordination of cellular metabolism and function during growth and differentiation of organs and tissues (9–13). Gap junctions are important in signal transduction, and in transport of nutrients within avascularized or poorly vascular tissues, such as the folli- cular granulosa layer or corpora hemorrhagica, since gap junctional channels allow for transfer of molecules <1000 M r (7,8,12,14–20). Thus, during follicular develop- ment, gap junctions are thought to be involved in regulating differentiation and maturation of the cumulus–oocyte com- plex (21–23). During development of CL, gap junctions mediate cellular interactions, which may be important for progesterone production and the luteolytic process (8,15). Gap junctions are composed of hemichannels (connexons), which consist of six-subunit proteins named connexins (9,24). Distribution of connexins is tissue- and cell-specific (9,24). Several connexins have been identified within ova- rian tissues of numerous species (5,8,14,15,18,21,25–37). The aim of the present study was to evaluate the pres- ence of Cx26, Cx32, and Cx43 in ovine ovaries at several stages of follicular and luteal development. Results Immunohistochemical staining demonstrated punctate staining for Cx26, Cx32, and Cx43 in several compart- ments of ovine ovaries throughout various stages of the Ovarian follicles from days 13, 14, 15, and 16 and corpora lutea (CL) from days 2, 4, 8, 12, and 15 of the estrous cycle were evaluated for the presence of connexins by immunohistochemistry. In addition, CL from days 5, 10, and 15 of the estrous cycle were used for immunofluorescent detection of Cx43 followed by image analysis, and for Western immunoblot. In all tissues, staining for all connexins appeared punctate, indicating the presence of assembled gap junctions. Cx26 was present in the ovarian surface epithelium, stroma, and blood vessels within the stroma and hilus, and in the CL. In healthy antral follicles, Cx26 was present only in the theca layer, whereas Cx43 was present in granulosa and theca layers. In the majority of atretic follicles, connexins were not detected, but in 13% of the atretic follicles, Cx43 was present in the theca layer. Cx32 was detected in the blood vessels of ovarian stroma and in the CL, and Cx43 was detected in the CL. Localization and/or expression of connexins depended on stage of luteal development. Western analysis demonstrated that expression of Cx32 in luteal tissues was similar across the estrous cycle. The area of positive staining for Cx43 and expression of Cx43 in luteal tissues decreased (p < 0.05) as the estrous cycle progressed. The pattern of expression of connexins indicates that gap junctional proteins may be impor- tant in the regulation of folliculogenesis and follicular atresia, as well as growth, differentiation, and regres- sion of the CL. Key Words: Gap junctions; Cx26; Cx32; Cx43; follicles; corpora lutea; ovaries; ewe.