Relationships among vasculature, mitotic activity, and endothelial nitric oxide synthase (eNOS) in bovine antral follicles of the first follicular wave T. Moonmanee a , C. Navanukraw a, b, * , S. Uriyapongson a , A. Kraisoon a , S. Aiumlamai c , S. Guntaprom d , T. Rittirod e , P.P. Borowicz f , D.A. Redmer f a Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand b Agricultural Biotechnology Research Center for Sustainable Economy (ABRCSE), Khon Kaen University, Khon Kaen, 40002, Thailand c Department of Surgery and Theriogenology, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand d Department of Animal Health Science, Agro Industrial Faculty, Rajamangala University of Technology Isan Kalasin Campus, Kalasin, 46000, Thailand e Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand f Department of Animal Sciences, North Dakota State University, Fargo, ND 58108, USA article info Article history: Received 14 August 2012 Received in revised form 9 March 2013 Accepted 10 March 2013 Keywords: Vasculature Mitotic activity eNOS protein Antral follicle Beef cows abstract To determine the relationships among vasculature, mitotic activity, and expression of endothelial nitric oxide synthase (eNOS) of antral follicles in Bos indicus, bovine ovaries were obtained on day 6 of the estrous cycle from 10 crossbred (Brahman to Thai native cows) after a synchronized estrus with prostaglandin F 2 a analogue. Ovaries were fixed, paraffin-embedded, and used for immunofluorescence detection of factor VIII (a marker of endothelial cells). Immunostaining of eNOS and proliferating cell nuclear antigen (PCNA) were performed with specific monoclonal antibodies. Vasculature and positive staining of eNOS and PCNA were quantitatively evaluated with the image analysis. Follicles were classified by size (small, medium, and large) and by structure as healthy and atretic follicles (n ¼ 82). The expression of factor VIII and eNOS were detected greater in the blood vessels of the theca layers of the healthy follicles than those in atretic follicles. The labeling indices (LIs) in granulosa and theca cells were greater (P < 0.05) in the healthy small and medium follicles than in the healthy large follicles. Vasculature, capillary area density, and capillary number density were positively correlated with eNOS expression and the LIs of granulosa and theca cells but were negatively correlated with the healthy follicle size. During the growing phase of antral follicle in Bos indicus, relationships among vasculature, mitotic activity, and eNOS were observed predominantly in healthy antral follicles. Thus, these data highlight the importance of vasculature, cell proliferation, and eNOS expression of growing and atretic follicles in the first follicular wave. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Bovine follicular growth and atresia are characterized by 2 or 3 follicular waves [1]. At each follicular wave, a cohort of follicles is initiated to grow, but only the dominant follicle continues to grow and ovulate or the follicle will be atretic [2]. Because the development and regression of follicles are associated with major structural and functional changes, it is important to classify follicles accurately as healthy or atretic at all stages of development [3,4]. Maintenance of follicular health depends on the presence of angiogenic factors and a functional vasculature [5]. Changes of vascu- larization and expression of some regulators, including angiogenic factors, are associated with follicular growth and/or atresia. Vascular network around developing folli- cles may be rate limiting for the selection of the dominant follicle(s), that is, the follicle(s) that is/are destined to ovulate. In turn, insufficient vascular supply could act as the * Corresponding author. Tel.: þ66 43 202 362; fax: þ66 43 202 362. E-mail address: chanav@kku.ac.th (C. Navanukraw). Contents lists available at SciVerse ScienceDirect Domestic Animal Endocrinology journal homepage: www.domesticanimalendo.com 0739-7240/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2013.03.002 Domestic Animal Endocrinology 45 (2013) 11–21