Multiple molecular forms of inhibin in buffalo (Bubalus bubalis) ovarian follicular fluid Anita Ganguly * , Indrajit Ganguly 1 , Sanat K. Meur Division of Biochemistry and Food Science, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India article info Article history: Accepted 20 January 2010 Keywords: Inhibin Buffalo Follicular fluid Ovary Gel filtration chromatography Proteolysis abstract Inhibin is a heterodimeric glycoprotein hormone involved in the regulation of FSH release from the ante- rior pituitary gland and it has been characterized from various animals. Although, multiple molecular forms of inhibin have been reported from different species, however, the molecular nature of inhibin has not been studied in buffaloes. In the present study, attempts were made to identify inhibin in buffalo ovarian follicular fluid. Buffalo ovaries were obtained from the local abattoir and follicular fluid was aspi- rated from surface follicle (with diameter P5 mm). A combination of techniques (viz., gel filtration, SDS– PAGE, Western blot etc.) was employed for identification and isolation of inhibin(s). Inhibin bands were detected at 129 and 63 kDa by Western blot analysis in non-reducing conditions. In reduced SDS–PAGE, 63 kDa fraction produced a single band while 129 kDa fraction resolved into four components of 63, 43, 29 and 20 kDa. Out of them only 29, 63 and the native 129 kDa fractions produced bands on Western blot analysis. In total five fractions (63, 54, 39, 29, 25 kDa) were obtained by trypsin digestion of 129 kDa form. However, only 63 and 29 kDa fractions showed immunoreactivity. In this study, for the first time, we have identified two major forms of inhibin (129 and 63 kDa) with little proteolytic cleavage/process- ing of the large precursor in the buffalo follicular fluid. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Inhibin is a gonadal-derived heterodimeric glycoprotein hor- mone, which selectively inhibits secretion of follicle stimulating hormone (FSH) from the anterior pituitary gland (Burger, 1988). Besides its action on FSH through hypothalamo–hypophyseal– ovarian axis, inhibin may also be playing an important role in reg- ulation of folliculogenesis through autocrine and paracrine control (Findlay, 1993). The heterodimer is composed of an a subunit linked with either a bA or bB subunits by disulfide bonds to form active dimers known as inhibin A and inhibin B, respectively (Burger and Igarashi, 1988). Both a- and b-subunits of inhibin are generated by proteolytic cleavage of two independently synthesized large precursor molecules (Mason et al., 1985, 1986; Forage et al., 1986). In several species, the most predominant form of biologically active inhibin identified has a molecular weight (MW) of approximately 31–32 kDa (Robertson et al., 1985, 1986; Rivier et al., 1985; Fukuda et al., 1986; Miyamoto et al., 1985; Ling et al., 1985). In addition, inhibin of different MW has been isolated from various species including cattle (Fukuda et al., 1986; Sugino et al., 1992). Ovarian follicular fluid (FF) is a rich source of inhibin (de Jong and Robertson, 1985). Treatment with follicular fluid (proteins) and its subsequent withdrawal increases FSH flux which in turns leads to greater ovulation rate and/or litter size in sheep and cattle (Findlay et al., 1993; O’Shea et al., 1994). It has also been reported that crude FF and highly purified bovine inhibin suppress FSH secretion in vivo (Ireland et al., 1983; Beard et al., 1990). In India, buffalo is the most important milk, meat and draught animal as compared to cattle and other farm animals. However, inhibin in buffalo follicular fluid (buFF) has not yet been purified or characterized. Initial studies in goat indicate a delayed onset of estrus and increased ovulation rate by direct administration of crude (Kumar, 1997) and partially purified (Ghosh et al., 2005) buFF. However, active immunization against 30 kDa and above buFF proteins does not affect the onset, duration of estrus or ovulation rate and large follicle population due to low antibody titre (Ghosh et al., 2005). Further, passive immunization against inhibin in cycling Murrah buffalo preferentially increases plasma FSH level (Chandrasekhar and Madan, 1998). A detailed study of characteriza- tion and biological activity of buFF proteins (viz., inhibin(s), activin and follistatin etc.) may further show the way to modulate ovarian function in farm animals. Accordingly, the present investigation was undertaken to explore the molecular nature of inhibin in buFF. 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.01.014 * Corresponding author. Present address: Department of Veterinary Biochemis- try, College of Veterinary Sciences, CCS Haryana Agricultural University, Hisar, Haryana 125004, India. Tel.: +91 01662 289497; fax: +91 0651 2450838. E-mail address: anitaganguly@gmail.com (A. Ganguly). 1 Present address: Animal Genetics and Breeding Section, Project Directorate on Cattle, Meerut, Uttar Pradesh 250001, India. Research in Veterinary Science 89 (2010) 14–19 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc