Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats Arash Kheradmand a,⇑ , Omid Dezfoulian b , Masoud Alirezaei c , Bahram Rasoulian d a Department of Clinical Sciences, School of Veterinary Medicine, Lorestan University, P.O. Box: 465, Khorram Abad, Iran b Department of Pathobiology, School of Veterinary Medicine, Lorestan University, Khorram Abad, Iran c Division of Biochemistry, School of Veterinary Medicine, Lorestan University, P.O. Box: 465, Khorram Abad, Iran d Razi Herbal Medicine Research Center, Lorestan University of Medical Sciences, Khorram Abad, Iran article info Article history: Received 29 January 2012 Available online 14 February 2012 Keywords: Ghrelin Rat Apoptosis Spermatogenesis Bax PCNA abstract Under normal condition in the most mammals, spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. The present study was designed to determine the effects of ghrelin treatment on in vivo quality and quantity expression of apoptosis and proliferation specific indices in rat testicular germ cells. Twenty eight adult normal rats were subdivided into equal control and treatment groups. Treatment group received 3 nmol of ghrelin as subcutaneous injection for 30 con- secutive days or vehicle to the control animals. The rats from each group (n = 7) were killed on days 10 and 30 and their testes were taken for immunocytochemical evaluation and caspase-3 assay. Immunohis- tochemical analysis indicated that the accumulations of Bax and PCNA peptides are generally more prominent in spermatocytes and spermatogonia of both groups. Likewise, the mean percentage of immu- noreactive spermatocytes against Bax increased (P < 0.01) in the ghrelin-treated group on day 10, while despite of 30% increment in the Bax level of spermatocytes in the treated rats on day 30, however, it was not statistically significant. During the experimental period, only a few spermatogonia represented Bax expression and the changes of Bax immunolabling cells were negligible upon ghrelin treatment. Likewise, there were immunostaining cells against Bcl-2 in each germ cell neither in the control nor in the treated animals. In fact, ghrelin balanced Bax/Bcl-2 ratio toward at increase of Bax level in the spermatocytes and therefore may stimulate apoptosis in these germ cells. In contrast, ghrelin administration signifi- cantly suppressed proliferation-associated peptide PCNA in the spermatocytes as well as spermatogonia (P < 0.05). Whereas, caspase-3 activity did not show any marked alteration during the experiment in both groups (P > 0.05). Upstream of Bax substance parallel to down-regulation of PCNA demonstrate that ghrelin may prevent massive accumulation of germ cells during normal spermatogenesis. These observa- tions also indicate that ghrelin may be considered as a modulator of spermatogenesis in normal adult rats and could be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Onset of spermatogenesis is associated with an apoptosis wave that limits its efficiency during the first cycles in the most mam- mals [1]. The seminiferous epithelium is a highly proliferating tis- sue in which germ cell degeneration is a constant feature [2]. During normal spermatogenesis, more than half of the germ cells undergo apoptosis [3]. Expression of ghrelin has been demonstrated in mature Leydig cells of rat and human. In addition, expression of the functional ghrelin receptor, GHS-R1a, has been shown in both Sertoli and Leydig cells. On the other hand, expression of GHS-R1a in the seminiferous tubules strongly suggests that the seminiferous epi- thelium might be a target for ghrelin action and directly regulates seminiferous tubules function [4]. Thus, it is expectable that ghre- lin may control the key gonadal functions, proliferation and apop- tosis, in the rat testis. Recently, numerous studies have documented the direct action of ghrelin in the modulation of apoptosis in different cell types. For example, it has been reported that ghrelin inhibits apoptosis in several cells such as cardiomyocytes [5], pancreatic b cells [6] and pituitary lactotrophs [7], but promotes it in chick ovarian gran- ulosa cells [8], endothelial cells [9], aldostroma and adenocarsino- ma derived cells [10]. However, the role of ghrelin in the testicular germ cells apoptosis and proliferation has not been yet studied. Therefore, the present work attempted to explore the possible involvement of ghrelin treatment on the apoptosis and prolifera- tion indices in the rat testicular tissue. 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.014 ⇑ Corresponding author. Fax: +98 661 6200109. E-mail address: arashkheradmand@yahoo.com (A. Kheradmand). Biochemical and Biophysical Research Communications 419 (2012) 299–304 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc