Cellular and intracellular distribution of growth hormone in the adult chicken testis C.G. Martínez-Moreno a , L. Palma b , M. Carranza a , S. Harvey c , C. Arámburo a , M. Luna a,⇑ a Departamento de Neurobiología Celular y Molecular, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro 76230, Mexico b Unidad de Microscopía, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro 76230, Mexico c Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G2H7 article info Article history: Received 4 January 2011 Revised 21 March 2011 Accepted 24 March 2011 Available online 31 March 2011 Keywords: Chicken growth hormone (GH) Growth hormone receptor (GHR) Testes Intracellular Spermatogonia Spermatocyte Spermatid abstract Endocrine actions of growth hormone (GH) have been implicated during the development of adult testic- ular function in several mammalian species, and recently intracrine, autocrine, and paracrine effects have been proposed for locally expressed GH. Previous reports have shown the distribution of GH mRNA and the molecular heterogeneity of GH protein in both adult chicken testes and vas deferens. This study pro- vides evidence of the presence and distribution of GH and its receptor (GHR) during all stages of sper- matogenesis in adult chicken testes. This hormone and its receptor are not restricted to the cytoplasm; they are also found in the nuclei of spermatogonia, spermatocytes, and spermatids. The pattern of GH iso- forms was characterized in the different, isolated germ cell subpopulations, and the major molecular var- iant in all subpopulations was 17 kDa GH, as reported in other chicken extra-pituitary tissues. Another molecular variant, the 29 kDa moiety, was found mainly in the enriched spermatocyte population, sug- gesting that it acts at specific developmental stages. The co-localization of GH with the proliferative cell nuclear antigen PCNA (a DNA replication marker present in spermatogonial cells) was demonstrated by immunohistochemistry. These results show for the first time that GH and GHR are present in the nuclei of adult chicken germinal cells, and suggest that GH could participate in proliferation and differentiation during the complex process of spermatogenesis. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Growth hormone (GH) is mainly produced by the pituitary in all vertebrates. This hormone is released into the bloodstream in order to stimulate growth and development and to participate in diverse differentiation processes in various target tissues. GH acts directly by binding to its receptor (GHR) or indirectly by means of insulin growth factor type 1 (IGF-1) [65]. Some studies have shown that GH plays a role in the reproductive function of males and females during growth and differentiation; it also participates in steroido- genesis and gametogenesis [33]. Currently, GH is known to be expressed also in extra-pituitary tissues of the immune [48,49,59,60], nervous [1,18,72], and repro- ductive [28,47] systems. The local expression of this hormone suggests an autocrine, paracrine, and/or intracrine activity [27]. Recent studies have shown that the GH gene is transcribed in the testis of human and chicken [10,28,69]. In chicken testis a variety of factors involved in GH expression and regulation are present, such as the Pit-1 factor [28] and growth hormone-releasing hormone and its receptor [71]. Other non-hypothalamic factors regulating GH release, such as ghrelin and its receptor (GHS-R1a) have been reported in rat testis [68]. Previously, we showed that GH mRNA is present in the testis and vas deferens of adult chickens, mainly localized in spermato- gonia and in primary and secondary spermatocytes, as determined by in situ hybridization [28]. GH immunoreactivity (GH-IR) was identified particularly in areas with primary and secondary sper- matocytes and spermatids, as well as in interstitial areas (Leydig cells) [47]. In conjunction with these results, the local GH concen- tration has been quantified by the ELISA method in the chicken tes- tis at various stages of development (embryo, neonate, juvenile, pubertal, and adult). GH concentrations were not significantly dif- ferent between weeks 4 and 36. However, a major increase relative to the embryonic stages was found at week 36, when the GH con- centration was approximately 25 times higher than on day 1 after birth [47]. At 36 weeks of age, chickens are already sexually ma- ture, and this stage correlates with an increase in testosterone lev- els and maximum sperm count [62]. It has also been shown that the GH present in testis exhibits a pattern of molecular heteroge- neity (variants of 17, 23, 26, 29, 32, 35, 40, 42, 45, and 52 kDa under reducing conditions) and that the 17-kDa GH isoform is the most abundant throughout development [47]. Indeed, GHR expression has been reported in muscle, adipocytes, lymphoid tissue, and in the nervous system during all stages of mammalian maturation [21,31,34,57,61]. GHR 0016-6480/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2011.03.023 ⇑ Corresponding author. Address: Instituto de Neurobiología, Campus Juriquilla, Universidad Nacional Autónoma de Mexico, Querétaro, Qro. 76230, Mexico. Fax: +52 442 238 1005. E-mail address: lunam@servidor.unam.mx (M. Luna). General and Comparative Endocrinology 172 (2011) 344–357 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen