Immunolocalization of estrogen receptor alpha, estrogen receptor beta and androgen receptor in the pre-, peri- and post-pubertal stallion testis

In various species, androgens and estrogens regulate the function of testicular Leydig. Sertoli, peritubular myoid, and germ cells by binding to their respective receptors and eliciting a cellular response. Androgen receptor (AR) is expressed in Sertoli cells, peritubular myoid cells, Leydig cells and perivascular smooth muscle cells in the testis depending on the species, but its presence in germ cells remains controversial. Two different estrogen receptors have been identified, estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta), and their localization and function in testicular cells varies depending on the species, developmental stage of the cell and type of receptor. The localization of AR in an immature and mature stallion has been reported but estrogen receptors have only been reported for the mature stallion. In the present study, the localizations of AR and ER alpha/ER beta were investigated in pre-pubertal, pen-pubertal and post-pubertal stallions. Testes were collected by routine castration from 21 horses, of light horse breeds (3 months-27 years). Animals were divided into the following age groups: pre-pubertal (3-11 months; n = 7), pen-pubertal (12-23 months; n = 7) and post-pubertal (2-27 years; n = 7). Testicular tissue samples were fixed and embedded, and the presence of AR, ER alpha and ER beta was investigated by immunohistochemistry (IHC) using procedures previously validated for the horse. Primary antibodies used were rabbit anti-human AR, mouse anti-human ER beta and rabbit anti-mouse ER alpha. Sections of each region were incubated with normal rabbit serum (NRS; AR and ER alpha) or mouse IgG (ER beta) instead of primary antibody to generate negative controls. Androgen receptors were localized in Leydig, Sertoli and peritubular myoid cells of all ages. Estrogen receptor alpha was localized in Leydig and germ cells of all ages but only in pre- and pen-pubertal Sertoli cells and post-pubertal peritubular myoid cells. Estrogen receptor beta was localized in Leydig and Sertoli cells of all ages but in only pre-pubertal germ cells and absent in peritubular myoid cells of all ages. Taken together, the data suggest that estrogen regulates steroidogenesis by acting through ER alpha and ER beta in the Leydig cells and promotes gametogenesis by acting through ER beta in the Sertoli cells and ER alpha in the germ cells. In contrast androgen receptors are not found in germ cells throughout development and thus are likely to support spermatogenesis by way of a paracrine/autocrine pathway via its receptors in Leydig, Sertoli and peritubular myoid cells. (C) 2011 Elsevier B.V. All rights reserved.