Prolactin Receptor Expression in Rat Spermatogenic Cells

BIOLOGY OF REPRODUCTION 52, 1284-1290 (1995) Prolactin Receptor Expression in Rat Spermatogenic Cells 2 EIICHI HONDO,1 ' 2 MASAMICHI KUROHMARU, 2 SENKITI SAKAI,3 KENJI OGAWA, 2 and YOSHIHIRO HAYASHI Department of Veterinary Anatomy2 and Department of Animal Breeding,3 Faculty of Agriculture The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ABSTRACT INTRODUCTION Prolactin (PRL), approximately 23 000 M,, is an anterior pituitary hormone present in many species from fishes to mammals, and it expresses various functions [1]. PRL regulates metamorphosis in amphibians [2]. In mammals, PRL stimulates gonadal tissues [3, 4] and mammary glands [5-9], and exerts immunomodulatory effects on the mammalian body [10, 11]. In vertebrates, PRL acts as an osmoregulatory hormone [12-16]. The effects of PRL on mammalian testes have been studied with various experimental models such as PRL-deficient hereditary dwarf mice [17-19], hypophysectomized rats [1922], and/or dopamine agonist-treated animals [23]. Bartke [24] measured quantitative changes in testes weight of hypophysectomized mice after PRL treatment and pointed out that PRL reduces testicular shrinkage in short-term hypophysectomized mice, while it increases testicular weight in long-term hypophysectomized mice. Although it is accepted that PRL binds to Leydig cells and increases the binding activity of hCG to Leydig cells [25-28], the exact role of PRL in the regulation of testicular function is still controversial [29]. It also remains unclear whether PRL acts directly on spermatogenic cells, because PRL binding activity has not been detected in membrane preparations of the seminiferous epithelium [28]. Prolactin receptor (PRL-R) mRNA was detected in rabbit testis [30], and recently, the localization of PRL-R mRNA in testes was reported by in situ hybridization using a radiolabeled probe [31]. According to that report, PRL-R mRNA was expressed not only in Leydig cells but also in the first layer of the seminiferous epithelium. In the present study, to determine the exact localiAccepted January 25, 1995. Received September 8, 1994. 'Correspondence. FAX: 81-3-5800-6896. zation of PRL-R mRNA in adult rat testes, we performed in situ hybridization using a different probe (digoxigenin-labeled cRNA). In recent years, two forms of the PRL-R gene were cloned from the ovary and liver of rats [32]. These genes have the same sequence in the extracellular and transmembranous domains but are not consistent in the intracellular domain. The short form of the PRL-R has a 57amino acid sequence in the cytoplasmic region, and the long form has 357 amino acids in the same region. The cRNA probe used in this study encodes a part of the extracellular domain of PRL-R and hybridizes with both shortand long-form PRL-R mRNA. Furthermore, to clarify the expression of PRL-R protein in testes, PRL binding to living testicular cells was investigated with use of colloidal gold. MATERIALS AND METHODS Animals and Tissue Preparationfor In Situ Hybridization Nine adult male rats (Wistar Imamichi strain, 12 wk old, 340-360 g BW) were purchased from the Imamichi Institute for Animal Reproduction (Ibaraki, Japan). Under pentobarbital anesthesia, five rats were perfused with Bouin's solution through the left ventricle after a brief wash with 0.9% saline. Specimens were then dehydrated in a graded series of ethanols, infiltrated in xylene, and embedded in paraffin wax. Sections of 4 pLm in thickness were cut and mounted onto slides coated with 1% gelatin in 0.1% chromium(III) potassium sulfate. Complementary RNA Probe Preparation Total RNA was extracted from female rat livers. Each female was intraperitoneally administered 173-estradiol (20 Wpg/kg BW) at a 12-h interval to increase the expression of PRL-R mRNA, and the liver was excised after the fifth injec1284 To identify target cells of prolactin (PRL) in the male gonad, the expression of prolactin receptor (PRL-R) mRNA in adult rat testes was investigated by in situ hybridization using a digoxigenin-labeled cRNA probe. We also investigated PRL binding to testicular cells in vitro. Signals for PRL-R mRNA were detected not only in interstitial cells but also in spermatogenic cells. Although the reaction was positive in all phases of spermatogonia and spermatocytes, it disappeared in early round spermatids. No signals were detected in elongated spermatids or spermatozoa. The signal intensity varied among each phase of spermatogenic cells. PRL-R mRNA was expressed in all stages of the cycle of the seminiferous epithelium. PRL-R were detected on the surface of Leydig cells, Sertoli cells, all phases of spermatogonia and spermatocytes, elongated spermatids, and spermatozoa. Ovine PRL did not bind to round spermatids. In Leydig cells, pachytene spermatocytes, and spermatozoa, PRL-R were observed in relatively large numbers. There were fewer receptors in other phases of spermatogenic cells. These results indicate that PRL-R mRNA expression is almost consistent with PRL binding sites except for elongated spermatids and spermatozoa, and suggest that PRL may have direct effects on spermatogenic cells. PROLACTIN RECEPTOR EXPRESSION IN RAT SPERMATOGENIC CELLS In Situ Hybridization Prior to hybridization, deparaffinized sections were treated with 4% paraformaldehyde/0.1 phosphate buffer, 0.1 M glycine/0.2 M Tris-HCI (pH 7.6), and 10 pg/ml proteinase K/ PBS. After treatment, sections were hybridized for 18 h at 50°C in the following solutions: 50% formamide, 5-strength SSC (standard saline citrate; 0.75 M NaCl, 0.075 M sodium citrate), single-strength Denhardt's solution (the following components diluted with 500 ml H20: 0.1 g Ficoll [Type 400; Pharmacia, Uppsala, Sweden], 0.1 g polyvinylpyrrolidone, and 0.1 g BSA), 100 g/ml heparin, 10 mM dithiothreitol (DTT), 100 jig/ml salmon sperm DNA, 100 jig/ml yeast tRNA, 10% dextran sulfate, and 5 ig/ml cRNA probe/ diethylpyrocarbonate-treated water). These sections were rinsed in 4-strength SSC at 42 0C and treated with RNase A (20 pg/ml RNase A/RNase buffer [10 mM Tris-HCl, pH 7.5, plus 0.5 M NaCl]) at 370C for 30 min. Sections were then rinsed in double-strength SSC at 600C, followed by 0.1strength SSC at 65°C. Specimens were then processed with immunohistochemical procedures. Each was incubated with horseradish peroxidase-conjugated anti-digoxigenin antibody for 1 h at room temperature and then developed with 0.025% diaminobenzidine with 0.01% hydrogen peroxide. Biotinylation of Ovine PRL (oPRL) Ovine PRL (NIADDK-oPrL-18) was kindly supplied by the NIADDK (Bethesda, MD). Biotinylation of oPRL was performed according to the procedure of Michel and Parsons [35]. N-Hydroxysuccinimidobiotin (B-NHS; Sigma, St. Louis, MO) dissolved in dimethylformamide at a concentration of 20 mg/ml was added to a solution of 20 mg oPRL in 0.5 M NaHCO 3 , pH 8.0, containing a trace quantity of 25I-oPRL (< 1% of oPRL available for reaction). The reaction was undertaken by incubating on ice f (...truncated)