Immunohistochemical Localization of Gonadotropin and Gonadal Steroid Receptors in Human Pineal Glands

0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society Vol. 82, No. 3 Printed in U.S.A. COMMENTS Immunohistochemical Localization of Gonadotropin and Gonadal Steroid Receptors in Human Pineal Glands* RAFAEL LUBOSHITZKY, MURALEE DHARAN, DALIA GOLDMAN, YEHUDA HISS, PAULA HERER, AND PERETZ LAVIE Departments of Endocrinology (R.L.) and Cytopathology (M.D., D.G.), Central Emek Hospital, Afula, The L. Grinberg Institute of Forensic Medicine, Tel-Aviv (Y.H.), and the B. Rappaport Faculty of Medicine (P.H., P.L.), Technion, Haifa, Israel ABSTRACT Recently, we demonstrated that melatonin secretion was increased in male patients with GnRH deficiency and decreased to normal levels during testosterone treatment. These data suggested that gonadal steroids modulate melatonin secretion, probably by activating specific receptors in the pineal gland. We used immunohistochemistry to localize gonadotropin (LH and FSH) and gonadal steroid (androgens and estrogens) receptors in human pineal glands. Tissues were obtained at autopsy from 25 males, aged 19 – 87 yr, and five prepubertal children, aged 0.2–10 yr. Positive staining for all four types of receptors (LH, FSH, androgen, and estrogen) in the pineal parenchymal cells, pinealocytes, was evident in all 30 glands examined. Double staining revealed that nuclear receptors (androgen or estrogen) coexisted with cytoplasmatic receptors (LH or FSH) in the same cells. The results demonstrate the presence of gonadotropin and gonadal steroid receptors in human pinealocytes from infancy to old age. (J Clin Endocrinol Metab 82: 977–981, 1997) I otropins modulate melatonin secretion via specific pineal receptors. Here, we used immunohistochemistry to localize gonadotropins and gonadal steroid receptors in human male pineal glands. N SEASONALLY breeding mammals, the antigonadal effects of melatonin are well established (1, 2). There is increasing evidence that melatonin plays an important role in human reproduction (1). The demonstration of nocturnal hypersecretion of melatonin in women with hypogonadotropic amenorrhea and in hypogonadal men (3–5) and its normalization during testosterone administration (6), on the one hand, and the demonstration of melatonin receptors in human granulosa cell membranes (7) and prostate (8), on the other hand, indicate that in addition to a possible role of melatonin in steroidogenesis, gonadal steroids may modulate melatonin secretion (6). As testosterone is aromatized to estradiol (E2) in the brain (9), it can be hypothesized that in male subjects, both testosterone and E2 exert their modulatory effects on melatonin secretion through specific receptors in the pineal gland. Animal studies had demonstrated androgen and estrogen receptors (AR and ER, respectively) in rat pinealocytes (10). A direct effect of testosterone and E2 on pineal melatonin release was demonstrated in male rats (11). The presence of LHRH in the monkey pineal gland and the ability of LHRH to influence the activity of protein synthesis in rat pinealocytes (12) suggest that gonadotropins may also participate in the modulation of melatonin secretion. We hypothesized that both gonadal steroids and gonad- Materials and Methods Tissues and fixation We have studied human pineal glands obtained at autopsy in 25 males (all had normal pubertal secondary sex signs), aged 19 – 87 yr (mean 6 sd, 44.2 6 19.8 yr), and in 5 male children, aged 2 months to 10 yr. All pineal glands were obtained over a 4-month period (May– August) from subjects who had died in Tel-Aviv, Israel (32°N). Causes of death were road accidents, gunshots, stabbing, and suicide. The autopsy was performed after obtaining permission from the family. Tissues were frozen in liquid nitrogen immediately upon removal. The postmortem interval was 18.0 6 4.1 h. Frozen specimens were stored at 270 C until sectioning. Prostate and testicular specimens were obtained at autopsy, and prostate specimens obtained at surgery were handled in the same fashion as the pineal glands and served as control tissues for the immunohistochemical staining. Immunohistochemistry Light microscope immunohistochemistry was performed for the demonstration of the following receptors in the pinealocytes: LH (LH-R), FSH (FSH-R), AR, and ER. To confirm the specificity of receptor localization in the pineal gland, the following procedures were performed. 1) As positive controls for the four types of receptors examined, we used prostate and testicular specimens, tissues known to be positively stained for these receptors (13). 2) Negative controls were used in two ways: 1) omission of the primary (receptor) antibody during the immunostaining and using phosphate-buffered saline (PBS) instead, and 2) substitution of the primary antibody with nonspecific IgG (normal mouse serum, Diagnostic Products Corp., Los Angeles, CA). Immunohistochemical reagents were obtained from Zymed Laboratories (San Francisco, CA). Staining was carried out using an avidin-biotin complex method (Histostatin-SP KI, Zymed Laboratories) (14). Briefly, the frozen tissues were Received August 16, 1996. Revision received November 18, 1996. Accepted November 25, 1996. Address all correspondence and requests for reprints to: Dr. R. Luboshitzky, Endocrine Institute, Central Emek Hospital, Afula 18101, Israel. * This work was supported by grants from the B. Rappaport Research Fund, The Maurice and Arlene King Fund (U.S.), and the Israel Ministry of Health (no. 181– 645 and 181–792). 977 978 JCE & M • 1997 Vol 82 • No 3 COMMENTS cut in 5-mm sections in a cryostat and mounted onto polylysine-coated slides. Slides were treated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and washed immediately. Serum blocking solution (goat serum) was added to each section and incubated for 10 min. Sections were incubated with the primary antibodies overnight at 4 C, a procedure that we have found in preliminary studies to be necessary for optimal results. Slides were subsequently washed three times in PBS (pH 7.4) and then incubated with secondary antibodies (goat antimouse IgG-IgA-IgM-biotin) for 10 min at 22 C. After washing in PBS, enzyme conjugate (streptavidin peroxidase) was applied to each slide for 10 min. After repeated washing in PBS, substratechromogen mixture (3-amino-9-ethylcarbazole) was applied to each slide and incubated for 10 min. Sections were counterstained with Mayer’s hematoxylin. We used mouse monoclonal antibodies for AR (clone 39.4.1, Euro-Diagnostica, The Netherlands) at a concentration of 5 mg/mL, mouse monoclonal antibodies for ER (clone 1D5, Zymed Laboratories) at a concentration of 5 mg/mL, mouse monoclonal antibodies for LH-R (clone ZSL11, Zymed Laboratories) at a concentration of 5 mg/mL, and mouse monoclonal antibodies for FSH-R (clone ZMFS1, Zymed Laboratories) at a concentration of 5 mg/mL. Positive staining was evident by a red-brown deposit, w (...truncated)