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Experimental Studies |
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
Address all correspondence and requests for reprints to: Dr. R. Luboshitzky, Endocrine Institute, Central Emek Hospital, Afula 18101, Israel.
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Abstract |
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Introduction |
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We hypothesized that both gonadal steroids and gonadotropins modulate melatonin secretion via specific pineal receptors. Here, we used immunohistochemistry to localize gonadotropins and gonadal steroid receptors in human male pineal glands.
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Materials and Methods |
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We have studied human pineal glands obtained at autopsy in 25 males (all had normal pubertal secondary sex signs), aged 19–87 yr (mean ± SD, 44.2 ± 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 ± 4.1 h. Frozen specimens were stored at -70 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 cut in 5-µm 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, substrate-chromogen 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 µg/mL, mouse monoclonal antibodies for ER (clone 1D5, Zymed Laboratories) at a concentration of 5 µg/mL, mouse monoclonal antibodies for LH-R (clone ZSL11, Zymed Laboratories) at a concentration of 5 µg/mL, and mouse monoclonal antibodies for FSH-R (clone ZMFS1, Zymed Laboratories) at a concentration of 5 µg/mL. Positive staining was evident by a red-brown deposit, which demonstrated the location of the receptor in the nucleus or cytoplasm. Identification of the tissues was carried out on sections stained only with hematoxylin at a magnification of x200. Immunohistochemistry on formalin-fixed tissues was performed as follows. The formalin-fixed specimens were removed, dehydrated, and embedded in paraffin, and 4-µm tissue sections were cut and mounted on microscope slides. Before staining, the slides were deparaffinized in xylene and rehydrated, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide and washed in distilled water and then in 0.01 mol/L sodium citrate buffer. The slides were then heated in a microwave at high power (780 watts) for 10 min. The microwave fixation was needed for better nuclear receptors staining (15). Staining was then performed as described for frozen tissue specimens. Prostate and testicular specimens were fixed in formalin and served as positive and negative controls. Double staining was performed using the following combinations: LH-R and AR, LH-R and ER, FSH-R and AR, FSH-R and ER, on testicular, prostate, and pineal gland specimens in frozen tissues and formalin-fixed specimens. As negative controls we used testicular and prostate specimens as described for single staining. As positive controls for the four combinations we used testicular and prostate specimens. Immunohistochemical reagents were obtained from Zymed Laboratories. Staining was carried out using the streptavidin-biotin amplification method (Histostatin-DS kit) (14). Briefly, slides were treated with peroxidase quenching solution for 10 min and washed with PBS. 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 (for AR, we used clone 39.4.1 at a concentration of 5 µg/mL; for ER, we used clone 1D5 at a concentration of 5 µg/mL). Slides were washed in PBS and incubated with secondary antibodies (goat antimouse). Streptavidin-alkaline phosphatase was added to each section, incubated for 10 min, and rinsed in PBS. Substrate-chromogen mixture (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium chloride; producing a black-purple stain) was applied to each slide and incubated for 10 min, and then a double staining enhancer was applied to each section and incubated for 30 min. Slides were washed with distilled water and then rinsed with PBS. Serum blocking solution (goat serum) was added and incubated for 10 min. The slides were then incubated with the primary antibodies overnight at 4 C (for LH-R, we used clone ZSL11 at a concentration of 5 µg/mL; for FSH-R, we used clone ZMFS1 at a concentration of 5 µg/mL). After incubation with biotinylated goat antimouse secondary antibodies for 10 min at 22 C, streptavidin-peroxidase was applied to each section and incubated for 10 min. Substrate-chromogen mixture (3-amino-9-ethylcarbazole; producing red-brown stain) was added to each section and incubated for 10 min. Sections were lightly counterstained with Mayer’s hematoxylin.
Semiquantification of single immunostaining
The percentage of positively stained cells was defined as the positivity index (PI) and was evaluated in a given microscopic area of 0.08 mm2 at a magnification of x200 (an average of 500 cells/field). Four fields were counted in each gland, and the mean value of the four readings was defined as the PI. As this study was carried out on pineal glands obtained at two different circadian phases from subjects who had died during the daytime (0800–2000 h) when pineal production of melatonin is low and from subjects who died at night (2000–0800 h) when pineal production of melatonin is at its peak (1), we compared the daytime with the nighttime PIs of these receptors (defined as the day-night differences).
Statistical analysis
PIs were compared using the two-independent sample Mann-Whitney test to determine differences between day and night values in adult subjects. Spearman’s correlations between the PI and age of the subjects were computed.
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Results |
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). LH-R was located
only in the cytoplasm, whereas FSH-R, ER, and AR were demonstrated in
the nucleus and cytoplasm. The PI and the location of the receptors are
given in Table 1. The PIs for LH-R and FSH-R (1.37
± 0.88% and 1.26 ± 0.78%, respectively) were greater than
those for cells positively stained for ER or AR (0.41 ± 0.46%
and 0.19 ± 0.20%, respectively).
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By double immunohistochemical staining, colocalization of nuclear AR or ER and cytoplasmatic LH-R or FSH-R was observed in the same cells. The formalin-fixed tissues showed the same LH-R, FSH-R, ER, and AR distribution pattern and the same PI as the frozen sections. However, the formalin-fixed specimens showed a diminished immunostaining intensity compared with corresponding frozen sections.
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Discussion |
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The immunolocalization of receptor proteins does not directly imply
that the entire receptor is present and functions. However, a direct
effect of E2 and testosterone on melatonin release (11, 17)
and the effect of LHRH on pinealocyte protein synthesis (12) have been
shown in male rats. Moreover, the demonstrations of AR and ER in rat
pinealocytes (18) and decreased 5-
dihydrotestosterone receptor
density in male rats during growth and development (10) support our
findings.
In our study, AR and ER immunostaining in all prepubertal children was confined to the cytoplasm, with no nuclear staining, which is in contrast to the widely accepted concept of the nuclear location of these receptors (19). In view of the positive nuclear staining of these receptors in adult subjects, it is likely that those differences are due to low circulating levels of androgens and estrogens in children and, hence, to the low availability of these hormones to their receptors in the pinealocytes. Recently, it was shown that AR-immunoreactive cells in prepubertal male ferrets were sensitive to circulating levels of androgens and demonstrated an increase in the number of AR during puberty (20). In addition, it was shown that circulating androgens regulate their own receptors and AR immunoreactivity became cytoplasmic 2–3 weeks after castration (19, 21). It is likely that as a result of low circulating levels of androgens and estrogens in our prepubertal children, AR and ER were ligand free and were associated in a complex with the abundant heat shock protein, hsp90 in the cytoplasm (22), thus accounting for the high percentage of positively stained cells in two of the prepubertal children.
The tendency for a nocturnal elevation of FSH-R in our study resembles the nocturnal rise of blood melatonin levels (1) and pineal melatonin content (23) observed in humans and the stimulating effects of testosterone and E2 on rat melatonin release (11).
Taken together, these data may suggest that in humans, the pituitary-gonadal hormones, via their receptors in the pineal gland, had evolved an adjustment to the rhythmicity of melatonin secretion. It is tempting to speculate that the function of this feedback mechanism is to inhibit the hypersecretion of melatonin.
In conclusion, the present findings support the idea that in humans, gonadal steroid and gonadotropin receptors are present in the pinealocytes from infancy to old age. The current observations do not permit drawing decisive conclusions as to the role of gonadotropins and gonadal steroids in the modulation of melatonin secretion from the pineal gland.
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Acknowledgments |
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Footnotes |
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Received August 16, 1996.
Revised November 18, 1996.
Accepted November 25, 1996.
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This article has been cited by other articles:
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  P. Lavie Melatonin: Role in Gating Nocturnal Rise in Sleep Propensity J Biol Rhythms, December 1, 1997; 12(6): 657 - 665. [Abstract] [PDF]
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 R. Luboshitzky Immunohistochemical Localization of Gonadotropin and Gonadal Steroid Receptors in Human Pineal Glands--Author's Responsee J. Clin. Endocrinol. Metab., August 1, 1997; 82(8): 2757 - 2757. [Full Text]
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