Localization of Immunoreactive Androgen in Testicular Tissue G. A. BUBENIK,+ G. M. BROWN,+ AND L. J. GROTA++ +
Neuroendocrinology Research Section, Clarke Institute of Psychiatry, Toronto, Canada, M5T 1R8, and ++University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 the interstitial tissue but a significant amount was also present in the seminiferous tubules in the layer adjacent to the tubular wall. Very small amounts of androgen were localized in the tubular wall. This restricted localization in the tubules might indicate either de novo synthesis or active uptake of steroids intermediate in the biosynthesis of testosterone in the seminiferous tubules. (Endocrinology 96: 63, 1975)
ABSTRACT. Immunohistochemical localization of androgen in the testes of both rat and squirrel monkey has been performed using an antiserum specific to testosterone and certain related androgens. The bound antibodies were visualized by coupling with antigammaglobulin labeled either with fluorescein isothiocyanate or with a horseradish peroxidase-diaminobenzidine technique. The highest concentration of androgen was found in
I
T was formerly thought that the interstitial tissue was the only site of androgen synthesis in the mammalian testis (1). However, based on recent evidence that tissue homogenates of seminiferous tubules were also capable of androgen production (2-4), Bell (5) suggested that there were at least two distinct sources of testicular steroid: a) the interstitium, which produced androgen for the systemic circulation and for maintenance of the accessory sex organs and b) the seminiferous tubules, which produced locally acting hormones controlling germ cell maturation. The ability of testosterone to maintain spermatogenesis in the hypophysectomized animal had been demonstrated in numerous experiments but the site of action of testosterone in the individual phases of sperm development is still controversial (6). Localization of androgens in testicular tissue has been performed in the majority of studies by biochemical investigation of tissue homogenates. The present study was undertaken in order to localize more exactly the presence of testosterone and related androgens in histological slices using a highly specific antiserum. Immunological methods have been widely used for precise localization of calcitonin (7), FSH and LH (8), ACTH (9) as well as androgens
(10-12) in cellular structures. In our investigation we have used antibodies labelled either by peroxidase or by fluorescein. The fluorescein method was faster and less complicated (antiserum conjugated with fluorescein is commercially available) but the fluorescence was not permanent and required the use of dark field UV microscopy. On the other hand, the enzyme labelled antibody technique allowed more precise and stable localization and is suitable for both light and electron microscopy. Both methods were used to determine where androgen is located in the tubule. This localization should in turn throw light on two related issues: 1) what is the site of androgen action in the tubules and 2) what is the origin of the tubular androgen? A preliminary abstract of the investigation has been published (13). Materials and Methods The testis and the epididymis of 3 male rats and 3 male squirrel monkeys in different stages of spermatogenic activity were frozen in the cryostat immediately after sacrifice. 10 /JL sections were fixed briefly in cold acetone, washed in 0.2M phosphate buffered saline (PBS, pH 7.4) and incubated 1 hr at room temperature with undiluted bovine serum albumin (BSA) absorbed antitestosterone rabbit antiserum. The antisera used were produced in rabbits by a single intrasplenic injection followed by biweekly intramuscular injections of V2 mg of
Received May 9, 1974.
63
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BUBENIK, BROWN AND GROTA
TABLE 1. Cross reactivity in testosterone radioimmunoassay using rabbit antibody to testosterone-3-BSA Equivalent of 100 pg testosterone
Percent activity
Testosterone
100 pg
100%
Dihydrotestosterone (5a-androstan-17/3ol-3-one)
100 pg
100%
4 Androsten-3i3-17/3-diol
700 pg
14%
5 Androsten-3j3-17/3-diol
2ng
4%
5 Androstan-3a-17/3-diol
5 ng
2%
20 ng
0.5%
Progesterone
>20 ng >20ng
Neuroendocrinology Research Section, Clarke Institute of Psychiatry, Toronto, Canada, M5T 1R8, and ++University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 the interstitial tissue but a significant amount was also present in the seminiferous tubules in the layer adjacent to the tubular wall. Very small amounts of androgen were localized in the tubular wall. This restricted localization in the tubules might indicate either de novo synthesis or active uptake of steroids intermediate in the biosynthesis of testosterone in the seminiferous tubules. (Endocrinology 96: 63, 1975)
ABSTRACT. Immunohistochemical localization of androgen in the testes of both rat and squirrel monkey has been performed using an antiserum specific to testosterone and certain related androgens. The bound antibodies were visualized by coupling with antigammaglobulin labeled either with fluorescein isothiocyanate or with a horseradish peroxidase-diaminobenzidine technique. The highest concentration of androgen was found in
I
T was formerly thought that the interstitial tissue was the only site of androgen synthesis in the mammalian testis (1). However, based on recent evidence that tissue homogenates of seminiferous tubules were also capable of androgen production (2-4), Bell (5) suggested that there were at least two distinct sources of testicular steroid: a) the interstitium, which produced androgen for the systemic circulation and for maintenance of the accessory sex organs and b) the seminiferous tubules, which produced locally acting hormones controlling germ cell maturation. The ability of testosterone to maintain spermatogenesis in the hypophysectomized animal had been demonstrated in numerous experiments but the site of action of testosterone in the individual phases of sperm development is still controversial (6). Localization of androgens in testicular tissue has been performed in the majority of studies by biochemical investigation of tissue homogenates. The present study was undertaken in order to localize more exactly the presence of testosterone and related androgens in histological slices using a highly specific antiserum. Immunological methods have been widely used for precise localization of calcitonin (7), FSH and LH (8), ACTH (9) as well as androgens
(10-12) in cellular structures. In our investigation we have used antibodies labelled either by peroxidase or by fluorescein. The fluorescein method was faster and less complicated (antiserum conjugated with fluorescein is commercially available) but the fluorescence was not permanent and required the use of dark field UV microscopy. On the other hand, the enzyme labelled antibody technique allowed more precise and stable localization and is suitable for both light and electron microscopy. Both methods were used to determine where androgen is located in the tubule. This localization should in turn throw light on two related issues: 1) what is the site of androgen action in the tubules and 2) what is the origin of the tubular androgen? A preliminary abstract of the investigation has been published (13). Materials and Methods The testis and the epididymis of 3 male rats and 3 male squirrel monkeys in different stages of spermatogenic activity were frozen in the cryostat immediately after sacrifice. 10 /JL sections were fixed briefly in cold acetone, washed in 0.2M phosphate buffered saline (PBS, pH 7.4) and incubated 1 hr at room temperature with undiluted bovine serum albumin (BSA) absorbed antitestosterone rabbit antiserum. The antisera used were produced in rabbits by a single intrasplenic injection followed by biweekly intramuscular injections of V2 mg of
Received May 9, 1974.
63
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 03:30 For personal use only. No other uses without permission. . All rights reserved.
64
BUBENIK, BROWN AND GROTA
TABLE 1. Cross reactivity in testosterone radioimmunoassay using rabbit antibody to testosterone-3-BSA Equivalent of 100 pg testosterone
Percent activity
Testosterone
100 pg
100%
Dihydrotestosterone (5a-androstan-17/3ol-3-one)
100 pg
100%
4 Androsten-3i3-17/3-diol
700 pg
14%
5 Androsten-3j3-17/3-diol
2ng
4%
5 Androstan-3a-17/3-diol
5 ng
2%
20 ng
0.5%
Progesterone
>20 ng >20ng
