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Immunocytochemical Localization of Androgen Receptor in the Comb, Uropygial Gland, Testis, and Epididymis in the Domestic Chicken
JOBNAME: 101#1 96 PAGE: 1 SESS: 4 OUTPUT: Wed Jun 12 20:18:54 1996 /xypage/worksmart/tsp000/66754g/18pu General and Comparative Endocrinology 101, 76–82 (1996) Article No. 0009
Immunocytochemical Localization of Androgen Receptor in the Comb, Uropygial Gland, Testis, and Epididymis in the Domestic Chicken Bhagyashri A. Shanbhag,1 and Peter J. Sharp Division of Development and Reproduction, Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS Accepted September 26, 1995
Nuclear, but not cytoplasmic androgen receptors (AR), were localized immunocytochemically in the comb, uropygial (preen) gland, testis, and epididymis of juvenile and adult cockerels. Androgen receptor immunoreactivity (AR-ir) was seen in the comb, in the stratum germinativum of the epidermis and in fibromucoid cells in the dermis in juvenile and adult cockerels. AR-ir was observed in the glandular epithelial (sebum-producing) cells lining the peripheral and middle sections of the tubules in the uropygial gland. AR-ir was not detected in innermost part of the tubules which conduct the sebum to the surface of the skin. AR-ir labeling was not observed in the uropygial gland of juveniles. In the testis, AR-ir was seen in the Leydig cells, in adults but not juveniles. The epithelial cells lining the tubules in the epididymis contained AR-ir in both juveniles and adults. © 1996 Academic Press, Inc.
In the chicken, androgens control the growth of the comb (Bolton, 1953; Munson and Sheps, 1956; Boris et al., 1970; Gow et al., 1991) and the epididymis (Lofts and Massa, 1980; Follett, 1984). There is also evidence that the uropygial or preen gland in birds is an androgen-dependent target organ. The gland responds to
testosterone treatment by increasing the production of sebum and by cellular proliferation (pigeon: Maiti and Ghosh, 1972; chicken: Ishida et al., 1974). Castration of the pigeon decreases the activity of the uropygial gland which can be restored by treatment with testosterone (Maiti and Ghosh, 1972; Bhattacharyya and Chowdhury, 1978). Androgen receptors (AR) have been demonstrated in the chicken comb (Dube and Tremblay, 1974) and the uropygial glands of ducks (Daniel, 1981) and quail (Amet et al., 1986a,b). In mammals, AR has been localized immunocytochemically in the human and rat testis (Takeda et al., 1990; DeWinter et al., 1991; Kimura et al., 1993; Vornberger et al., 1994) and in rat epididymal tubules (Takeda et al., 1990). There is limited evidence in birds that androgens may have a role in the regulation of testicular function (Lofts and Murton, 1973; Lofts and Massa, 1980; Follett, 1984). There are no reports on the cellular localization of AR in the chicken comb, or in the avian uropygial gland, testis, or epididymis. This study was undertaken immunocytochemically to examine AR in these tissues.
MATERIALS AND METHODS 1 Present address: Department of Zoology, Karnatak University, Dharwad-580003, India.
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Two-week-old-old juvenile (n = 6) and 28-week-old adult cockerels (n = 3, ISA Brown, ISA Poultry Ser0016-6480/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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vices, Peterborough, England) were killed by cervical dislocation. The comb, testis, epididymis, and uropygial gland were rapidly frozen in isopentene precooled in liquid nitrogen and then embedded in O.C.T. (Merck Ltd., Poole, England). Sections (1.5 mm) were cut on a cryostat maintained at −30°, collected on chrome alum gelatine-coated slides and air dried at 20° for 5 min. They were then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4) containing 15% sucrose for 10 min followed by a wash in PBS. Androgen receptor was localized using a Vectastain Elite avidin–biotin–peroxidase kit (Code: PK6101, Vector Laboratories, Peterborough, England) using nickel-3,39- diaminobenzidine (DAB) as chromogen. The androgen receptor antibody was produced in a rabbit immunized with an N-terminal peptide sequence from the human androgen receptor (Code AR70, NCl-ARp) and was purchased from Novocastra Laboratories Ltd. (Newcastle-upon-Tyne, England). Sections were treated with 0.3% hydrogen peroxide (10 min) to block endogenous peroxidase and then with 5% goat serum (45 min) to inhibit nonspecific background staining. Sections of the testis and epididymis were treated with avidin–biotin blocking complex (Vector Laboratories) to block endogenous biotin. They were then incubated at 4° in a humidity chamber with androgen receptor antibody (1:50) for about 40 hr. Following a wash in PBS, the sections were incubated in biotinylated goat-anti-rabbit IgG for 1 hr at 20° and then with avidin–biotin complex reagent for 30 min. The sections were then treated with nickelDAB for 6–8 min. Between steps, the sections were thoroughly rinsed with PBS. The sections were finally rinsed in distilled water, air dried, rinsed in CNP, and mounted in Euparol. Control sections for each tissue were treated the same way except that the primary antibody was omitted and replaced by nonimmune rabbit IgG (0.5 mg/ ml; Vector Laboratories).
Comb The comb heights were 620 ± 37 mm and 2.3 ± 0.27 mm in the adult and juvenile cockerels, respectively. Androgen receptor immunolabeling (AR-ir) occurred in the nuclei of cells in the basal and intermediate layers of the stratum germinativum of the epidermis in the combs of adult (Fig. 1) and juvenile cockerels (Fig. 2a). It was also seen in the fibromucoid cells in the dermis (Figs. 1 and 2a).
Uropygial Gland AR-ir was observed in the glandular epithelial cells lining the peripheral (Zone 1, Figs. 3, 3a and 9) and intermediary (Zone II, Fig. 9) sections of the tubules in the uropygial gland of the adult cockerel. No AR-ir was detected in epithelial cells lining the central section of the tubules (Zone III, Figs. 4 and 9). No AR-ir was detected in the uropygial glands of juvenile cockerels.
Testis Testicular weights were 20.59 ± 1.96 g and 0.44 ± 0.08 g in adult and juvenile cockerels, respectively. No AR-ir was detected in testis of juveniles. In the testis of adults, intense immunolabeling for AR-ir was seen in Leydig cells; no AR-ir was seen in seminiferous tubules (Figs. 5 and 6).
Epididymis AR-ir was observed in the nuclei of epithelial cells lining the efferent ductules and epididymal duct in the epididymis of juvenile (Figs 7 and 7a) and adult cockerels (Fig. 8).
DISCUSSION RESULTS Immunolabeling for AR occurred in cell nuclei as a black-brown deposits and did not occur in the cytoplasm (Figs. 1, 2a, 3, 3a, 5, 7, and 8). No immunolabeling was observed in the control sections incubated with nonimmune rabbit IgG (Figs. 2, 6, and 7a).
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
The amino acid sequence of the chicken androgen receptor has not been reported but is likely to be highly conserved evolutionarily as illustrated by the canary androgen receptor (Nastiuk and Clayton, 1994). The amino acid sequences of the chicken estrogen and progesterone receptors are known (Maxwell
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FIGS. 1–4. Immunocytochemical localization of androgen receptor in the comb of (1) adult and (2a) juvenile cockerels. No immunolabeling occurred in the adult comb when (2) the primary antibody was omitted from the immunocytochemical procedure. Immunocytochemical localization of androgen receptor at (3) low and (3a) high magnification in the epithelial lining (arrow) of the peripheral zone (Zone 1) in the uropygial gland of the adult cockerel are shown. No immunolabeling occurred in (4) the central zone of the tubules of the uropygial gland. SG, stratum germinativum of the epidermis; FM, fibromucoid cells.
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIGS. 5–8. Immunocytochemical localization of androgen receptor in (5) the testis of an adult cockerel. No immunolabeling was observed in the testis when (6) the primary antibody was omitted from the immunocytochemical procedure. Immunocytochemical localization of androgen receptor in the efferent ductules in the epididymis of (7) juvenile and (8) adult cockerels. No immunolabeling was seen in the efferent ductules in (7a) juvenile birds when the primary antibody was omitted from the immunocytochemical procedure. Arrows in (7) and (8) show immunolabeled nuclei in the epithelial cells lining the efferent ductules. L, Leydig cells.
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FIG. 9. Diagrammatic representation of a tubule from the uropygial gland of the domestic chicken. Dots represent the localization of androgen receptor in the glandular epithelial cells lining its peripheral (Zone 1) and intermediate (Zone II) but not central (Zone III) sections. P, peripheral region; L, lumen of the tubule (Based on Jacob, 1978).
et al., 1987; Conneely et al., 1987; Gronemyer et al., 1987) and do not contain sequences corresponding to those at the N-terminus of the mammalian AR receptor (Chang et al., 1988; Lubahn et al., 1988) used to produce the primary antibody purchased for this study. This was used, as recommended by the commercial suppliers, at the same dilution as used for mammalian tissues. It is therefore likely, as claimed by the suppliers, that the antibody is specific for the immunocytochemical localization of androgen receptors in both mammals and the chicken. In preliminary studies, attempts were made to improve tissue morphology using different fixatives and more prolonged fixation. No method was found to give results as satisfactory as achieved using briefly fixed fresh frozen sections. The present study reports for the first time, the subcellular distribution of AR-ir in the chicken’s comb, and in the uropygial gland, testis, and epididymis in any species of bird. The cockerel’s comb consists of an epidermis with a keratinized outer layer, a superficial dermis containing large blood vessels and a deep dermis with elastic and collagen fibers and fibromucoid cells (Lucas and Shettenheim, 1972). The central core consists of adipose tissue. The basal and intermediate layers of the stratum germinativum of the epidermis are proliferative with keratinization starting in the intermediate layer. The occurrence of androgen receptor in the basal and intermediate layers of the epidermis is consistent with a stimulatory action of androgens on cellular proliferation and possibly the process of kera-
tinization. The fibromucoid cells in the dermis are responsible for formation of fibers and presumably mucoid, thereby controlling its size (Lucas and Shettenheim, 1972). The presence of AR-ir in fibromucoid cells suggests that androgens are required for this function. The uropygial gland in the chicken is a bilobed organ containing a large number of tubules in which sebum is produced by a holocrine mechanism and transported to a central cavity (Jacob, 1978). Each tubule is divided, using histological and histochemical criteria, into peripheral (Zone I), intermediary (Zone II), and central (Zone III) sections (Jacob, 1978). Zone I is characterized by a well-developed stratum germinativum and with several layers of holocrine secretory cells. Zone II contains a reduced layer of glandular epithelial cells while Zone III contains no secretory cells. The function of Zones I and II is to produce sebum (Jacob and Ziswiler, 1982), while Zone III transports sebum to the central cavity of the uropygial gland. The presence of androgen receptor in the glandular epithelial cells lining Zones I and II of the tubules in the uropygial gland support earlier studies suggesting a stimulatory effect of androgen on sebum production (Maiti and Ghosh, 1972; Ishida et al., 1974; Bhattacharyya and Chowdhury, 1978). The observation that AR-ir could not be found in the uropygial gland of juvenile birds may be a reflection of low circulating concentrations of androgens (Culbert et al., 1977) which may not be adequate to induce uropygial androgen receptors sufficiently to be detected immunocytochemically.
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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AR-ir has been localized immunocytochemically in Sertoli cells, peritubular myoid cells, and Leydig cells in rat and human testis (Sar et al., 1990; Takeda et al., 1990; DeWinter et al., 1991). A study on the rat testis (Vornberger et al., 1994) also reported AR-ir in nuclei of stage XI elongated spermatids. Further, AR-ir in Sertoli cells was found to be stage dependent being most evident at stage VII of the cycle of spermatogenesis. In the present study, no AR-ir was detected in the testis of juveniles or in the seminiferous tubules of adults. It may be that sensitivity of the immunocytochemical procedure was insufficient to detect weak AR-ir in the seminiferous tubules or in the juvenile testis. However, the presence of AR-ir in the nuclei of Leydig cells in adult cockerels is consistent with the presence of AR in the same cell type as mammals. This observation suggests that in birds as in mammals, androgen may modify Leydig cell activity in an autocrine fashion (Vornberger et al., 1994). The presence of AR-ir in nuclei of epithelial cells lining the efferent ductules and epididymal tubules in the cockerel epididymis confirms the androgen dependency of these structures suggested in earlier studies (Lofts and Murton, 1973; Lofts and Massa, 1980).
ACKNOWLEDGMENT Dr. B. A. Shanbhag was supported by a Commonwealth Staff Academic Research Fellowship.
REFERENCES Amet, Y., Abalain, J. H., Daniel, J. Y., Di Stefano, S., and Floch, H. (1986a). Testosterone regulation of androgen receptor levels in the uropygial gland of quails (Coturnix coturnix): A further proof for the androgen dependency of the uropygial gland. Gen. Comp. Endocrinol. 62, 210–212. Amet, Y., Abalain, J. H., Di Stefano, S., Daniel, J. Y., Tea, K., Floch, H. H., and Robel, P. (1986b). Androgen regulation of the androgen receptor of the quail uropygial gland: Application of a [3H] mibolerone exchange assay. J. Endocrinol. 109, 299–306. Bhattacharyya, S. P., and Chowdhury, M. (1978). The effect of androgen on the composition of lipid materials of the preen gland of pigeons. Folia Biol. (Krakow) 26, 15–23. Boris, A., Cox, D. C., and Hurley, J. E. (1970). Comparison of the effects of six antiandrogens on chick comb stimulation by testosterone. Proc. Soc. Exp. Biol. Med. 134, 985–987.
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
81 Bolton, W. (1953). The effect of sex hormones on comb growth in immature pullets. J. Endocrinol. 9, 440–445. Chang, C., Kokontis, J., and Liao, S. (1988). Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors. Proc. Natl. Sci. USA 85, 7211–7215. Conneely, O. M., Dobson, A. D. W., Tsai, M.-J., Beattie, W. G., Toft, D. O., Huckaby, C. S., Zarucki, T., Schrader, W. T., and O’Malley, B. W. (1987). Sequence and expression of a functional chicken progesterone receptor. Mol. Endocrinol. 1, 517–525. Culbert, J., Sharp, P. J., and Wells, J. W. (1977). Concentrations of androstenedione, testosterone and LH in the blood before and after the onset of spermatogenesis in the cockerel. J. Reprod. Fert. 51, 153–154. Daniel, J. Y. (1981). Characterization and regulation of androgen receptors in the preen gland of adult male ducks. In “Adv. Phys. Sci: Recent Advances in Avian Endocrinology” (G. Pethes, P. Peczely, and P. Rudas, Eds.), Vol. 33, pp. 153–161, Pergamon Press, Oxford. DeWinter, J. A. R., Trapman, J., Vermey, M., Mulder, E., Zegers, N. D., and Vanderkwast, T. H. (1991). Androgen receptor expression in human tissue—An immunohistochemical study. J. Histochem. Cytochem. 39, 927–936. Dube, J. Y., and Tremblay, R. R. (1974). Androgen binding proteins in cock’s tissue: Properties of ear lobe protein and determination of binding sites in head appendages and other tissues. Endocrinology 95, 1105–1112. Follett, B. K. (1984). Birds. In “Marshall’s Physiology of Reproduction Vol. I, Reproductive cycles of Vertebrates” (G. E. Lamming, Ed.), pp. 283–350. Churchill Livingstone, New York. Gow, C. B., Scaramuzzi, R. J., Carter, N. B., and Sharp, P. J. (1991). Plasma concentrations of luteinizing hormone and body weights during somatic maturation in intact and castrated Australorp cockerels from a line of hens selected for increased ovulation rate. Br. Poult. Sci. 32, 799–808. Gronemyer, H., Turcotte, B., Quirin-Stricker, C., Bocquel, M. T., Meyer, M. E., Krozowski, Z., Jeltsch, J. M., Lerouge, T., Garnier, J. M., and Chambon, P. (1987). The chicken progesterone receptor sequence: Expression and functional analysis. EMBO J. 6, 3985– 3994. Jacob, J. (1978). Uropygial gland secretions and feather waxes. In “Chemical Zoology” (A. H. Brush, Ed.), Vol. X pp. 165–211. Academic Press, New York. Jacob, J., and Ziswiler, V. (1982). The uropygial gland. In “Avian Biology” (D. S. Farner, A. S. King, and K. C. Parkes, Eds.), Vol. VI pp. 199–324. Academic Press, New York. Ishida, K., Terai, Y., Kusuhura, S., and Yamaguchi, M. (1974). Hormone dominance in the chicken uropygial gland. Nippon Chikusan Gakkai Ho 45, 465–466. Kimura, N., Mizokami, A., Oonura, T., Sasano, H., and Nagura, H. (1993). Immunocytochemical localization of androgen receptor with polyclonal antibody in paraffin-embedded human tissues. J. Histochem. Cytochem. 41, 671–678. Lofts, B., and Murton, R. K. (1973). Reproduction in Birds. In “Avian Biology” (D. S. Farner, and J. R. King, Eds.), Vol. III pp. 1–107. Academic Press, New York.
JOBNAME: 101#1 96 PAGE: 7 SESS: 2 OUTPUT: Wed Jun 12 20:18:54 1996 /xypage/worksmart/tsp000/66754g/18pu 82 Lofts, B., and Massa, R. (1980). Male reproduction. In “Avian Endocrinology” (A. Epple, and M. H. Stetson, Eds.), pp. 413–434. Academic Press, New York. Lucas, A. M., and Shettenheim, P. R. (1972). Avian Anatomy: Integument-Part II. Agri. Handbook 362, pp. 331–547. U.S. Dept. Agri. and Michigan State Univ. Lubahn, D. B., Joseph, D. R., Sullivan, P. M., Willard, H. F., French, F. S., and Wilson, E. M. (1988). Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science 240, 327–330. Maiti, B. R., and Ghosh, A. (1972). Probable role of androgen in the regulation of the uropygial gland. Gen. Comp. Endocrinol. 19, 527– 536. Maxwell, B. L., McDonnell, D. P., Conneely, O. M., Schutz, T. Z., Greene, G. L., and O’Malley, B. W. (1987). Structural organization and regulation of the chicken estrogen receptor. Mol. Endocrinol. 1, 25–35.
Shanbhag and Sharp Munson, P. L., and Sheps, M. C. (1958). An improved procedure for the biological assay of androgens by direct application to the comb of baby chicks. Endocrinology 126, 17–26. Nastiuk, K. L., and Clayton, D. F. (1994). Seasonal and tissue specific regulation of canary androgen receptor messenger ribonucleic acid. Endocrinology 134, 640–649. Sar, M., Lubahn, D. B., French, F. S., and Wilson, E. M. (1990). Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 127, 3180–3186. Takeda, H., Chodak, G., Mutchnik, S., Nakamoto, T., and Chang, C. (1990). Immunohistochemical localization of androgen receptors with mono-and polyclonal antibodies to androgen receptor. J. Endocrinol. 126, 17–26. Vornberger, W., Prins, G., Musto, N., and Suarez-Quian, C. A. (1994). Androgen receptor distribution in rat testis-new implications for androgen regulation of spermatogenesis. Endocrinology 134, 2307–2316.
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Immunocytochemical Localization of Androgen Receptor in the Comb, Uropygial Gland, Testis, and Epididymis in the Domestic Chicken Bhagyashri A. Shanbhag,1 and Peter J. Sharp Division of Development and Reproduction, Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS Accepted September 26, 1995
Nuclear, but not cytoplasmic androgen receptors (AR), were localized immunocytochemically in the comb, uropygial (preen) gland, testis, and epididymis of juvenile and adult cockerels. Androgen receptor immunoreactivity (AR-ir) was seen in the comb, in the stratum germinativum of the epidermis and in fibromucoid cells in the dermis in juvenile and adult cockerels. AR-ir was observed in the glandular epithelial (sebum-producing) cells lining the peripheral and middle sections of the tubules in the uropygial gland. AR-ir was not detected in innermost part of the tubules which conduct the sebum to the surface of the skin. AR-ir labeling was not observed in the uropygial gland of juveniles. In the testis, AR-ir was seen in the Leydig cells, in adults but not juveniles. The epithelial cells lining the tubules in the epididymis contained AR-ir in both juveniles and adults. © 1996 Academic Press, Inc.
In the chicken, androgens control the growth of the comb (Bolton, 1953; Munson and Sheps, 1956; Boris et al., 1970; Gow et al., 1991) and the epididymis (Lofts and Massa, 1980; Follett, 1984). There is also evidence that the uropygial or preen gland in birds is an androgen-dependent target organ. The gland responds to
testosterone treatment by increasing the production of sebum and by cellular proliferation (pigeon: Maiti and Ghosh, 1972; chicken: Ishida et al., 1974). Castration of the pigeon decreases the activity of the uropygial gland which can be restored by treatment with testosterone (Maiti and Ghosh, 1972; Bhattacharyya and Chowdhury, 1978). Androgen receptors (AR) have been demonstrated in the chicken comb (Dube and Tremblay, 1974) and the uropygial glands of ducks (Daniel, 1981) and quail (Amet et al., 1986a,b). In mammals, AR has been localized immunocytochemically in the human and rat testis (Takeda et al., 1990; DeWinter et al., 1991; Kimura et al., 1993; Vornberger et al., 1994) and in rat epididymal tubules (Takeda et al., 1990). There is limited evidence in birds that androgens may have a role in the regulation of testicular function (Lofts and Murton, 1973; Lofts and Massa, 1980; Follett, 1984). There are no reports on the cellular localization of AR in the chicken comb, or in the avian uropygial gland, testis, or epididymis. This study was undertaken immunocytochemically to examine AR in these tissues.
MATERIALS AND METHODS 1 Present address: Department of Zoology, Karnatak University, Dharwad-580003, India.
76
Two-week-old-old juvenile (n = 6) and 28-week-old adult cockerels (n = 3, ISA Brown, ISA Poultry Ser0016-6480/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
JOBNAME: 101#1 96 PAGE: 2 SESS: 2 OUTPUT: Wed Jun 12 20:18:54 1996 /xypage/worksmart/tsp000/66754g/18pu 77
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vices, Peterborough, England) were killed by cervical dislocation. The comb, testis, epididymis, and uropygial gland were rapidly frozen in isopentene precooled in liquid nitrogen and then embedded in O.C.T. (Merck Ltd., Poole, England). Sections (1.5 mm) were cut on a cryostat maintained at −30°, collected on chrome alum gelatine-coated slides and air dried at 20° for 5 min. They were then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4) containing 15% sucrose for 10 min followed by a wash in PBS. Androgen receptor was localized using a Vectastain Elite avidin–biotin–peroxidase kit (Code: PK6101, Vector Laboratories, Peterborough, England) using nickel-3,39- diaminobenzidine (DAB) as chromogen. The androgen receptor antibody was produced in a rabbit immunized with an N-terminal peptide sequence from the human androgen receptor (Code AR70, NCl-ARp) and was purchased from Novocastra Laboratories Ltd. (Newcastle-upon-Tyne, England). Sections were treated with 0.3% hydrogen peroxide (10 min) to block endogenous peroxidase and then with 5% goat serum (45 min) to inhibit nonspecific background staining. Sections of the testis and epididymis were treated with avidin–biotin blocking complex (Vector Laboratories) to block endogenous biotin. They were then incubated at 4° in a humidity chamber with androgen receptor antibody (1:50) for about 40 hr. Following a wash in PBS, the sections were incubated in biotinylated goat-anti-rabbit IgG for 1 hr at 20° and then with avidin–biotin complex reagent for 30 min. The sections were then treated with nickelDAB for 6–8 min. Between steps, the sections were thoroughly rinsed with PBS. The sections were finally rinsed in distilled water, air dried, rinsed in CNP, and mounted in Euparol. Control sections for each tissue were treated the same way except that the primary antibody was omitted and replaced by nonimmune rabbit IgG (0.5 mg/ ml; Vector Laboratories).
Comb The comb heights were 620 ± 37 mm and 2.3 ± 0.27 mm in the adult and juvenile cockerels, respectively. Androgen receptor immunolabeling (AR-ir) occurred in the nuclei of cells in the basal and intermediate layers of the stratum germinativum of the epidermis in the combs of adult (Fig. 1) and juvenile cockerels (Fig. 2a). It was also seen in the fibromucoid cells in the dermis (Figs. 1 and 2a).
Uropygial Gland AR-ir was observed in the glandular epithelial cells lining the peripheral (Zone 1, Figs. 3, 3a and 9) and intermediary (Zone II, Fig. 9) sections of the tubules in the uropygial gland of the adult cockerel. No AR-ir was detected in epithelial cells lining the central section of the tubules (Zone III, Figs. 4 and 9). No AR-ir was detected in the uropygial glands of juvenile cockerels.
Testis Testicular weights were 20.59 ± 1.96 g and 0.44 ± 0.08 g in adult and juvenile cockerels, respectively. No AR-ir was detected in testis of juveniles. In the testis of adults, intense immunolabeling for AR-ir was seen in Leydig cells; no AR-ir was seen in seminiferous tubules (Figs. 5 and 6).
Epididymis AR-ir was observed in the nuclei of epithelial cells lining the efferent ductules and epididymal duct in the epididymis of juvenile (Figs 7 and 7a) and adult cockerels (Fig. 8).
DISCUSSION RESULTS Immunolabeling for AR occurred in cell nuclei as a black-brown deposits and did not occur in the cytoplasm (Figs. 1, 2a, 3, 3a, 5, 7, and 8). No immunolabeling was observed in the control sections incubated with nonimmune rabbit IgG (Figs. 2, 6, and 7a).
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
The amino acid sequence of the chicken androgen receptor has not been reported but is likely to be highly conserved evolutionarily as illustrated by the canary androgen receptor (Nastiuk and Clayton, 1994). The amino acid sequences of the chicken estrogen and progesterone receptors are known (Maxwell
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FIGS. 1–4. Immunocytochemical localization of androgen receptor in the comb of (1) adult and (2a) juvenile cockerels. No immunolabeling occurred in the adult comb when (2) the primary antibody was omitted from the immunocytochemical procedure. Immunocytochemical localization of androgen receptor at (3) low and (3a) high magnification in the epithelial lining (arrow) of the peripheral zone (Zone 1) in the uropygial gland of the adult cockerel are shown. No immunolabeling occurred in (4) the central zone of the tubules of the uropygial gland. SG, stratum germinativum of the epidermis; FM, fibromucoid cells.
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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79
FIGS. 5–8. Immunocytochemical localization of androgen receptor in (5) the testis of an adult cockerel. No immunolabeling was observed in the testis when (6) the primary antibody was omitted from the immunocytochemical procedure. Immunocytochemical localization of androgen receptor in the efferent ductules in the epididymis of (7) juvenile and (8) adult cockerels. No immunolabeling was seen in the efferent ductules in (7a) juvenile birds when the primary antibody was omitted from the immunocytochemical procedure. Arrows in (7) and (8) show immunolabeled nuclei in the epithelial cells lining the efferent ductules. L, Leydig cells.
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Shanbhag and Sharp
FIG. 9. Diagrammatic representation of a tubule from the uropygial gland of the domestic chicken. Dots represent the localization of androgen receptor in the glandular epithelial cells lining its peripheral (Zone 1) and intermediate (Zone II) but not central (Zone III) sections. P, peripheral region; L, lumen of the tubule (Based on Jacob, 1978).
et al., 1987; Conneely et al., 1987; Gronemyer et al., 1987) and do not contain sequences corresponding to those at the N-terminus of the mammalian AR receptor (Chang et al., 1988; Lubahn et al., 1988) used to produce the primary antibody purchased for this study. This was used, as recommended by the commercial suppliers, at the same dilution as used for mammalian tissues. It is therefore likely, as claimed by the suppliers, that the antibody is specific for the immunocytochemical localization of androgen receptors in both mammals and the chicken. In preliminary studies, attempts were made to improve tissue morphology using different fixatives and more prolonged fixation. No method was found to give results as satisfactory as achieved using briefly fixed fresh frozen sections. The present study reports for the first time, the subcellular distribution of AR-ir in the chicken’s comb, and in the uropygial gland, testis, and epididymis in any species of bird. The cockerel’s comb consists of an epidermis with a keratinized outer layer, a superficial dermis containing large blood vessels and a deep dermis with elastic and collagen fibers and fibromucoid cells (Lucas and Shettenheim, 1972). The central core consists of adipose tissue. The basal and intermediate layers of the stratum germinativum of the epidermis are proliferative with keratinization starting in the intermediate layer. The occurrence of androgen receptor in the basal and intermediate layers of the epidermis is consistent with a stimulatory action of androgens on cellular proliferation and possibly the process of kera-
tinization. The fibromucoid cells in the dermis are responsible for formation of fibers and presumably mucoid, thereby controlling its size (Lucas and Shettenheim, 1972). The presence of AR-ir in fibromucoid cells suggests that androgens are required for this function. The uropygial gland in the chicken is a bilobed organ containing a large number of tubules in which sebum is produced by a holocrine mechanism and transported to a central cavity (Jacob, 1978). Each tubule is divided, using histological and histochemical criteria, into peripheral (Zone I), intermediary (Zone II), and central (Zone III) sections (Jacob, 1978). Zone I is characterized by a well-developed stratum germinativum and with several layers of holocrine secretory cells. Zone II contains a reduced layer of glandular epithelial cells while Zone III contains no secretory cells. The function of Zones I and II is to produce sebum (Jacob and Ziswiler, 1982), while Zone III transports sebum to the central cavity of the uropygial gland. The presence of androgen receptor in the glandular epithelial cells lining Zones I and II of the tubules in the uropygial gland support earlier studies suggesting a stimulatory effect of androgen on sebum production (Maiti and Ghosh, 1972; Ishida et al., 1974; Bhattacharyya and Chowdhury, 1978). The observation that AR-ir could not be found in the uropygial gland of juvenile birds may be a reflection of low circulating concentrations of androgens (Culbert et al., 1977) which may not be adequate to induce uropygial androgen receptors sufficiently to be detected immunocytochemically.
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JOBNAME: 101#1 96 PAGE: 6 SESS: 2 OUTPUT: Wed Jun 12 20:18:54 1996 /xypage/worksmart/tsp000/66754g/18pu Androgen Receptors in Chicken
AR-ir has been localized immunocytochemically in Sertoli cells, peritubular myoid cells, and Leydig cells in rat and human testis (Sar et al., 1990; Takeda et al., 1990; DeWinter et al., 1991). A study on the rat testis (Vornberger et al., 1994) also reported AR-ir in nuclei of stage XI elongated spermatids. Further, AR-ir in Sertoli cells was found to be stage dependent being most evident at stage VII of the cycle of spermatogenesis. In the present study, no AR-ir was detected in the testis of juveniles or in the seminiferous tubules of adults. It may be that sensitivity of the immunocytochemical procedure was insufficient to detect weak AR-ir in the seminiferous tubules or in the juvenile testis. However, the presence of AR-ir in the nuclei of Leydig cells in adult cockerels is consistent with the presence of AR in the same cell type as mammals. This observation suggests that in birds as in mammals, androgen may modify Leydig cell activity in an autocrine fashion (Vornberger et al., 1994). The presence of AR-ir in nuclei of epithelial cells lining the efferent ductules and epididymal tubules in the cockerel epididymis confirms the androgen dependency of these structures suggested in earlier studies (Lofts and Murton, 1973; Lofts and Massa, 1980).
ACKNOWLEDGMENT Dr. B. A. Shanbhag was supported by a Commonwealth Staff Academic Research Fellowship.
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Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.