0013-7227/91/1281-0383$02.00/0 Endocrinology Copyright© 1991 by The Endocrine Society
Vol. 128, No. 1 Printed in U.S.A.
Localization of the Androgen Receptor in the Developing Rat Gubernaculum* D. A. HUSMANNf AND M. J. M C P H A U L J Department of Surgery, Division of Urology, and the Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas, Texas 75235-9031
development. However, during the morphological alterations of the gubernaculum into a muscular structure, the level of androgen receptor detected in these cells declines, with minimal immunoreactivity present 2 weeks postpartum. Whether the loss of staining of the mesenchyme is related to decreases in androgen receptor expression, death of the mesenchymal cells, or differentiation of these cells into myoblasts remains to be determined. {Endocrinology 128: 383-387, 1991)
ABSTRACT. The distribution of androgen receptor expression within the developing rat gubernaculum was examined at different times during fetal and neonatal development using polyclonal antibodies directed at the amino-terminus (amino acids 1-21) and the carboxy-terminus (amino acids 898-917) of the androgen receptor. These studies reveal a high level expression of androgen receptor in the undifferentiated cells that comprise the mesenchymal core of the gubernaculum in early
T
completely disappears by the third postpartum day. Histological studies of the rat gubernaculum reveal minimal changes in its appearance from postnatal day 3 to the time of testicular descent on postnatal day 21 (2). Although the available evidence has suggested that the gubernaculum is an androgen-sensitive structure and that alterations in its morphology are androgen mediated (1, 5), it was not until recently that ligand binding studies revealed the presence of the androgen receptor in tissue homogenates of the gubernaculum (2, 6, 7). To date, however, the precise anatomical localization of the androgen receptor within this structure has not been possible due to the lack of specific antibodies that recognize the receptor protein. To examine the pattern of androgen receptor expression in the rat gubernaculum we have used polyclonal antibodies directed at the amino-terminus (amino acids 1-21) and the carboxy-terminus (amino acids 898-917) of the rat androgen receptor (5). Our developmental studies demonstrate that high levels of androgen receptor are expressed in the embryonic gubernaculum and that the pattern of expression changes drastically during the immediate postpartum period.
HE GUBERNACULUM testes connects the caudal portion of the testes, epididymis, and vas deferens to the scrotum and is a structure common to all animals that undergo testicular descent (1, 2). Despite its well known anatomical structure, whether the gubernaculum performs an essential role in testicular descent is unknown. The available evidence, however, supports a functional role for the gubernaculum, since its surgical removal or the incision of its distal attachments to the scrotum causes failure of the testicle to descend (3). Histologically, the gubernaculum can be readily defined by the 17th day of gestation in the developing rat, at which time it is comprised of undifferentiated mesenchymal cells rich in mucopolysaccharides (1, 2, 4). As it matures, the gubernaculum alters its morphological appearance, developing a thin muscular coat at 20 days gestation. This outer layer of muscle is comprised of both smooth and skeletal components that rapidly proliferate, resulting in a well defined external muscular layer of the gubernaculum by first postnatal day (1, 2). Concurrent with the growth of the muscular layer, the mesenchymal core of the gubernaculum decreases in volume and almost Received July 20,1990. Address all correspondence and requests for reprints to: Douglas A. Husmann, M.D., Division of Urology, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9031. * This work was supported by grants from the NIH (DK-03892), the Perot Family Foundation, the Culpepper Foundation, the March of Dimes (Basil O'Connor Award Grant 5-694), the Life and Health Insurance Medical Research Fund, and the Welch University of Texas Foundation (Grant 1-1990). t Scholar of the American Urological Association. X Culpepper Medical Scholar.
Materials and Methods Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) with timed pregnancies were maintained under standard conditions. Day 1 of pregnancy was the day on which sperm was detected in the vagina. Gubernacular tissues were obtained from animals on days 18 and 20 of gestation and on days 1, 3, 5, 7,14, and 21 postnatally. Birth occurs on gestational day 21. 383
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR LOCALIZATION IN GUBERNACULUM
384
Endo • 1991 Vol 128* No 1
FlG. 1. Immunohistological pattern of staining in the A, caudal epididymis and gubernaculum,
gestational
day
20,
amino-terminal antisera (note the thin layer of muscular investment on the right; magnification, X60); B, gubernaculum, postnatal day 1 (mesenchymal core in the center), control slide with amino-terminal antisera with 10-fold excess peptide (magnification, X60); C, gubernaculum, postnatal day 1, amino-terminal antisera (magnification, x60); and D, gubernaculum, postnatal day 1, amino-terminal antisera (magnification, X150). a, Muscular coat; b, mesenchymal core; c, caudal epididymis.
Polyclonal antibodies directed at the amino-terminus were derived from rabbit R402, and the anticarboxyl-terminal antibodies were purified from serum from rabbit R489. These sera have been documented to be specific for the androgen receptor protein by Western blot analysis and in immunohistological assays (8). Animals were killed at the ages noted above, and the penis, urogenital sinus, and testicle along with the intact gubernaculum were removed, embedded in Tissue-Tek OCT compound (Miles Scientific, Nagerville, IL), and frozen in a dry ice acetone slurry. Specimens were maintained at —137 C until processed for histology. Immunohistochemical stains were performed on tissues specimens cut at 10 /xm thickness, fixed
with 10% neutral buffered formaldehyde for 10 min, and rinsed with 50 mM phosphate buffer (pH 7.2) for 10 min three times. After the washes, localization of androgen receptor was determined with the use of polyclonal antibodies directed at either the amino-terminal or carboxy-terminal of the androgen receptor diluted 1:1000 with 3% rat sera. In all experiments control slides with preimmune sera and immune sera preblocked by a 10-fold excess of the peptide antigen were run in parallel with specimens stained with primary antibody. Slides from each age group along with positive control slides using the animal's urogenital sinus and penis were run simultaneously in each experiment. Primary antibody incubations were performed
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR LOCALIZATION IN GUBERNACULUM
385
FIG. 2. Comparison of immunohistological staining patterns in the third postnatal day gubernaculum between the carboxy-terminal antisera (A and B; magnification, X60 and X150, respectively) and amino-terminal antisera (C and D; magnification, X60 and X150, respectively), a, Muscular coat; b, mesenchymal core.
overnight at 4 C, and slides were rinsed with phosphate buffer for 10 min three times and developed using the peroxidasestreptavidin biotin method with diaminobenzedrine tetrachloride as a chromogen (9). No counterstains were applied.
Results The gubernaculum at 18 and 20 days gestation is composed largely of an abundant ground substance and numerous fibroblast-like cells. These cells stain intensely with immune antiserum directed at both amino- and carboxy-terminal segments of the androgen receptor pro-
tein in the gubernaculum of the 18 (data not shown) and 20 day gestation rat (see Fig. 1A). This immunoreactivity is predominantly nuclear in location and is not visualized when antiamino- or anticarboxy-terminal antisera are preblocked by peptide (Fig. IB). As the animal matures the gubernaculum becomes more cellular, and a thin layer of muscle cells appears (Fig. 1A); these cells proliferate at the periphery (Fig. 1, B-D). While the androgen receptor protein is originally detected in the outer muscular layer at early time points (see Fig. 1, C and D, postnatal day 1), the majority of
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
386
ANDROGEN RECEPTOR LOCALIZATION IN GUBERNACULUM
Endo• 1991 Vol 128 • No 1
FIG. 3. Immunohistological staining pattern with the amino-terminal antisera in the postnatal day 14 rat (magnification, X60). a, Muscular coat; b, mesenchymal core.
this immunoreactivity is lost as the muscle layer matures (see Fig. 2), leaving only a few isolated immunoreactive cells within the muscular coat after postnatal day 14 (see Fig. 3). During the normal course of development in the gubernaculum, the mesenchymal core becomes fragmented, and numerous lacunae appear and coalesce to form a large central lumenal area. As the mesenchymal core regresses, the level of androgen receptor detected in the remaining cells declines. This decrease in androgen receptor expression is synchronous with the development of the outer muscular coat and dissolution of the mesenchymal core, and reaches a nadir by the fourteenth postnatal day. Immunohistological stains of the gubernaculum on postnatal day 14 and in specimens obtained from older animals show minimal but persistent immunoreactivity within isolated cells of the mesenchymal core and muscular coat.
Discussion The role of androgen in testicular descent has been a subject of controversy since Engle's induction of testicular descent in the monkey by administration of pregnancy urine and pituitary extracts in 1932 (10). At present the most compelling evidence that androgens mediate the descent of the testes is derived from clinical studies that reveal an increased incidence of cryptorchidism in children with abnormal gonadotropic and/or abnormal androgen secretion and function (11, 12) and from laboratory investigations that reveal the ability of dihydrotestosterone or hCG to overcome the estradiol inhibition of testicular descent induced in the rat or rabbit (5, 13).
Further substantiation that androgens play at least a synergistic role in the descent of the testes is found by their ability to facilitate intraabdominal pressure in the descent of a testicular silicon prosthesis in the rat (14). Despite documentation by these studies that androgens have a role in testicular descent, the exact target for their activity remains in question. For decades the gubernaculum had been theorized to be androgen sensitive (4); however, it was not until recently that ligand binding studies confirmed the presence of the androgen receptor in the gubernaculum (2, 6, 7). Our present study confirms that the cells within the gubernaculum express high levels of androgen receptor early in gubernacular development. However, during the early postnatal period, the levels of androgen receptor rapidly decline, so that minimal receptor immunoreactivity is present within the gubernaculum at the time of testicular descent in the rat (postnatal day 21). These changes in androgen receptor expression found immunohistologically in the gubernaculum are consistent with previous ligand binding assays that demonstrated a loss of androgen-binding activity in the gubernaculum between gestational day 18 and postnatal day 3 in the rat (2). The concordance of these two methods reinforces an important conclusion of these studies, namely that the level of androgen receptor expression reaches a nadir on postnatal day 14, far in advance of actual testicular descent. These findings suggest that the androgen-sensitive gubernaculum may play an important permissive role in testicular descent, while the final descent of the testicle is probably a result of other factors, i.e. intraabdominal pressure (3, 14). The exact role that androgens may play in the histological development of the gubernaculum and descent of the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR LOCALIZATION IN GUBERNACULUM testicle, therefore, remains unknown. Although we have attempted to alter the histological development of the gubernaculum and subsequent testicular descent by 5areductase inhibitors and/or flutamide given from birth to postnatal day 20, we have been unable to either inhibit testicular descent or alter the postnatal morphological changes that occur in the gubernaculum (2) (Husmann, D. A., and M. J. McPhaul, unpublished observations). Attempts to use these inhibitors during antenatal development have been complicated by their ability to prevent parturition, and further studies are presently being performed to clarify what role androgen inhibition may have on antenatal gubernacular development and subsequent testicular descent. The rapid decline in androgen receptor expression seen within the developing gubernaculum is in contrast to the developmental pattern of androgen receptor expression in the rat penis and rat prostate (Husman, D. A., and M. J. McPhaul, unpublished observations). In these latter structures the androgen receptor expression persists in the mesenchyme throughout the neonatal period. Further studies will be required to determine whether this loss of androgen receptor from the mesenchyme of the gubernaculum is caused by androgen-mediated alterations in androgen receptor expression, death of the mesenchymal cells that express the receptor, or differentiation of these cells into the muscular outer layers.
References 1. Elder JS, Isaacs JT, Walsh PC 1982 Androgenic sensitivity of the gubernaculum testis: evidence for hormonal/mechanical interac-
387
tions in testicular descent. J Urol 127:170-176 2. George FW 1989 Developmental pattern of 5a-reductase activity in the rat gubernaculum. Endocrinology 124:727-732 3. Frey HL, Rajfer J 1984 Role of the gubernaculum and intraabdominal pressure in the process of testicular descent. J Urol 131:574579 4. Backhouse KM, Butler H 1960 The gubernaculum testes of the pig. J Anat (Lond) 94:107-120 5. Rajfer J 1982 Endocrinologic study of testicular descent in the rabbit. J Surg Res 33:158-163 6. George FW, Peterson KG 1988 Partial characterization of the androgen receptor of the newborn rat gubernaculum. Biol Reprod 39:536-539 7. Oprins AC, Fentene van Clissinger JM, Blankenster MA 1988 Testicular descent: androgen receptors in cultured porcine gubernaculum cells. J Steroid Biochem 31:387-391 8. Husmann DA, Wilson CM, McPhaul MJ, Tilley WD, Wilson JD 1990 Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein in the nuclei of target cells in the rat and human prostate. Endocrinology 126:2359-2368 9. Hsu S, Raine C, Fanger H 1989 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580 10. Engle ET 1931 Experimentally-induced descent of the testes in the Macacos monkey by hormones from the anterior pituitary and pregnancy urine. The role of gonadokinetic hormones in pregnancy blood in the hormonal descent of the testes in man. Endocrinology 16:513-520 11. Bardin CW, Ross GT, Rifkind AB, Cargille CM, Lipsett MB 1969 Studies of the pituitary-Leydig cell axis in young men with hypogonadotropic hypogonadism and hyposmia: comparison with normal men, prepubertal boys and hypopituitary patients. J Clin Invest 48:2046-2056 12. Santen RJ, Paulsen CA 1973 Hypogonadotropic eunuchoidism. II. Gonadal responsiveness to exogenous gonadotropins. J Clin Endocrinol Metab 36:55 13. Rajfer J, Walsh PC 1977 Hormonal regulation of testicular descent, experimental and clinical observations. J Urol 118:985-990 14. Frey HL, Peng S, Rajfer J 1983 Synergy of abdominal pressure and androgens in testicular descent. Biol Reprod 29:1233-1239
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
Vol. 128, No. 1 Printed in U.S.A.
Localization of the Androgen Receptor in the Developing Rat Gubernaculum* D. A. HUSMANNf AND M. J. M C P H A U L J Department of Surgery, Division of Urology, and the Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas, Texas 75235-9031
development. However, during the morphological alterations of the gubernaculum into a muscular structure, the level of androgen receptor detected in these cells declines, with minimal immunoreactivity present 2 weeks postpartum. Whether the loss of staining of the mesenchyme is related to decreases in androgen receptor expression, death of the mesenchymal cells, or differentiation of these cells into myoblasts remains to be determined. {Endocrinology 128: 383-387, 1991)
ABSTRACT. The distribution of androgen receptor expression within the developing rat gubernaculum was examined at different times during fetal and neonatal development using polyclonal antibodies directed at the amino-terminus (amino acids 1-21) and the carboxy-terminus (amino acids 898-917) of the androgen receptor. These studies reveal a high level expression of androgen receptor in the undifferentiated cells that comprise the mesenchymal core of the gubernaculum in early
T
completely disappears by the third postpartum day. Histological studies of the rat gubernaculum reveal minimal changes in its appearance from postnatal day 3 to the time of testicular descent on postnatal day 21 (2). Although the available evidence has suggested that the gubernaculum is an androgen-sensitive structure and that alterations in its morphology are androgen mediated (1, 5), it was not until recently that ligand binding studies revealed the presence of the androgen receptor in tissue homogenates of the gubernaculum (2, 6, 7). To date, however, the precise anatomical localization of the androgen receptor within this structure has not been possible due to the lack of specific antibodies that recognize the receptor protein. To examine the pattern of androgen receptor expression in the rat gubernaculum we have used polyclonal antibodies directed at the amino-terminus (amino acids 1-21) and the carboxy-terminus (amino acids 898-917) of the rat androgen receptor (5). Our developmental studies demonstrate that high levels of androgen receptor are expressed in the embryonic gubernaculum and that the pattern of expression changes drastically during the immediate postpartum period.
HE GUBERNACULUM testes connects the caudal portion of the testes, epididymis, and vas deferens to the scrotum and is a structure common to all animals that undergo testicular descent (1, 2). Despite its well known anatomical structure, whether the gubernaculum performs an essential role in testicular descent is unknown. The available evidence, however, supports a functional role for the gubernaculum, since its surgical removal or the incision of its distal attachments to the scrotum causes failure of the testicle to descend (3). Histologically, the gubernaculum can be readily defined by the 17th day of gestation in the developing rat, at which time it is comprised of undifferentiated mesenchymal cells rich in mucopolysaccharides (1, 2, 4). As it matures, the gubernaculum alters its morphological appearance, developing a thin muscular coat at 20 days gestation. This outer layer of muscle is comprised of both smooth and skeletal components that rapidly proliferate, resulting in a well defined external muscular layer of the gubernaculum by first postnatal day (1, 2). Concurrent with the growth of the muscular layer, the mesenchymal core of the gubernaculum decreases in volume and almost Received July 20,1990. Address all correspondence and requests for reprints to: Douglas A. Husmann, M.D., Division of Urology, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9031. * This work was supported by grants from the NIH (DK-03892), the Perot Family Foundation, the Culpepper Foundation, the March of Dimes (Basil O'Connor Award Grant 5-694), the Life and Health Insurance Medical Research Fund, and the Welch University of Texas Foundation (Grant 1-1990). t Scholar of the American Urological Association. X Culpepper Medical Scholar.
Materials and Methods Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) with timed pregnancies were maintained under standard conditions. Day 1 of pregnancy was the day on which sperm was detected in the vagina. Gubernacular tissues were obtained from animals on days 18 and 20 of gestation and on days 1, 3, 5, 7,14, and 21 postnatally. Birth occurs on gestational day 21. 383
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR LOCALIZATION IN GUBERNACULUM
384
Endo • 1991 Vol 128* No 1
FlG. 1. Immunohistological pattern of staining in the A, caudal epididymis and gubernaculum,
gestational
day
20,
amino-terminal antisera (note the thin layer of muscular investment on the right; magnification, X60); B, gubernaculum, postnatal day 1 (mesenchymal core in the center), control slide with amino-terminal antisera with 10-fold excess peptide (magnification, X60); C, gubernaculum, postnatal day 1, amino-terminal antisera (magnification, x60); and D, gubernaculum, postnatal day 1, amino-terminal antisera (magnification, X150). a, Muscular coat; b, mesenchymal core; c, caudal epididymis.
Polyclonal antibodies directed at the amino-terminus were derived from rabbit R402, and the anticarboxyl-terminal antibodies were purified from serum from rabbit R489. These sera have been documented to be specific for the androgen receptor protein by Western blot analysis and in immunohistological assays (8). Animals were killed at the ages noted above, and the penis, urogenital sinus, and testicle along with the intact gubernaculum were removed, embedded in Tissue-Tek OCT compound (Miles Scientific, Nagerville, IL), and frozen in a dry ice acetone slurry. Specimens were maintained at —137 C until processed for histology. Immunohistochemical stains were performed on tissues specimens cut at 10 /xm thickness, fixed
with 10% neutral buffered formaldehyde for 10 min, and rinsed with 50 mM phosphate buffer (pH 7.2) for 10 min three times. After the washes, localization of androgen receptor was determined with the use of polyclonal antibodies directed at either the amino-terminal or carboxy-terminal of the androgen receptor diluted 1:1000 with 3% rat sera. In all experiments control slides with preimmune sera and immune sera preblocked by a 10-fold excess of the peptide antigen were run in parallel with specimens stained with primary antibody. Slides from each age group along with positive control slides using the animal's urogenital sinus and penis were run simultaneously in each experiment. Primary antibody incubations were performed
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR LOCALIZATION IN GUBERNACULUM
385
FIG. 2. Comparison of immunohistological staining patterns in the third postnatal day gubernaculum between the carboxy-terminal antisera (A and B; magnification, X60 and X150, respectively) and amino-terminal antisera (C and D; magnification, X60 and X150, respectively), a, Muscular coat; b, mesenchymal core.
overnight at 4 C, and slides were rinsed with phosphate buffer for 10 min three times and developed using the peroxidasestreptavidin biotin method with diaminobenzedrine tetrachloride as a chromogen (9). No counterstains were applied.
Results The gubernaculum at 18 and 20 days gestation is composed largely of an abundant ground substance and numerous fibroblast-like cells. These cells stain intensely with immune antiserum directed at both amino- and carboxy-terminal segments of the androgen receptor pro-
tein in the gubernaculum of the 18 (data not shown) and 20 day gestation rat (see Fig. 1A). This immunoreactivity is predominantly nuclear in location and is not visualized when antiamino- or anticarboxy-terminal antisera are preblocked by peptide (Fig. IB). As the animal matures the gubernaculum becomes more cellular, and a thin layer of muscle cells appears (Fig. 1A); these cells proliferate at the periphery (Fig. 1, B-D). While the androgen receptor protein is originally detected in the outer muscular layer at early time points (see Fig. 1, C and D, postnatal day 1), the majority of
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
386
ANDROGEN RECEPTOR LOCALIZATION IN GUBERNACULUM
Endo• 1991 Vol 128 • No 1
FIG. 3. Immunohistological staining pattern with the amino-terminal antisera in the postnatal day 14 rat (magnification, X60). a, Muscular coat; b, mesenchymal core.
this immunoreactivity is lost as the muscle layer matures (see Fig. 2), leaving only a few isolated immunoreactive cells within the muscular coat after postnatal day 14 (see Fig. 3). During the normal course of development in the gubernaculum, the mesenchymal core becomes fragmented, and numerous lacunae appear and coalesce to form a large central lumenal area. As the mesenchymal core regresses, the level of androgen receptor detected in the remaining cells declines. This decrease in androgen receptor expression is synchronous with the development of the outer muscular coat and dissolution of the mesenchymal core, and reaches a nadir by the fourteenth postnatal day. Immunohistological stains of the gubernaculum on postnatal day 14 and in specimens obtained from older animals show minimal but persistent immunoreactivity within isolated cells of the mesenchymal core and muscular coat.
Discussion The role of androgen in testicular descent has been a subject of controversy since Engle's induction of testicular descent in the monkey by administration of pregnancy urine and pituitary extracts in 1932 (10). At present the most compelling evidence that androgens mediate the descent of the testes is derived from clinical studies that reveal an increased incidence of cryptorchidism in children with abnormal gonadotropic and/or abnormal androgen secretion and function (11, 12) and from laboratory investigations that reveal the ability of dihydrotestosterone or hCG to overcome the estradiol inhibition of testicular descent induced in the rat or rabbit (5, 13).
Further substantiation that androgens play at least a synergistic role in the descent of the testes is found by their ability to facilitate intraabdominal pressure in the descent of a testicular silicon prosthesis in the rat (14). Despite documentation by these studies that androgens have a role in testicular descent, the exact target for their activity remains in question. For decades the gubernaculum had been theorized to be androgen sensitive (4); however, it was not until recently that ligand binding studies confirmed the presence of the androgen receptor in the gubernaculum (2, 6, 7). Our present study confirms that the cells within the gubernaculum express high levels of androgen receptor early in gubernacular development. However, during the early postnatal period, the levels of androgen receptor rapidly decline, so that minimal receptor immunoreactivity is present within the gubernaculum at the time of testicular descent in the rat (postnatal day 21). These changes in androgen receptor expression found immunohistologically in the gubernaculum are consistent with previous ligand binding assays that demonstrated a loss of androgen-binding activity in the gubernaculum between gestational day 18 and postnatal day 3 in the rat (2). The concordance of these two methods reinforces an important conclusion of these studies, namely that the level of androgen receptor expression reaches a nadir on postnatal day 14, far in advance of actual testicular descent. These findings suggest that the androgen-sensitive gubernaculum may play an important permissive role in testicular descent, while the final descent of the testicle is probably a result of other factors, i.e. intraabdominal pressure (3, 14). The exact role that androgens may play in the histological development of the gubernaculum and descent of the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR LOCALIZATION IN GUBERNACULUM testicle, therefore, remains unknown. Although we have attempted to alter the histological development of the gubernaculum and subsequent testicular descent by 5areductase inhibitors and/or flutamide given from birth to postnatal day 20, we have been unable to either inhibit testicular descent or alter the postnatal morphological changes that occur in the gubernaculum (2) (Husmann, D. A., and M. J. McPhaul, unpublished observations). Attempts to use these inhibitors during antenatal development have been complicated by their ability to prevent parturition, and further studies are presently being performed to clarify what role androgen inhibition may have on antenatal gubernacular development and subsequent testicular descent. The rapid decline in androgen receptor expression seen within the developing gubernaculum is in contrast to the developmental pattern of androgen receptor expression in the rat penis and rat prostate (Husman, D. A., and M. J. McPhaul, unpublished observations). In these latter structures the androgen receptor expression persists in the mesenchyme throughout the neonatal period. Further studies will be required to determine whether this loss of androgen receptor from the mesenchyme of the gubernaculum is caused by androgen-mediated alterations in androgen receptor expression, death of the mesenchymal cells that express the receptor, or differentiation of these cells into the muscular outer layers.
References 1. Elder JS, Isaacs JT, Walsh PC 1982 Androgenic sensitivity of the gubernaculum testis: evidence for hormonal/mechanical interac-
387
tions in testicular descent. J Urol 127:170-176 2. George FW 1989 Developmental pattern of 5a-reductase activity in the rat gubernaculum. Endocrinology 124:727-732 3. Frey HL, Rajfer J 1984 Role of the gubernaculum and intraabdominal pressure in the process of testicular descent. J Urol 131:574579 4. Backhouse KM, Butler H 1960 The gubernaculum testes of the pig. J Anat (Lond) 94:107-120 5. Rajfer J 1982 Endocrinologic study of testicular descent in the rabbit. J Surg Res 33:158-163 6. George FW, Peterson KG 1988 Partial characterization of the androgen receptor of the newborn rat gubernaculum. Biol Reprod 39:536-539 7. Oprins AC, Fentene van Clissinger JM, Blankenster MA 1988 Testicular descent: androgen receptors in cultured porcine gubernaculum cells. J Steroid Biochem 31:387-391 8. Husmann DA, Wilson CM, McPhaul MJ, Tilley WD, Wilson JD 1990 Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein in the nuclei of target cells in the rat and human prostate. Endocrinology 126:2359-2368 9. Hsu S, Raine C, Fanger H 1989 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580 10. Engle ET 1931 Experimentally-induced descent of the testes in the Macacos monkey by hormones from the anterior pituitary and pregnancy urine. The role of gonadokinetic hormones in pregnancy blood in the hormonal descent of the testes in man. Endocrinology 16:513-520 11. Bardin CW, Ross GT, Rifkind AB, Cargille CM, Lipsett MB 1969 Studies of the pituitary-Leydig cell axis in young men with hypogonadotropic hypogonadism and hyposmia: comparison with normal men, prepubertal boys and hypopituitary patients. J Clin Invest 48:2046-2056 12. Santen RJ, Paulsen CA 1973 Hypogonadotropic eunuchoidism. II. Gonadal responsiveness to exogenous gonadotropins. J Clin Endocrinol Metab 36:55 13. Rajfer J, Walsh PC 1977 Hormonal regulation of testicular descent, experimental and clinical observations. J Urol 118:985-990 14. Frey HL, Peng S, Rajfer J 1983 Synergy of abdominal pressure and androgens in testicular descent. Biol Reprod 29:1233-1239
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 06:56 For personal use only. No other uses without permission. . All rights reserved.
