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Pubertal changes in androgen composition of rat rete testis and cauda epididymal fluids
0022.4731/g1/0101-0109102.0010
Journal of Srrroid Biochemistry Vol. 14. pp. 109 to I IO Pergamon Press Ltd 1981. Printed in Great Britain
SHORT COMMUNICATION PUBERTAL CHANGES IN ANDROGEN COMPOSITION OF RAT RETE TESTIS AND CAUDA EPIDIDYMAL FLUIDS R. G. FOLDESY* and J. H. LEATHEM Bureau of Biological Research, Rutgers-The State University, New Brunswick, NJ 08903, U.S.A. (Received 15 April
1980)
SUMMARY The concentrations of testosterone, dihydrotestosterone (DHT), 5a-androstane-3a,l7/l-diol, and 5a-androstane-38,17/l-diol were measured by radio-immunoassay in rete testis fluid (RTF) and luminal fluid of the cauda epididy-midis (CLF) in pubertal rats. Fluids were collected prior to and after spermatozoa enter the epididymis (45 and 55 days of age). Testosterone concentrations rose 2.5-fold in RTF and DHT concentrations increased 67% in CLF. These represented the only changes observed and it is proposed that the increase in DHT is an important concomitant to the acquisition of fertility. Testosterone and several Sa-reduced androgens, notably (dihydrotestosterone, 17P-hydroxy-5a-androstan-3-one DHT) and 5a-androstane-3a.l7f?-diol (3a-dial) can stimulate adult epididymal function <14], b;t little’is known of their role in this regard during sexual maturation. Previous studies have shown that the amount of testosterone available systemically to the epididymis increases in the maturing rat [S, 63 as does the endogenous tissue content of androgens [7]. Then too, epididymal androgen-metabolizing activity also displays significant changes [8]. In an attempt to correlate changes in local androgen concentrations with the onset of fertility, the concentrations of testosterone, DHT, 3a-dial, and 5a-androstane-3/?,17,!%diol (3/l-diol) in the rete testis fluid (RTF) and the luminal fluid of the cauda epididymidis (CLF) of the rat were measured by radioimmunoassay just prior to and after sperm entry into the epididymis. Samples were obtained from 45 and 55 day old LongEvans rats of the Rutgers University colony between 9:00 and 10:30a.m. during May through July. Details of animal husbandry have been previously described [9]. RTF, collected in 2 ~1 capillary tubes (Drummond Scientific) under light ether anesthesia according to the method of Harris and Bartke[lO], was transferred to a culture tube and stored at -20°C until assayed. A sufficient number of samples was collected to assay four 9-15 ~1 pools of fluid for each age. CLF was collected from excised tissues of decapitated animals by cannulating the proximal portion of the vas deferens with PE 10 polyethylene tubing (ClayAdams) after straightening 5-8 cm of tubule from the terminal portion of the epididymis. The luminal fluid gently massaged from the cauda tubule into the cannula was stored at -20°C until assayed. Samples were pooled by flushing the cannulae with approximately 20 volumes of water to obtain four pools containing 20-45 ~1 CLF for each age. Samples were assayed in duplicate following ether extraction and chromatographic separation of the four androgens on columns of Sephadex LH-20. Details and validation of the procedures have been described elsewhere [9]. Differences between means were considered significant when P < 0.05according to Student’s t-test. The fact that spermatozoa first enter the rat epididymis around 50 days of age was confirmed for our colony by
* Present address and address to which all correspondence should be sent: R. G. Foldesy, Department of Obstetrics & Gynecology, Vanderbilt University, School of Medicine, Room C-2304, Nashville, TN 37232, U.S.A.
histological examination of tissues from several animals at 45 and 55 days of age. No spermatozoa were present in the epididymides of the former group while those of the latter were occupied throughout. Radioimmunoassay of RTF revealed relatively high levels of 3a-diol and testosterone in both age groups while those of DHT and 3/3-diol were comparatively low. However, of the four, only testosterone concentrations rose significantly with increasing age (P < 0.025; Fig. IA). During the IO-day period, total androgen content of the RTF increased considerably, from 53.8 to 81.2 ng/ml, nearly all of which consisted of testosterone. Because RTF is an important source of androgen for the caput epididymidis [ 111, the rise in testosterone suggests an important role, via its conversion to DHT[l2], in stimulating the immature tubular epithelium. The androgen profile of CLF was very different from that of RTF (Fig. 1B). Although 3a-diol was again a major androgen at both ages, concentrations of testosterone were relatively unchanged and this was the lowest of the four at 55 days of age. Unlike those in RTF. both 3B-diol and DHT concen;ations comprised substantial poriions of the total androgen content measured. In fact, DHT concentrations rose 67% (4.3 vs 7.2ng/ml), an increase that represented the only statistically significant change (P < 0.025) in CLF androgen composition during the time when spermatozoa fill the epididymis. It is unlikely, however, that the spermatozoa themselves have contributed directly to this increase since little androgen-metabolizing activity is associated with these cells [13]. On the other hand, the rat epididymis possesses several androgen-metabolizing enzymes and the reported maturational increases in their activity [8] may account for the rise in DHT. Alternatively, the question of accumulation or retention of DHT within the tubule may be more pertinent to the observed changes. [3H]-Testosterone readily enters the tubular lumen of the cauda epididymidis to be converted largely to DHT [14]. Furthermore, this entry is facilitated by luminal proteins [ 151. Hence, specific compositional changes in the tubular milieu during puberty, together with the rise in the systemic supply of testosterone to the epididymis, may facilitate the accumulation of DHT in the caudal fluid. The sum of the four androgens in CLF was unaltered during the IO-day period (29.0 vs 28.4ng/ml, day 45 and 55, respectively) in contrast to that found in RTF. The ratio of DHT: testosterone, which increases from 0.9 to 1.8 in the present study, is lower than the 3.1 reported by Pujol et a[.[ 161 for cauda epididymal fluid in adult rats, suggesting that a rise in DHT concentrations may be a prerequi-
109
110
Short communication A.
Rete
Testis
Fluid
& Caudo Luminol
Fluid
Fig. I. Androgen concentrations in (A) rete testis fluid and (B) luminal fluid of the cauda epididymidis in 45 and 55 day old rats. Each bar represents the mean +SE of four pools of fluid. T = testosterone; DHT = dihydrotestosterone~ 3a = 3a-dial; 3p = 3p-diol. site for the onset of fertility. DHT, in fact, appears to be the key androgen for the epididymis; it is bound preferentially by the cytoplasmic receptor [17] and comprises most of the steroid associated with the cell nuclei [18]. Furthermore, most of the testosterone accumulating in the epididymal tubules in situ from the peripheral circulation is converted to DHT [ 14, 151 and this latter androgen is the only one which will support development of sperm maturation in cultured tubules[3]. Because mammalian sperm maturation and storage are androgen-dependent functions [19], it is proposed that increasing DHT concentrations in CLF represent an important concomitant to the acquisition of fertility. Acknowledgemmts-Helpful comments on the manuscript offered by Drs Bedford. Saling and Rodger are much appreciated. This work was supported by the Busch Fund of Rutgers University. REFERENCES 1. Blaquier J. A., Cameo M. S. and Burgos M. H.: The role of androgens in the maturation of epididymal spermatozoa in the guinea pig. Endocrinology 90 (1972) 839-842. 2. Dyson A. L. M. B. and Orgebin-Crist M.-C.: Effect of hypophysectomy, castration and androgen replacement upon the fertilizing ability of rat ep~didymal spermatozoa. Endocrinology 93 (1973) 391402. 3. Orgebin-Crist M.-C., Jahad N. and Hoffman L. H.: The effects of testosterone, Sa-dihydrotestosterone, 3x-androstanediol and 3/l-androstanediol on the maturation of rabbit epididymal spermatozoa in organ culture. Cell Tin. Res. 167 (1976) 515-525. 4. Lubicz-Nawrocki C. M.: The effect of metabolites of testosterone on the viability of hamster epididymal spermatozoa. J. Endoer. Ss, (1973) 193-198. 5. Gupta D., Zarzycki J. and Rager K.: Plasma testosterone and dihydrotestosterone in male rats during sexual maturation and following orchidectomy and experimental-bilateral cryptorchidism. Steroids 25 (1975) 33-42. 6. Dohler K. D. and Wuttke W.: Changes with age in levels of serum gonadotropins, prolactin and gonadal steroids in prepubertal male and female rats. ~~~ocr~~ology 97 (1975) 898-907. 7 Calandra R. S.. Podesta E. J., Rivarola M. A. and Blaquier J. A. : Tissue androgens and androphilic proteins
in rat epididymis during sexual development. Steroids 24 (1974) 507-518. 8. Scheer H. and Robaire B.: Developmental study of epididymal A’-Sa-reductase and 3a-hydroxysteroid dehydrogenase. Am. Sot. Andrology Fourth Annual Meet., Houston, Texas (1979) Abstract. 9. Foldesy R. G. and Leathem J. H.: Simultaneous measurements of testosterone and three 5c+reduced androgens in the venous effluent of immature rat testes in situ. Steroids 35 (1980) 621-631, 10. Harris M. E. and Bartke A.: Concentration of testosterone in testis fluid of the rat. Endocrinology 95 (1974) 701-706. I I. Setchell B. P. The Mammalian Testis. Cornell Univ. Press, Ithaca (1978) p. 138. 12. Vreeburg J. T. M.: Distribution of testosterone and So-dihydrotestosterone in rat epididymis and their concentrations in efferent duct fluid. J. Endocr. 67 (1975) 203-210. 13. Robaire B., Ewing L. L., Zirkin B. R. and frby D. C.: Steroid A’-5s-reductase and 3a-hydroxysteroid dehydrogenase in the rat epididymis. Endocrinology JO1 (1977) 1379-1390. 14 Back D. J.: The presence of metabolites of ‘H-testosterone in the lumen of the cauda epididymidis of the rat. Steroids 25 (1975) 413-420. t 5. Cooper T. G. and Waites G. M. H.: Factors affecting the entry of testosterone into the lumen of cauda epididymidis of the anaesthetized rat. J. Reprod. Fert. 56 (1979) 165-174. 16 Pujol A., Bayard F., Louvet J.-P. and Boulard C.: Testosterone and dihydrotestosterone concentrations in plasma, epididymal tissues, and seminal fluid of adult rats. Endocrinology 98 ( 1976) 1 I I-I I 3. I7 Blaquier J. A.: Selective uptake and meta~lism of androgens by rat epididymis. The presence of a cytoplasmic receptor. Biocheni. Biophys. Res. Commun. 45 (1971) 1076-1082.
18 Blaquier J. A. and Calandra R. S.: Intranuclear recep tor for androgens in rat epididymis. Endocrinology 93 (1973) 51-60.
19. Orgebin-Crist M.-C., Danzo 9. J. and Davies J.: Endocrine control of the development and maintenance of sperm fertilizing ability in the epididymis. In Handbook of ~~y~io~ff~_~ (Edited by D. W. Hamilton and R. 0. Greep). American Physiological Society, Washington, D.C., Sec. 7. Vol. V (1975) p, 319.
Journal of Srrroid Biochemistry Vol. 14. pp. 109 to I IO Pergamon Press Ltd 1981. Printed in Great Britain
SHORT COMMUNICATION PUBERTAL CHANGES IN ANDROGEN COMPOSITION OF RAT RETE TESTIS AND CAUDA EPIDIDYMAL FLUIDS R. G. FOLDESY* and J. H. LEATHEM Bureau of Biological Research, Rutgers-The State University, New Brunswick, NJ 08903, U.S.A. (Received 15 April
1980)
SUMMARY The concentrations of testosterone, dihydrotestosterone (DHT), 5a-androstane-3a,l7/l-diol, and 5a-androstane-38,17/l-diol were measured by radio-immunoassay in rete testis fluid (RTF) and luminal fluid of the cauda epididy-midis (CLF) in pubertal rats. Fluids were collected prior to and after spermatozoa enter the epididymis (45 and 55 days of age). Testosterone concentrations rose 2.5-fold in RTF and DHT concentrations increased 67% in CLF. These represented the only changes observed and it is proposed that the increase in DHT is an important concomitant to the acquisition of fertility. Testosterone and several Sa-reduced androgens, notably (dihydrotestosterone, 17P-hydroxy-5a-androstan-3-one DHT) and 5a-androstane-3a.l7f?-diol (3a-dial) can stimulate adult epididymal function <14], b;t little’is known of their role in this regard during sexual maturation. Previous studies have shown that the amount of testosterone available systemically to the epididymis increases in the maturing rat [S, 63 as does the endogenous tissue content of androgens [7]. Then too, epididymal androgen-metabolizing activity also displays significant changes [8]. In an attempt to correlate changes in local androgen concentrations with the onset of fertility, the concentrations of testosterone, DHT, 3a-dial, and 5a-androstane-3/?,17,!%diol (3/l-diol) in the rete testis fluid (RTF) and the luminal fluid of the cauda epididymidis (CLF) of the rat were measured by radioimmunoassay just prior to and after sperm entry into the epididymis. Samples were obtained from 45 and 55 day old LongEvans rats of the Rutgers University colony between 9:00 and 10:30a.m. during May through July. Details of animal husbandry have been previously described [9]. RTF, collected in 2 ~1 capillary tubes (Drummond Scientific) under light ether anesthesia according to the method of Harris and Bartke[lO], was transferred to a culture tube and stored at -20°C until assayed. A sufficient number of samples was collected to assay four 9-15 ~1 pools of fluid for each age. CLF was collected from excised tissues of decapitated animals by cannulating the proximal portion of the vas deferens with PE 10 polyethylene tubing (ClayAdams) after straightening 5-8 cm of tubule from the terminal portion of the epididymis. The luminal fluid gently massaged from the cauda tubule into the cannula was stored at -20°C until assayed. Samples were pooled by flushing the cannulae with approximately 20 volumes of water to obtain four pools containing 20-45 ~1 CLF for each age. Samples were assayed in duplicate following ether extraction and chromatographic separation of the four androgens on columns of Sephadex LH-20. Details and validation of the procedures have been described elsewhere [9]. Differences between means were considered significant when P < 0.05according to Student’s t-test. The fact that spermatozoa first enter the rat epididymis around 50 days of age was confirmed for our colony by
* Present address and address to which all correspondence should be sent: R. G. Foldesy, Department of Obstetrics & Gynecology, Vanderbilt University, School of Medicine, Room C-2304, Nashville, TN 37232, U.S.A.
histological examination of tissues from several animals at 45 and 55 days of age. No spermatozoa were present in the epididymides of the former group while those of the latter were occupied throughout. Radioimmunoassay of RTF revealed relatively high levels of 3a-diol and testosterone in both age groups while those of DHT and 3/3-diol were comparatively low. However, of the four, only testosterone concentrations rose significantly with increasing age (P < 0.025; Fig. IA). During the IO-day period, total androgen content of the RTF increased considerably, from 53.8 to 81.2 ng/ml, nearly all of which consisted of testosterone. Because RTF is an important source of androgen for the caput epididymidis [ 111, the rise in testosterone suggests an important role, via its conversion to DHT[l2], in stimulating the immature tubular epithelium. The androgen profile of CLF was very different from that of RTF (Fig. 1B). Although 3a-diol was again a major androgen at both ages, concentrations of testosterone were relatively unchanged and this was the lowest of the four at 55 days of age. Unlike those in RTF. both 3B-diol and DHT concen;ations comprised substantial poriions of the total androgen content measured. In fact, DHT concentrations rose 67% (4.3 vs 7.2ng/ml), an increase that represented the only statistically significant change (P < 0.025) in CLF androgen composition during the time when spermatozoa fill the epididymis. It is unlikely, however, that the spermatozoa themselves have contributed directly to this increase since little androgen-metabolizing activity is associated with these cells [13]. On the other hand, the rat epididymis possesses several androgen-metabolizing enzymes and the reported maturational increases in their activity [8] may account for the rise in DHT. Alternatively, the question of accumulation or retention of DHT within the tubule may be more pertinent to the observed changes. [3H]-Testosterone readily enters the tubular lumen of the cauda epididymidis to be converted largely to DHT [14]. Furthermore, this entry is facilitated by luminal proteins [ 151. Hence, specific compositional changes in the tubular milieu during puberty, together with the rise in the systemic supply of testosterone to the epididymis, may facilitate the accumulation of DHT in the caudal fluid. The sum of the four androgens in CLF was unaltered during the IO-day period (29.0 vs 28.4ng/ml, day 45 and 55, respectively) in contrast to that found in RTF. The ratio of DHT: testosterone, which increases from 0.9 to 1.8 in the present study, is lower than the 3.1 reported by Pujol et a[.[ 161 for cauda epididymal fluid in adult rats, suggesting that a rise in DHT concentrations may be a prerequi-
109
110
Short communication A.
Rete
Testis
Fluid
& Caudo Luminol
Fluid
Fig. I. Androgen concentrations in (A) rete testis fluid and (B) luminal fluid of the cauda epididymidis in 45 and 55 day old rats. Each bar represents the mean +SE of four pools of fluid. T = testosterone; DHT = dihydrotestosterone~ 3a = 3a-dial; 3p = 3p-diol. site for the onset of fertility. DHT, in fact, appears to be the key androgen for the epididymis; it is bound preferentially by the cytoplasmic receptor [17] and comprises most of the steroid associated with the cell nuclei [18]. Furthermore, most of the testosterone accumulating in the epididymal tubules in situ from the peripheral circulation is converted to DHT [ 14, 151 and this latter androgen is the only one which will support development of sperm maturation in cultured tubules[3]. Because mammalian sperm maturation and storage are androgen-dependent functions [19], it is proposed that increasing DHT concentrations in CLF represent an important concomitant to the acquisition of fertility. Acknowledgemmts-Helpful comments on the manuscript offered by Drs Bedford. Saling and Rodger are much appreciated. This work was supported by the Busch Fund of Rutgers University. REFERENCES 1. Blaquier J. A., Cameo M. S. and Burgos M. H.: The role of androgens in the maturation of epididymal spermatozoa in the guinea pig. Endocrinology 90 (1972) 839-842. 2. Dyson A. L. M. B. and Orgebin-Crist M.-C.: Effect of hypophysectomy, castration and androgen replacement upon the fertilizing ability of rat ep~didymal spermatozoa. Endocrinology 93 (1973) 391402. 3. Orgebin-Crist M.-C., Jahad N. and Hoffman L. H.: The effects of testosterone, Sa-dihydrotestosterone, 3x-androstanediol and 3/l-androstanediol on the maturation of rabbit epididymal spermatozoa in organ culture. Cell Tin. Res. 167 (1976) 515-525. 4. Lubicz-Nawrocki C. M.: The effect of metabolites of testosterone on the viability of hamster epididymal spermatozoa. J. Endoer. Ss, (1973) 193-198. 5. Gupta D., Zarzycki J. and Rager K.: Plasma testosterone and dihydrotestosterone in male rats during sexual maturation and following orchidectomy and experimental-bilateral cryptorchidism. Steroids 25 (1975) 33-42. 6. Dohler K. D. and Wuttke W.: Changes with age in levels of serum gonadotropins, prolactin and gonadal steroids in prepubertal male and female rats. ~~~ocr~~ology 97 (1975) 898-907. 7 Calandra R. S.. Podesta E. J., Rivarola M. A. and Blaquier J. A. : Tissue androgens and androphilic proteins
in rat epididymis during sexual development. Steroids 24 (1974) 507-518. 8. Scheer H. and Robaire B.: Developmental study of epididymal A’-Sa-reductase and 3a-hydroxysteroid dehydrogenase. Am. Sot. Andrology Fourth Annual Meet., Houston, Texas (1979) Abstract. 9. Foldesy R. G. and Leathem J. H.: Simultaneous measurements of testosterone and three 5c+reduced androgens in the venous effluent of immature rat testes in situ. Steroids 35 (1980) 621-631, 10. Harris M. E. and Bartke A.: Concentration of testosterone in testis fluid of the rat. Endocrinology 95 (1974) 701-706. I I. Setchell B. P. The Mammalian Testis. Cornell Univ. Press, Ithaca (1978) p. 138. 12. Vreeburg J. T. M.: Distribution of testosterone and So-dihydrotestosterone in rat epididymis and their concentrations in efferent duct fluid. J. Endocr. 67 (1975) 203-210. 13. Robaire B., Ewing L. L., Zirkin B. R. and frby D. C.: Steroid A’-5s-reductase and 3a-hydroxysteroid dehydrogenase in the rat epididymis. Endocrinology JO1 (1977) 1379-1390. 14 Back D. J.: The presence of metabolites of ‘H-testosterone in the lumen of the cauda epididymidis of the rat. Steroids 25 (1975) 413-420. t 5. Cooper T. G. and Waites G. M. H.: Factors affecting the entry of testosterone into the lumen of cauda epididymidis of the anaesthetized rat. J. Reprod. Fert. 56 (1979) 165-174. 16 Pujol A., Bayard F., Louvet J.-P. and Boulard C.: Testosterone and dihydrotestosterone concentrations in plasma, epididymal tissues, and seminal fluid of adult rats. Endocrinology 98 ( 1976) 1 I I-I I 3. I7 Blaquier J. A.: Selective uptake and meta~lism of androgens by rat epididymis. The presence of a cytoplasmic receptor. Biocheni. Biophys. Res. Commun. 45 (1971) 1076-1082.
18 Blaquier J. A. and Calandra R. S.: Intranuclear recep tor for androgens in rat epididymis. Endocrinology 93 (1973) 51-60.
19. Orgebin-Crist M.-C., Danzo 9. J. and Davies J.: Endocrine control of the development and maintenance of sperm fertilizing ability in the epididymis. In Handbook of ~~y~io~ff~_~ (Edited by D. W. Hamilton and R. 0. Greep). American Physiological Society, Washington, D.C., Sec. 7. Vol. V (1975) p, 319.