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Androgen biosynthesis by marmoset testes in vitro
GENERAL
AND
COMPARATIVE
Androgen
31, 101-105 (1977)
ENDOCRINOLOGY
Biosynthesis JAMES
P. PRESLOCK
Department University
by Marmoset AND EMIL
of Reproductive Medicine of Texas Medical School Houston, Texas 77025
Testes in Vitro
STEINBERGER and Biology, at Hoaston.
Accepted July 26, 1976 These studies were undertaken to determine the major steroid metabolites formed from selected androgen precursors by the testis of the marmoset, Saguinus Oedipus. a New World primate of the family Callitricadae. Testicular fragments (50 mg) were incubated for 3.0 hr with pregnenolone-7-3H or with progesterone-7-3H. The major metabolites formed from pregnenolone were 17a-hydroxyprogesterone (42.7%). testosterone (20.5%), androstenedione (I 1.4%) and progesterone (9.2%). Nonmetabolized substrate was 6.8% of radioactivity. For porgesterone incubations, 17a-hydroxyprogesterone was the major metabolite (49.0%), with testosterone (21.2%) and androstenedione (10.7%) as lesser metabolites. Unreacted progesterone accounted for 14.9% of all radioactivity. The unusually high levels of 17cr-hydroxyprogesterone in marmosets is in contrast to that observed in other mammalian species.
The vertebrate testis converts various nonsteroidal and steroidal precursors into biologically active androgens (Steinberger and Ficher, 1968, 1969; Coffey et al., 1971; Bardin and Peterson, 1967: Inano and Tamaoki, 1966; Nayfeh et al., 1966) with further reduction of androgens into estrogens, and into So-reduced metabolites (Sowell et al., 1974; Yamada and Matsumoto, 1974). Although the rat testis has been used as a model for testicular steroidogenesis, there are apparently appreciable differences in the metabolism of steroid hormones and in the elimination of metabolites among vertebrate species (Barry et al., 1952; Bradlow et al., 1954; Gallagher et al., 1954; Saunberg and Slaunwhite, 1956; Acevedo er al., 1961; Taylor and Scratcher, 1966; Kulkarni er al., 1970; Steinberger et al., 1973). However, although considerable body of data exists concerned with testicular steroidogenesis in rodents, comparatively little is known in nonhuman primates. Marmosets are small New World primates of the family Callitricadae which have been referred to as the most primitive primate
(Hershkowitz, 1969, 1970). Although marmosets are unique in that their most frequent form of birth is chimeric twins of biovular origin, thus offering an important model for study of reproductive physiology, no information is available regarding the biogenesis of steroids by the testis of marmosets. The study reported here was undertaken to investigate the in vitro formation of androgens from selected radiolabeled precursors by the testis of Saguinus oedipus, a marmoset species. MATERIALS Materials
All organic solvents were nanograde and were obtained from Mallinckrodt Chemical Works, St. Louis, MO. The cofactors from Sigma Chemical Co., St. Louis, MO. The paper for chromatography (Whatman No. I;87 s/m*) was obtained from Whatmann Paper Co. and was washed with methanol prior to use. The silica gel (silicar TLC-7GfJ purchased from Mallinckrodt Chemical Works, St. Louis, MO.. was washed with methanol prior to use. All nonradioactive steroids were obtained from Steraloids. Inc., Rawling, NY, and the radioactive steroids from Amersham Searle. Inc., Arlington Heights. III. All steroids were checked for purity by thin-layer chromatography prior to use. 101
Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
102 Experimental
PRESLOCK
AND
Animals
An adult male S. oedipus was caged at constant temperature and humidity with lighting maintained at lOL:14D. and fed a commercial diet with water provided ad libirrrm. The right testis was surgically removed and placed into ice cold 0.25 M sucrose-tris buffer, pH 7.4. It was weighed and cut into four approximately equal 50 mg fragments. Duplicate fragments were incubated with radiolabeled precursors.
Incubations Incubations were carried out in Krebs-Ringer bicarbonate buffer (pH 7.4), at 37” in a 95% 0,/j% CO, atmosphere. The buffer contained NADH (0.927 mg/ incubate), NADP (1.005 mg/incubate), glucose (6 mg/ incubate), glucose-6-phosphate (3.885 mg/incubate), pyruvate (0.99 mg/incubate), nicotinamide (10.99 mg/ incubate), magnesium chloride hexahydrate (0.915 mgiincubate). glucose-6-phosphate dehydrogenase. and lactic dehydrogenase. Duplicate tissue fragments were incubated for 3 hr with pregnenolone-7-3H (2 PCi; 20 Ci/mmole) or progesterone-7-3H (2 &I; 24 Ciimmole). The reaction was terminated by addition of 0.5 ml of I N HCI and the incubates were immediately frozen.
STEINBERGER
and carbon-14 radioactivity was determined by double label counting in a Packard liquid scintillation spectrometer.
Identification
of Metabolites
The identity of metabolites was established by comparison of their chromatographic mobilities with that of authentic standards. Final identity was established by recrystallization of each metabolite to constant specific activity and 3H/W ratio through five successive solvent combinations of acetone, chloroform, cyclohexane. hexane, and isooctane. Each metabolite received approximately 15 mg of cold mass for recrystallization. Percentage conversions were calculated by determining the total 3H dpm for each metabolite from recrystallization data. The total 3H dpm was corrected for procedural losses, divided by the 3H dpm of the total incubate, and expressed as a percentage of the total 3H dpm of the incubate. % conversion =
3H dpm,,,,,, x % recovery 3H dpmcincu,xm
RESULTS
The major metabolites formed in incubates containing pregnenolone-7-3H as the precursors were progesterone, 17~~ Prior to extraction the incubates received approhydroxyprogesterone, androstenedione, and priate W-labeled and unlabeled tracers. Incubates were extracted ten times with cold diethyl ether: testosterone. 17~Hydroxy-progesterone chloroform, 4: 1. Extracts were evaporated under nit- was the major metabolite, representing rogen, the residue concentrated, and aliquots re42.7% of the total radioactivity. Testosmoved for recovery estimates prior to paper terone represented 20.5% of conversions, chromatography. while androstenedione and progesterone Separation and Purification of Steroids were 11.4% and 9.2%, respectively. The pregnenolone substrate Residues were applied to paper strips (2.5 x 50 cm) non-metabolized impregnated with formamide, and chromatographed represented 6.8% of radioactivity. The in hexane, followed by rechromatography in hexane: remaining radioactivity represented mibenzene, 1: 1. Radioactive peaks were located with a nor, unidentified metabolites. The recrysPackard Model 385 Recording ratemeter. Peaks with tallization data are presented in Table 1. Rf values close to those of testosterone, 17~ When progesterone-7-3H was used as the hydroxyprogesterone, androstenedione, progesterone and pregnenolone were predominant. These peaks substrate, 17cr-hydroxyprogesterone, anwere eluted with methanol (80 ml), and individual ste- drostenedione, and testosterone were roids isolated by thin-layer chromatography (tic) in also formed in amounts similar to that selected solvent systems. These solvent systems conobserved in incubates containing pregsisted of benzene:ethyl acetate (80:20, 60:40). bennenolone-7-3H (17~~hydroxyprogesteronezene:methanol (98:2, 95:5), and chloroform acetone (90: IO, 80:20). Testosterone and 17a-hydroxyand tes49%, androstenedione-10.7%, progesterone were separated by tic. acetylation of tosterone-21.2%). Approximately 15% of testosterone to testosterone acetate. followed by a progesterone remained unmetabolized. Sumfinal tic separation. Aliquots, for estimation of recovermary of the recrystallization and conies, were obtained prior to each tic separation, and prior to recrystallization of each metabolite. Tritium version data is presented in Table 2. Extraction
ANDROGEN
SYNTHESIS
IN
TABLE STEROID
Metabolite” Testost. Andros. 17~OH Prog. Prog. Preg.*
METABOLITES FROM PREc~NENOLONE-%~H(~
” t + ” +
1
INCUBATION OF MARMOSET TESTICULAR FRAGMENTS(SO mg) WITH &i) FOR 3 HR IN KREBS-RINGER BICARBONATE BUFFER
3H dpmlmg*d 37.2 20.7 74.6 24.7 12.1
103
MARMOSETS
3.3 1.7 3.8 2.1 1.1
r4C dpm/mg*d
3H/‘4C
3H dpmd (cm
Conversione (%)
8.3 k 0.6 4.2 + 0.4 4.0 f 0.3 3.9 + 0.3
4.4 + 0.2 4.8 i 0.3 6.1 k 0.4 3.0 k 0.2
862.3 479.4 1803.5 575.4 289.4
20.4 11.4 42.7 13.6 6.8
’ The following abbreviations are used in the table: Testost., testosterone as testosterone acetate; Andros., androstenedione; 17~OH Prog., 17a-hydroxyprogesterone; Prog., progesterone: Preg., pregnenolone. * Nonmetabolized pregnenolone substrate. c Data as mean of five successive recrystallizations (; k SD). d Data represented as mean dpm (x 101) of duplicate samples. p Mean of duplicate determinations. TABLE STEROID
Metabolite’ Testost. Andros. 17kOH Prop. Pr0g.b
METABOLITES FROM INCUBATION PROGESTERONE-?% (2 &~)FOR
3H dpmlmgcsd 28.1 18.4 89.6 28.6
+ + c t
0.5 1.1 6.4 1.8
2
OF MARMOSET TESTICULAR FRAGMENTS (50 ~~)WITH 3 HR IN KREBS-RINGER BICARBONATE BUFFER
r4C dpmlmgc*d
3H/*4C
3H dpmd (Corr)
Conversione (%)
6.0 h 0.4 4.1 t 0.2 4.3 k 0.2
4.5 2 0.2 4.4 + 0.2 6.4 k 0.3
890.4 447.5 2055.3 627.5
21.2 10.6 49.0 14.9
’ The following abbreviations are used in the table: Testost., testosterone as testosterone acetate; Andros., androstenedione; 17~OH Prog., 17a-hydroxyprogesterone; Prog., progesterone. b Nonmetabolized progesterone substrate. c Data as mean of five successive recrystallizations (i -r- SD). d Data represented as mean dpm (x 101) of duplicate samples. ’ Mean of duplicate determinations.
DISCUSSION
The results presented in this paper demonstrate that testicular tissue of the marmoset, S. Oedipus, converts radiolabeled pregnenolone and progesterone to testosterone in vitro. However, the major metabolite representing nearly 50% of all radioactivity is 17&-hydroxyprogesterone. The accumulation of 17a-hydroxyprogesterone is associated with diminished conversion to testosterone and androstenedione. This is in contrast to data obtained in the rat (Steinberger and Ficher, 1968, 1969; Slaunwhite and Burgett, 1965),
the mouse (Ellis and Berliner, 1965), the rabbit (Rosner et al., 1964) and the dog (Eik-Nes and Kekre, 1963) where testosterone is the primary metabolite formed from incubations of testicular tissue with similar precursors. Under the experimental conditions described in this paper, therefore, the marmoset testis appears to be unique among mammalian species in accumulating primarily 17a-hydroxyprogesterone from pregnenolone and progesterone. It is not possible from the present studies with Saguinus Oedipus to conclusively de-
104
PRESLOCK
AND STEINBERGER
termine whether the A4 or the A5 pathway is the principal pathway for androgen biosynthesis in the marmoset testis. However, the conversion of pregnenolone primarily to 17a-hydroxyprogesterone, and then to testosterone through formation of androstenedione suggests that the A4 pathway may be predominant in marmosets. The only other nonhuman primate in which the testicular biosynthesis of androgens has been detailed is in rhesus monkeys. In this primate, Hoschoian and Brownie (1967) reported that pregnenolone and 17a-hydroxypregnenolone were more efficiently converted to testosterone than were progesterone and 17a-hydroxyprogesterone, suggesting formation of testosterone through a pathway (A5) independent of androstenedione. Conversely, Sharma et al. (1967) reported that 17a-hydroxyprogesterone was a better substrate than progesterone for testosterone synthesis, suggesting testosterone formation via progesterone + 17a-hydroxyprogesterone + androstenedione + testosterone. From the present studies with marmosets and previous studies with rhesus monkeys, it is apparent that there is little information available regarding testicular steroidogenesis in nonhuman primates. Therefore, the determination of the primary pathways of androgen biosynthesis in these primates awaits further investigations. REFERENCES Acevedo, H. F., Axelrod, L. R., Ishikawa, E., and Takaki, F. (1961). Steroidogenesis in the human fetal testis: the conversion of pregnenolone-7&H to dehydroepiandrosterone. testosterone and 4-androstene-3,17-dione. J. Clin. Endocrinol. 21, 1611-1613. Bardin, C. W., and Peterson, R. F. (1967). Studies of androgen production by the rat: testosterone and androstenedione content of blood. Endocrinology 80, 38-44. Barry, M. C., Eidinoff, M. D., Dobriner, K., and Gallagher, T. F. (1952). The fate of ‘%testosterone and i4C-progesterone in mice and rats. Endocrinology
Bradlow,
50, 587-599.
H. L., Dobriner,
K., and Gallagher, T. F.
(1954). The fate of cortisone-t crinology
in mice. E&o-
54, 343-352.
Coffey, J. C.. French. (1971). Metabolism ticular homogenates. tosterone formation Endocrinology
F. S., and Nayfeh, S. N. of progesterone by rat tesIV. Further studies of tesin immature testis in vitro.
89, 865-872.
Eik-Nes, K. B., and Kekre, M. (1963). Metabolism in viva of steroids by the canine testes. Biochim. Biophys.
Acta
78, 449-456.
Ellis, L. C., and Berliner, D. L. (1965). Sequential biotransformation of 5-pregnenolone-7&H and progesteone-4-“C into androgens by mouse testes. Endocrinology 76, 591-599. Gallagher. T. F., Bradlow, H. L., Fukushima, D. K., Beer, C. T., Kritchevsky, T. H., Stokem, M., Eidihoff, M. L., Hellman. L.. and Dobriner, R. K. (1954). Studies of the metabolites of isotopic hormones in man. Recent Progr. Horm. Res. 9, 411-433. Hershkowitz, P. (1969). The evolution of mammals on the southern continent. VI. The recent mammals on the neotropical region: a zoogeographic and ecological review. Quart. Rev. Biol. 44, l-70. Hershkowitz, P. (1970). Notes on tertiary platyrrine monkeys and description of a new genus from the late miocene of Colombia. Folia Primat. (Basel) 12, l-37. Hoschoian, J. C., and Brownie, A. C. (1967). Pathways for androgen synthesis in monkey testis. Steroids 10, 49-69. Inano, H.. and Tamaoki, B. (1966). Bioconversion of steroids in immature rat testes in vitro. Endocrinology
79, 579-590.
Kulkami, B. D.. Kammer, C. S.. and Goldzieher, J. W. (1970). Tracer studies of the fate of steroid hormones in the baboon. Gen. Comp. Endocrinol. 14, 68-71. Nayfeh, S. N., Barefoot, S. W., Jr., and Baggett, B. (1966). Metabolism of progesterone by rat testitular homogenates. II. Changes with age. Endocrinology 78, 1041-1048. Rosner, J. M., Horita, S., and Forsham, P. H. (1964). Androstenediol, a possible intermediate in the in vitro conversion of dehydroepiandrosterone to testosterone by the rabbit testis. Endocrinology 75, 299-303. Saunberg, A. A., and Slaunwhite, W. R. (1956). Metabolism of 4-14C-testosterone in human subjects. J. Clin. Invest. 35, 1331-1339. Sharma, D. C., Joshi, S. G., and Dorfman, R. I. (1967). Biosynthesis of testosterone by monkey testes in vitro. Endocrinology 80, 499-504. Slaunwhite, W. R., Jr.. and Burgett, M. T. (1965). In vitro testosterone synthesis by rat testicular tissue. Steroids 6, 721-735. Sowell, J. G., Folman, Y.. and Eik-Nes, K. B.
ANDROGEN
SYNTHESIS
(1974). Androgen metabolism in rat testicular tissue. Endocrinology
94,
346-353.
Steinberger, E., and Ficher, M. (1968). Conversion of progesterone to testosterone by testicular tissue at different stages of maturation. Steroids 11, 351-368. Steinberger, E., and Ficher, M. (1969). Differentiation of steroid bisynthetic pathways in developing testes. Biof. Reprod. 1, 119-133. Steinberger, E., Smith, K. D., Tcholakian, R. K., Chowdhury, M., Steinberger, A., Ficher, M.,
IN MARMOSETS
105
and Paulsen, C. A. (1973). Steroidogenesis in human testes. In “Male Fertility and Sterility” (R. E. Mancini and L. Martini, eds.), Academic Press, New York. Taylor, W., and Scratcher, T. (1967). Steroid metabolism in the rabbit. Biochem. J. 104, 250-253. Yamada. Y., and Matsumoto, K. (1974). Pathway from progesterone to So-reduced C,, steroids not involving androstenedione and testosterone in immature rat testes in vitro. Endocrinology 94, 777-784.
AND
COMPARATIVE
Androgen
31, 101-105 (1977)
ENDOCRINOLOGY
Biosynthesis JAMES
P. PRESLOCK
Department University
by Marmoset AND EMIL
of Reproductive Medicine of Texas Medical School Houston, Texas 77025
Testes in Vitro
STEINBERGER and Biology, at Hoaston.
Accepted July 26, 1976 These studies were undertaken to determine the major steroid metabolites formed from selected androgen precursors by the testis of the marmoset, Saguinus Oedipus. a New World primate of the family Callitricadae. Testicular fragments (50 mg) were incubated for 3.0 hr with pregnenolone-7-3H or with progesterone-7-3H. The major metabolites formed from pregnenolone were 17a-hydroxyprogesterone (42.7%). testosterone (20.5%), androstenedione (I 1.4%) and progesterone (9.2%). Nonmetabolized substrate was 6.8% of radioactivity. For porgesterone incubations, 17a-hydroxyprogesterone was the major metabolite (49.0%), with testosterone (21.2%) and androstenedione (10.7%) as lesser metabolites. Unreacted progesterone accounted for 14.9% of all radioactivity. The unusually high levels of 17cr-hydroxyprogesterone in marmosets is in contrast to that observed in other mammalian species.
The vertebrate testis converts various nonsteroidal and steroidal precursors into biologically active androgens (Steinberger and Ficher, 1968, 1969; Coffey et al., 1971; Bardin and Peterson, 1967: Inano and Tamaoki, 1966; Nayfeh et al., 1966) with further reduction of androgens into estrogens, and into So-reduced metabolites (Sowell et al., 1974; Yamada and Matsumoto, 1974). Although the rat testis has been used as a model for testicular steroidogenesis, there are apparently appreciable differences in the metabolism of steroid hormones and in the elimination of metabolites among vertebrate species (Barry et al., 1952; Bradlow et al., 1954; Gallagher et al., 1954; Saunberg and Slaunwhite, 1956; Acevedo er al., 1961; Taylor and Scratcher, 1966; Kulkarni er al., 1970; Steinberger et al., 1973). However, although considerable body of data exists concerned with testicular steroidogenesis in rodents, comparatively little is known in nonhuman primates. Marmosets are small New World primates of the family Callitricadae which have been referred to as the most primitive primate
(Hershkowitz, 1969, 1970). Although marmosets are unique in that their most frequent form of birth is chimeric twins of biovular origin, thus offering an important model for study of reproductive physiology, no information is available regarding the biogenesis of steroids by the testis of marmosets. The study reported here was undertaken to investigate the in vitro formation of androgens from selected radiolabeled precursors by the testis of Saguinus oedipus, a marmoset species. MATERIALS Materials
All organic solvents were nanograde and were obtained from Mallinckrodt Chemical Works, St. Louis, MO. The cofactors from Sigma Chemical Co., St. Louis, MO. The paper for chromatography (Whatman No. I;87 s/m*) was obtained from Whatmann Paper Co. and was washed with methanol prior to use. The silica gel (silicar TLC-7GfJ purchased from Mallinckrodt Chemical Works, St. Louis, MO.. was washed with methanol prior to use. All nonradioactive steroids were obtained from Steraloids. Inc., Rawling, NY, and the radioactive steroids from Amersham Searle. Inc., Arlington Heights. III. All steroids were checked for purity by thin-layer chromatography prior to use. 101
Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
102 Experimental
PRESLOCK
AND
Animals
An adult male S. oedipus was caged at constant temperature and humidity with lighting maintained at lOL:14D. and fed a commercial diet with water provided ad libirrrm. The right testis was surgically removed and placed into ice cold 0.25 M sucrose-tris buffer, pH 7.4. It was weighed and cut into four approximately equal 50 mg fragments. Duplicate fragments were incubated with radiolabeled precursors.
Incubations Incubations were carried out in Krebs-Ringer bicarbonate buffer (pH 7.4), at 37” in a 95% 0,/j% CO, atmosphere. The buffer contained NADH (0.927 mg/ incubate), NADP (1.005 mg/incubate), glucose (6 mg/ incubate), glucose-6-phosphate (3.885 mg/incubate), pyruvate (0.99 mg/incubate), nicotinamide (10.99 mg/ incubate), magnesium chloride hexahydrate (0.915 mgiincubate). glucose-6-phosphate dehydrogenase. and lactic dehydrogenase. Duplicate tissue fragments were incubated for 3 hr with pregnenolone-7-3H (2 PCi; 20 Ci/mmole) or progesterone-7-3H (2 &I; 24 Ciimmole). The reaction was terminated by addition of 0.5 ml of I N HCI and the incubates were immediately frozen.
STEINBERGER
and carbon-14 radioactivity was determined by double label counting in a Packard liquid scintillation spectrometer.
Identification
of Metabolites
The identity of metabolites was established by comparison of their chromatographic mobilities with that of authentic standards. Final identity was established by recrystallization of each metabolite to constant specific activity and 3H/W ratio through five successive solvent combinations of acetone, chloroform, cyclohexane. hexane, and isooctane. Each metabolite received approximately 15 mg of cold mass for recrystallization. Percentage conversions were calculated by determining the total 3H dpm for each metabolite from recrystallization data. The total 3H dpm was corrected for procedural losses, divided by the 3H dpm of the total incubate, and expressed as a percentage of the total 3H dpm of the incubate. % conversion =
3H dpm,,,,,, x % recovery 3H dpmcincu,xm
RESULTS
The major metabolites formed in incubates containing pregnenolone-7-3H as the precursors were progesterone, 17~~ Prior to extraction the incubates received approhydroxyprogesterone, androstenedione, and priate W-labeled and unlabeled tracers. Incubates were extracted ten times with cold diethyl ether: testosterone. 17~Hydroxy-progesterone chloroform, 4: 1. Extracts were evaporated under nit- was the major metabolite, representing rogen, the residue concentrated, and aliquots re42.7% of the total radioactivity. Testosmoved for recovery estimates prior to paper terone represented 20.5% of conversions, chromatography. while androstenedione and progesterone Separation and Purification of Steroids were 11.4% and 9.2%, respectively. The pregnenolone substrate Residues were applied to paper strips (2.5 x 50 cm) non-metabolized impregnated with formamide, and chromatographed represented 6.8% of radioactivity. The in hexane, followed by rechromatography in hexane: remaining radioactivity represented mibenzene, 1: 1. Radioactive peaks were located with a nor, unidentified metabolites. The recrysPackard Model 385 Recording ratemeter. Peaks with tallization data are presented in Table 1. Rf values close to those of testosterone, 17~ When progesterone-7-3H was used as the hydroxyprogesterone, androstenedione, progesterone and pregnenolone were predominant. These peaks substrate, 17cr-hydroxyprogesterone, anwere eluted with methanol (80 ml), and individual ste- drostenedione, and testosterone were roids isolated by thin-layer chromatography (tic) in also formed in amounts similar to that selected solvent systems. These solvent systems conobserved in incubates containing pregsisted of benzene:ethyl acetate (80:20, 60:40). bennenolone-7-3H (17~~hydroxyprogesteronezene:methanol (98:2, 95:5), and chloroform acetone (90: IO, 80:20). Testosterone and 17a-hydroxyand tes49%, androstenedione-10.7%, progesterone were separated by tic. acetylation of tosterone-21.2%). Approximately 15% of testosterone to testosterone acetate. followed by a progesterone remained unmetabolized. Sumfinal tic separation. Aliquots, for estimation of recovermary of the recrystallization and conies, were obtained prior to each tic separation, and prior to recrystallization of each metabolite. Tritium version data is presented in Table 2. Extraction
ANDROGEN
SYNTHESIS
IN
TABLE STEROID
Metabolite” Testost. Andros. 17~OH Prog. Prog. Preg.*
METABOLITES FROM PREc~NENOLONE-%~H(~
” t + ” +
1
INCUBATION OF MARMOSET TESTICULAR FRAGMENTS(SO mg) WITH &i) FOR 3 HR IN KREBS-RINGER BICARBONATE BUFFER
3H dpmlmg*d 37.2 20.7 74.6 24.7 12.1
103
MARMOSETS
3.3 1.7 3.8 2.1 1.1
r4C dpm/mg*d
3H/‘4C
3H dpmd (cm
Conversione (%)
8.3 k 0.6 4.2 + 0.4 4.0 f 0.3 3.9 + 0.3
4.4 + 0.2 4.8 i 0.3 6.1 k 0.4 3.0 k 0.2
862.3 479.4 1803.5 575.4 289.4
20.4 11.4 42.7 13.6 6.8
’ The following abbreviations are used in the table: Testost., testosterone as testosterone acetate; Andros., androstenedione; 17~OH Prog., 17a-hydroxyprogesterone; Prog., progesterone: Preg., pregnenolone. * Nonmetabolized pregnenolone substrate. c Data as mean of five successive recrystallizations (; k SD). d Data represented as mean dpm (x 101) of duplicate samples. p Mean of duplicate determinations. TABLE STEROID
Metabolite’ Testost. Andros. 17kOH Prop. Pr0g.b
METABOLITES FROM INCUBATION PROGESTERONE-?% (2 &~)FOR
3H dpmlmgcsd 28.1 18.4 89.6 28.6
+ + c t
0.5 1.1 6.4 1.8
2
OF MARMOSET TESTICULAR FRAGMENTS (50 ~~)WITH 3 HR IN KREBS-RINGER BICARBONATE BUFFER
r4C dpmlmgc*d
3H/*4C
3H dpmd (Corr)
Conversione (%)
6.0 h 0.4 4.1 t 0.2 4.3 k 0.2
4.5 2 0.2 4.4 + 0.2 6.4 k 0.3
890.4 447.5 2055.3 627.5
21.2 10.6 49.0 14.9
’ The following abbreviations are used in the table: Testost., testosterone as testosterone acetate; Andros., androstenedione; 17~OH Prog., 17a-hydroxyprogesterone; Prog., progesterone. b Nonmetabolized progesterone substrate. c Data as mean of five successive recrystallizations (i -r- SD). d Data represented as mean dpm (x 101) of duplicate samples. ’ Mean of duplicate determinations.
DISCUSSION
The results presented in this paper demonstrate that testicular tissue of the marmoset, S. Oedipus, converts radiolabeled pregnenolone and progesterone to testosterone in vitro. However, the major metabolite representing nearly 50% of all radioactivity is 17&-hydroxyprogesterone. The accumulation of 17a-hydroxyprogesterone is associated with diminished conversion to testosterone and androstenedione. This is in contrast to data obtained in the rat (Steinberger and Ficher, 1968, 1969; Slaunwhite and Burgett, 1965),
the mouse (Ellis and Berliner, 1965), the rabbit (Rosner et al., 1964) and the dog (Eik-Nes and Kekre, 1963) where testosterone is the primary metabolite formed from incubations of testicular tissue with similar precursors. Under the experimental conditions described in this paper, therefore, the marmoset testis appears to be unique among mammalian species in accumulating primarily 17a-hydroxyprogesterone from pregnenolone and progesterone. It is not possible from the present studies with Saguinus Oedipus to conclusively de-
104
PRESLOCK
AND STEINBERGER
termine whether the A4 or the A5 pathway is the principal pathway for androgen biosynthesis in the marmoset testis. However, the conversion of pregnenolone primarily to 17a-hydroxyprogesterone, and then to testosterone through formation of androstenedione suggests that the A4 pathway may be predominant in marmosets. The only other nonhuman primate in which the testicular biosynthesis of androgens has been detailed is in rhesus monkeys. In this primate, Hoschoian and Brownie (1967) reported that pregnenolone and 17a-hydroxypregnenolone were more efficiently converted to testosterone than were progesterone and 17a-hydroxyprogesterone, suggesting formation of testosterone through a pathway (A5) independent of androstenedione. Conversely, Sharma et al. (1967) reported that 17a-hydroxyprogesterone was a better substrate than progesterone for testosterone synthesis, suggesting testosterone formation via progesterone + 17a-hydroxyprogesterone + androstenedione + testosterone. From the present studies with marmosets and previous studies with rhesus monkeys, it is apparent that there is little information available regarding testicular steroidogenesis in nonhuman primates. Therefore, the determination of the primary pathways of androgen biosynthesis in these primates awaits further investigations. REFERENCES Acevedo, H. F., Axelrod, L. R., Ishikawa, E., and Takaki, F. (1961). Steroidogenesis in the human fetal testis: the conversion of pregnenolone-7&H to dehydroepiandrosterone. testosterone and 4-androstene-3,17-dione. J. Clin. Endocrinol. 21, 1611-1613. Bardin, C. W., and Peterson, R. F. (1967). Studies of androgen production by the rat: testosterone and androstenedione content of blood. Endocrinology 80, 38-44. Barry, M. C., Eidinoff, M. D., Dobriner, K., and Gallagher, T. F. (1952). The fate of ‘%testosterone and i4C-progesterone in mice and rats. Endocrinology
Bradlow,
50, 587-599.
H. L., Dobriner,
K., and Gallagher, T. F.
(1954). The fate of cortisone-t crinology
in mice. E&o-
54, 343-352.
Coffey, J. C.. French. (1971). Metabolism ticular homogenates. tosterone formation Endocrinology
F. S., and Nayfeh, S. N. of progesterone by rat tesIV. Further studies of tesin immature testis in vitro.
89, 865-872.
Eik-Nes, K. B., and Kekre, M. (1963). Metabolism in viva of steroids by the canine testes. Biochim. Biophys.
Acta
78, 449-456.
Ellis, L. C., and Berliner, D. L. (1965). Sequential biotransformation of 5-pregnenolone-7&H and progesteone-4-“C into androgens by mouse testes. Endocrinology 76, 591-599. Gallagher. T. F., Bradlow, H. L., Fukushima, D. K., Beer, C. T., Kritchevsky, T. H., Stokem, M., Eidihoff, M. L., Hellman. L.. and Dobriner, R. K. (1954). Studies of the metabolites of isotopic hormones in man. Recent Progr. Horm. Res. 9, 411-433. Hershkowitz, P. (1969). The evolution of mammals on the southern continent. VI. The recent mammals on the neotropical region: a zoogeographic and ecological review. Quart. Rev. Biol. 44, l-70. Hershkowitz, P. (1970). Notes on tertiary platyrrine monkeys and description of a new genus from the late miocene of Colombia. Folia Primat. (Basel) 12, l-37. Hoschoian, J. C., and Brownie, A. C. (1967). Pathways for androgen synthesis in monkey testis. Steroids 10, 49-69. Inano, H.. and Tamaoki, B. (1966). Bioconversion of steroids in immature rat testes in vitro. Endocrinology
79, 579-590.
Kulkami, B. D.. Kammer, C. S.. and Goldzieher, J. W. (1970). Tracer studies of the fate of steroid hormones in the baboon. Gen. Comp. Endocrinol. 14, 68-71. Nayfeh, S. N., Barefoot, S. W., Jr., and Baggett, B. (1966). Metabolism of progesterone by rat testitular homogenates. II. Changes with age. Endocrinology 78, 1041-1048. Rosner, J. M., Horita, S., and Forsham, P. H. (1964). Androstenediol, a possible intermediate in the in vitro conversion of dehydroepiandrosterone to testosterone by the rabbit testis. Endocrinology 75, 299-303. Saunberg, A. A., and Slaunwhite, W. R. (1956). Metabolism of 4-14C-testosterone in human subjects. J. Clin. Invest. 35, 1331-1339. Sharma, D. C., Joshi, S. G., and Dorfman, R. I. (1967). Biosynthesis of testosterone by monkey testes in vitro. Endocrinology 80, 499-504. Slaunwhite, W. R., Jr.. and Burgett, M. T. (1965). In vitro testosterone synthesis by rat testicular tissue. Steroids 6, 721-735. Sowell, J. G., Folman, Y.. and Eik-Nes, K. B.
ANDROGEN
SYNTHESIS
(1974). Androgen metabolism in rat testicular tissue. Endocrinology
94,
346-353.
Steinberger, E., and Ficher, M. (1968). Conversion of progesterone to testosterone by testicular tissue at different stages of maturation. Steroids 11, 351-368. Steinberger, E., and Ficher, M. (1969). Differentiation of steroid bisynthetic pathways in developing testes. Biof. Reprod. 1, 119-133. Steinberger, E., Smith, K. D., Tcholakian, R. K., Chowdhury, M., Steinberger, A., Ficher, M.,
IN MARMOSETS
105
and Paulsen, C. A. (1973). Steroidogenesis in human testes. In “Male Fertility and Sterility” (R. E. Mancini and L. Martini, eds.), Academic Press, New York. Taylor, W., and Scratcher, T. (1967). Steroid metabolism in the rabbit. Biochem. J. 104, 250-253. Yamada. Y., and Matsumoto, K. (1974). Pathway from progesterone to So-reduced C,, steroids not involving androstenedione and testosterone in immature rat testes in vitro. Endocrinology 94, 777-784.