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In vitro 16α-hydroxylation of progesterone by testes of crab-eating monkey (Macaca irus): A characteristic of primates?
Comp. Biochem. PhysioLVol. 81B, No. 1, pp. 55-57, 1985 Printed in Great Britain
0305-0491/85 $3.00+0.00 © 1985 Pergamon Press Ltd
IN VITRO 16~-HYDROXYLATION OF PROGESTERONE BY TESTES OF CRAB-EATING MONKEY (MACACA IRUS): A CHARACTERISTIC OF P R I M A T E S ? HIROSHI INANO, AYA KIKUCHI* and BUN-ICHI TAMAOKI National Institute of Radiological Sciences, 9-I, Anagawa-4-chome, Chiba-shi 260, Japan
(Received 21 August 1984) Akstract--1. Radioactive 16~-hydroxyprogesterone was obtained as one of the major metabolites of [14C]pregnenolone and progesterone by testicular homogenates of an adult crab-eating monkey (Macaca irus). 2. 16c~-hydroxylation of progesterone occurred by incubation oftestes of men and the monkey, but not by those of rodents and other vertebrates examined.
INTRODUCTION
Extraction, separation and identification 16ct-Hydroxyprogesterone and other steroids were extracted with dichloromethane from the incubation mixture. To an aliquot of the extract, the five standard reference steroids, progesterone, 17~t-hydroxyprogesterone, 11-deoxycortisol, androstenedione and testosterone were added, and then the mixture was subjected to silica gel-thin layer chromatography. Radioactive metabolites were detected by autoradiographic technique. Metabolites were identified as by isopolarity on thin layer chromatograms developed in different solvent systems, by chemical derivation and finally by constant specific activity throughout repeated crystallization with authentic preparation, as previously reported (Oshima et al., 1967).
Occurrence of 16ct-hydroxyprogesterone from progesterone by testicular homogenates of a rhesus monkey was reported by Sharma et al. (1967). By testicular tissue of a crab-eating monkey (Macaca irus), in vitro formation of 16~-hydroxyprogesterone was further confirmed (Mizutani et al., 1977). However, they paid little attention to this metabolite. As testicular tissue of M. irus became recently available to us, we incubated several radioactive steroids with its testicular homogenates, and identified the metabolites. In this paper, we report the results, focusing to 16ct-hydroxylation of the C21-steroid.
Measurement of radioactivity After adding 5 ml of liquid scintillator (ACS-II, Amersham International) to each sample in a counting vial, radioactivities were measured by a liquid scintillation spectrometer (725 System, Nuclear Chicago, IL) with a counting efficiency of radiocarbon, 70~.
MATERIALS AND METHODS
Tissue preparation Testicular glands of an adult crab-eating monkey (M. irus) were isolated immediately after killing, and the tissue (11 g) was homogenized in a 0.25 M sucrose-10 mM Tris-HCl solution at pH 7.4. The supernatant fluid (13 ml) obtained by centrifugation of the homogenates at 800 g for 20 min was used as its cell-free homogenates.
RESULTS
On the initial thin layer chromatogram of the metabolites developed in a benzene-acetone system (4:1 v/v), one of the major radioactive spots was detected at a position more polar than the spot of authentic ll-deoxycortisol in both cases of metabolism ofpregnenolone and progesterone. This fraction was further separable into two metabolites by second thin layer chromatography developed in a benzenechloroform-ether-methanol system (2: 2: 2: 1, v/v). A metabolite with Rr = 0.58 coincided with that of 16~-hydroxyprogesterone and the other with Rf = 0.55 was 17~t-hydroxy-20~-dihydroprogesterone. The former was acetylatable by a mixture of pyridine and acetic anhydride (1: 1, v/v), resulting in the identical mobility on thin layer chromatograms with that of authentic 16ct-hydroxyprogesterone. After repeated crystallization of the metabolite with authentic 16~-hydroxyprogesterone, practically constant specific radioactivities were obtained (Table 1). These identification procedures were almost the same as we reported on 16ct-hydroxyprogesterone obtained by incubation with human testes (Oshima et al., 1967).
Chemicals [4-14C]Labeled pregnenolone and progesterone (50-60 mCi/mmole, each) were obtained from New England Nuclear Corp., Boston. After dilution of the radioactive steroids with the corresponding non-radioactive steroids, respectively, these steroids were used as the substrates. 16c~-Hydroxyprogesterone, other steroid preparations which were used for identification of the metabolites, cofactors and other chemicals were commercially obtained. Incubation In an incubation flask, pregnenolone (200 nmole, 15 x 104 cpm) was aerobically incubated with the testicular homogenates (126.3 mg protein), NAD + and NADPH (2.4/tmole, each) at 37°C for 60 min. Similarly, progesterone (200 nmole, 15 x 104 cpm) was incubated in the presence of NADPH (2.4 #mole).
* Visiting scientist from Kyoritsu College of Pharmacy, Shiba Park, Minato-ku, Tokyo 105, Japan. 55
HIROSHI INANO AYA KIKUCH1 and BUN ICH1 TAMAOKI
56
Table 1. Recrystallization of a radioactive metabolite obtained by incubation of pregnenolone and progesterone with the testicular homogenates of M. irus with authentic 16~-hydroxyprogesterone
No. crystallization
Specific activity (cpm/mg)
1 2 3 4
147 154 119 130
Solventsused CH2Cl2-n-heptane CH2C12-n-heptane CH2Cl2-n-heptane CH2Cl2-n-heptane
Calculated specificactivity before crystallization: 183.
Table 2, Metabolism of pregnenolone and progesterone in the testis of M. irus Substrate Pregnenolone Progesterone (nmole) (nmole)
Metabolites Recovered substrate Progesterone 17~t-Hydroxypregnenolone 16ct-Hydroxyprogesterone 170t-Hydroxyprogesterone 17ct-Hydroxy-20ct-dihydroprogesterone Androstenedione Testosterone Dehydroepiandrosterone
20 14 13 20 77 12 4 34 6
genesis in the testis of rat ( T a m a o k i et al., 1969), mouse ( M u r o t a et al., 1965), guinea pig ( I n a n o et al., 1967), horse (Oh a n d T a m a o k i , 1970), cat (Oh a n d T a m a o k i , 1973a), dog (Oh a n d T a m a o k i , 1973b), goat ( M o r i et al., 1980) a n d n o n - m a m m a l i a n vertebrates (Tamaoki, 1981), no 16ct-hydroxylation o f progesterone has been observed. Therefore, we propose here t h a t 16~-hydroxyation o f progesterone would be a characteristic o f testicular function of primates, interestingly from the aspect of c o m p a r a t i v e biochemistry of steroidogenesis. By adrenal h o m o g e n a t e s o f the same monkey, we also confirmed f o r m a t i o n o f 16ct-hydroxyprogesterone as a metabolite. W h o l e in vitro metabolism of pregnenolone, progesterone a n d a n d r o s t e n e d i o n e in the testicular a n d adrenal h o m o g e n a t e s of M . irus is to be reported in detail elsewhere. Acknowledgements--We express our gratitude to Dr Junji Yamagiwa in this Institute for his kind offer of the experimental material. This work was partly supported by a Grant-in-Aid for Scientific Research from Ministry of Edution, Science and Culture of Japan.
28 19 115 30 1 6
After other metabolites o b t a i n e d were similarly identified, yield of each metabolite was calculated as s h o w n in Table 2. DISCUSSION Previously, 16ct-hydroxyprogesterone was identified as a metabolite obtained by i n c u b a t i o n with h u m a n testicular homogenates. 16ct-Hydroxylase activity was localized exclusively in the microsomal fraction of h u m a n testes (Oshima et al., 1967). The role of 16ct-hydroxyprogesterone was studied u p o n h u m a n testicular enzyme activity in vitro, a n d this metabolite was found as a competitive inhibitor to C-17-C-20 lyase a n d a non-competitive inhibitor to 20~-hydroxysteroid d e h y d r o g e n a s e ( I n a n o a n d T a m a o k i , 1978). In the case of rhesus monkey, p r o d u c t i o n of 16ct-hydroxyprogesterone was reported ( S h a r m a et al., 1967; M i z u t a n i et al., 1977). 16~-Hydroxylase activity in the testes o f M . irus seems to be higher before puberty t h a n t h a t at m a t u r a t i o n a n d susceptible to storage in - 2 0 ° C (Mizutani et al., 1977). O n the o t h e r h a n d , t h o u g h testicular m e t a b o l i s m o f progesterone has been studied in adult rhesus m o n k e y ( M a c a c a mulatta) ( H o s c h o i a n a n d Brownie, 1967; Preslock a n d Steinberger, 1979; Higashi et al., 1984) a n d its fetus (Coffey a n d J o h n s o n b a u g h , 1979), a n d a lower primate, the m a r m o s e t (Saguinus oedipurs) (Preslock a n d Steinberger, 1976), no occurrence of 16ct-hydroxyprogesterone has been reported. It is not clear whether they did n o t detect any significant p r o d u c t i o n o f the metabolite after t h o r o u g h analysis, or overlooked it. As far as we examined steroido-
REFERENCES Coffey J. C. and Johnsonbaugh R. E. (1979) The in vitro metabolism of progesterone, 4-androstene-3,17-dione, testosterone and 17fl-hydroxy-5~-androstan-3-one by fetal rhesus monkey testes. Steroids 33, 427 434. Higashi Y., Takahashi J., Yoshida K., Winters S. J., Oshima H. and Troen P. (1984) Seasonal changes in steroidogenesis in the testis of the rhesus monkey (Macaca mulatta). J. Androl. 5, 70-79. Hoschoian J. C. and Brownie A. C. (1967) Pathways for androgen biosynthesis in monkey testes. Steroids 10, 49-69. Inano H. and Tamaoki B. (1978) In vitro effect of 16~-hydroxyprogesterone on the enzyme activities related to androgen production in human testes. Acta endocr. Copenh. 88, 768 777. Inano H., Egusa M. and Tamaoki B. (1967) Studies on the enzymes related to steroidogenesis in testicular tissue of guinea pig. Biochim. biophys. Acta 144, 165-167. Mizutani S., Tsujimura T., Akashi S. and Matsumoto K. (1977) Lack of metabolism of progesterone, testosterone and pregnenolone to 5~-products in monkey and human testes compared with rodent testes. J. clin. Endocr. Metab. 44, 1023 1031. Mori M., Matsukura S., Kawakura K. and Tamaoki B. (1980) In vitro synthesis of androgen from pregnenolone in the testes of the goat (Capra hircus) and identification of 5-pregnen-3fl, 17ct,20ct-triol as an intermediate in metabolic pathway of pregnenolone. J. Endocr. 84, 381-390. Murota S., Shikita M. and Tamaoki B. (1965) Intracellular distribution of the enzymes related to androgen formation in mouse testes. Steroids 5, 409-413. Oh R. and Tamaoki B. (1970) Steroidogenesis in equine testis. Acta endocr. Copenh. 64, 1 16. Oh R. and Tamaoki B. (1973a) In vitro steroidogenesis in feline testes. Biochim. biophys. Acta 316, 395402. Oh R. and Tamaoki B. (1973b) In vitro biosynthesis of androgen in canine testes. Acta endocr. Copenh. 74, 615-624. Oshima H., Sarada T., Ochi-ai K. and Tamaoki B. (1967) Intracellular distribution and properties of steroid 16ct-hydroxylase in human testes. J. clin. Endocr. Metab. 27, 1249-1254. Preslock J. P. and Steinberger E. (1976) Pathway of testosterone biosynthesis in the testis of the marmoset Sanguinus oedipus. Steroids 28, 775-784. Preslock J. P. and Steinberger E. (1979) Testicular steroido-
16ct-Hydroxylation by monkey testes genesis in the rhesus monkey Macaca mulatta. Steroids 34, 52%537. Sharma D. C., Joshi S. G. and Dorfman R. I. (1967) Biosynthesis of testosterone by monkey testes in vitro. Endocrinology 80, 499-504. Tamaoki B. (1981)Gonadal steroids: their species-specificities and biogenesis. In Advances in Physiological Sciences, Vol. 20, Advances in Animal and Comparative Physiology (Edited by Pethes G. and Frenyo V. L.), pp. 32%336. Pergamon Press, London. Tamaoki B., Inano H. and Nakano H. (1969) In vitro synthesis and conversion of androgens in testicular tissue. In The Gonads (Edited by McKerns K.W.), pp. 547-614. Appleton-Century-Crofts, New York.
57 ADDENDUM
From our personal communication with Dr Hiroyuki Oshima (Yokohama City University School of Medicine), they found that a part of progesterone was converted by testicular preparations of the rhesus monkey (Macaca mulatta) into 16~-hydroxyprogesterone which was tentatively identified, even though unpublished. Higashi et al., (1984) in References [and Yoshida K., Takahashi J., Winters S. J., Oshima H. and Troen P. (1984) Steroidogenesis in the monkey testis: relationship of enzyme organization to endogenous steroids, steroidogenesis and gonadotropin treatment. J. Steroid Biochem. 21, (49-58)].
0305-0491/85 $3.00+0.00 © 1985 Pergamon Press Ltd
IN VITRO 16~-HYDROXYLATION OF PROGESTERONE BY TESTES OF CRAB-EATING MONKEY (MACACA IRUS): A CHARACTERISTIC OF P R I M A T E S ? HIROSHI INANO, AYA KIKUCHI* and BUN-ICHI TAMAOKI National Institute of Radiological Sciences, 9-I, Anagawa-4-chome, Chiba-shi 260, Japan
(Received 21 August 1984) Akstract--1. Radioactive 16~-hydroxyprogesterone was obtained as one of the major metabolites of [14C]pregnenolone and progesterone by testicular homogenates of an adult crab-eating monkey (Macaca irus). 2. 16c~-hydroxylation of progesterone occurred by incubation oftestes of men and the monkey, but not by those of rodents and other vertebrates examined.
INTRODUCTION
Extraction, separation and identification 16ct-Hydroxyprogesterone and other steroids were extracted with dichloromethane from the incubation mixture. To an aliquot of the extract, the five standard reference steroids, progesterone, 17~t-hydroxyprogesterone, 11-deoxycortisol, androstenedione and testosterone were added, and then the mixture was subjected to silica gel-thin layer chromatography. Radioactive metabolites were detected by autoradiographic technique. Metabolites were identified as by isopolarity on thin layer chromatograms developed in different solvent systems, by chemical derivation and finally by constant specific activity throughout repeated crystallization with authentic preparation, as previously reported (Oshima et al., 1967).
Occurrence of 16ct-hydroxyprogesterone from progesterone by testicular homogenates of a rhesus monkey was reported by Sharma et al. (1967). By testicular tissue of a crab-eating monkey (Macaca irus), in vitro formation of 16~-hydroxyprogesterone was further confirmed (Mizutani et al., 1977). However, they paid little attention to this metabolite. As testicular tissue of M. irus became recently available to us, we incubated several radioactive steroids with its testicular homogenates, and identified the metabolites. In this paper, we report the results, focusing to 16ct-hydroxylation of the C21-steroid.
Measurement of radioactivity After adding 5 ml of liquid scintillator (ACS-II, Amersham International) to each sample in a counting vial, radioactivities were measured by a liquid scintillation spectrometer (725 System, Nuclear Chicago, IL) with a counting efficiency of radiocarbon, 70~.
MATERIALS AND METHODS
Tissue preparation Testicular glands of an adult crab-eating monkey (M. irus) were isolated immediately after killing, and the tissue (11 g) was homogenized in a 0.25 M sucrose-10 mM Tris-HCl solution at pH 7.4. The supernatant fluid (13 ml) obtained by centrifugation of the homogenates at 800 g for 20 min was used as its cell-free homogenates.
RESULTS
On the initial thin layer chromatogram of the metabolites developed in a benzene-acetone system (4:1 v/v), one of the major radioactive spots was detected at a position more polar than the spot of authentic ll-deoxycortisol in both cases of metabolism ofpregnenolone and progesterone. This fraction was further separable into two metabolites by second thin layer chromatography developed in a benzenechloroform-ether-methanol system (2: 2: 2: 1, v/v). A metabolite with Rr = 0.58 coincided with that of 16~-hydroxyprogesterone and the other with Rf = 0.55 was 17~t-hydroxy-20~-dihydroprogesterone. The former was acetylatable by a mixture of pyridine and acetic anhydride (1: 1, v/v), resulting in the identical mobility on thin layer chromatograms with that of authentic 16ct-hydroxyprogesterone. After repeated crystallization of the metabolite with authentic 16~-hydroxyprogesterone, practically constant specific radioactivities were obtained (Table 1). These identification procedures were almost the same as we reported on 16ct-hydroxyprogesterone obtained by incubation with human testes (Oshima et al., 1967).
Chemicals [4-14C]Labeled pregnenolone and progesterone (50-60 mCi/mmole, each) were obtained from New England Nuclear Corp., Boston. After dilution of the radioactive steroids with the corresponding non-radioactive steroids, respectively, these steroids were used as the substrates. 16c~-Hydroxyprogesterone, other steroid preparations which were used for identification of the metabolites, cofactors and other chemicals were commercially obtained. Incubation In an incubation flask, pregnenolone (200 nmole, 15 x 104 cpm) was aerobically incubated with the testicular homogenates (126.3 mg protein), NAD + and NADPH (2.4/tmole, each) at 37°C for 60 min. Similarly, progesterone (200 nmole, 15 x 104 cpm) was incubated in the presence of NADPH (2.4 #mole).
* Visiting scientist from Kyoritsu College of Pharmacy, Shiba Park, Minato-ku, Tokyo 105, Japan. 55
HIROSHI INANO AYA KIKUCH1 and BUN ICH1 TAMAOKI
56
Table 1. Recrystallization of a radioactive metabolite obtained by incubation of pregnenolone and progesterone with the testicular homogenates of M. irus with authentic 16~-hydroxyprogesterone
No. crystallization
Specific activity (cpm/mg)
1 2 3 4
147 154 119 130
Solventsused CH2Cl2-n-heptane CH2C12-n-heptane CH2Cl2-n-heptane CH2Cl2-n-heptane
Calculated specificactivity before crystallization: 183.
Table 2, Metabolism of pregnenolone and progesterone in the testis of M. irus Substrate Pregnenolone Progesterone (nmole) (nmole)
Metabolites Recovered substrate Progesterone 17~t-Hydroxypregnenolone 16ct-Hydroxyprogesterone 170t-Hydroxyprogesterone 17ct-Hydroxy-20ct-dihydroprogesterone Androstenedione Testosterone Dehydroepiandrosterone
20 14 13 20 77 12 4 34 6
genesis in the testis of rat ( T a m a o k i et al., 1969), mouse ( M u r o t a et al., 1965), guinea pig ( I n a n o et al., 1967), horse (Oh a n d T a m a o k i , 1970), cat (Oh a n d T a m a o k i , 1973a), dog (Oh a n d T a m a o k i , 1973b), goat ( M o r i et al., 1980) a n d n o n - m a m m a l i a n vertebrates (Tamaoki, 1981), no 16ct-hydroxylation o f progesterone has been observed. Therefore, we propose here t h a t 16~-hydroxyation o f progesterone would be a characteristic o f testicular function of primates, interestingly from the aspect of c o m p a r a t i v e biochemistry of steroidogenesis. By adrenal h o m o g e n a t e s o f the same monkey, we also confirmed f o r m a t i o n o f 16ct-hydroxyprogesterone as a metabolite. W h o l e in vitro metabolism of pregnenolone, progesterone a n d a n d r o s t e n e d i o n e in the testicular a n d adrenal h o m o g e n a t e s of M . irus is to be reported in detail elsewhere. Acknowledgements--We express our gratitude to Dr Junji Yamagiwa in this Institute for his kind offer of the experimental material. This work was partly supported by a Grant-in-Aid for Scientific Research from Ministry of Edution, Science and Culture of Japan.
28 19 115 30 1 6
After other metabolites o b t a i n e d were similarly identified, yield of each metabolite was calculated as s h o w n in Table 2. DISCUSSION Previously, 16ct-hydroxyprogesterone was identified as a metabolite obtained by i n c u b a t i o n with h u m a n testicular homogenates. 16ct-Hydroxylase activity was localized exclusively in the microsomal fraction of h u m a n testes (Oshima et al., 1967). The role of 16ct-hydroxyprogesterone was studied u p o n h u m a n testicular enzyme activity in vitro, a n d this metabolite was found as a competitive inhibitor to C-17-C-20 lyase a n d a non-competitive inhibitor to 20~-hydroxysteroid d e h y d r o g e n a s e ( I n a n o a n d T a m a o k i , 1978). In the case of rhesus monkey, p r o d u c t i o n of 16ct-hydroxyprogesterone was reported ( S h a r m a et al., 1967; M i z u t a n i et al., 1977). 16~-Hydroxylase activity in the testes o f M . irus seems to be higher before puberty t h a n t h a t at m a t u r a t i o n a n d susceptible to storage in - 2 0 ° C (Mizutani et al., 1977). O n the o t h e r h a n d , t h o u g h testicular m e t a b o l i s m o f progesterone has been studied in adult rhesus m o n k e y ( M a c a c a mulatta) ( H o s c h o i a n a n d Brownie, 1967; Preslock a n d Steinberger, 1979; Higashi et al., 1984) a n d its fetus (Coffey a n d J o h n s o n b a u g h , 1979), a n d a lower primate, the m a r m o s e t (Saguinus oedipurs) (Preslock a n d Steinberger, 1976), no occurrence of 16ct-hydroxyprogesterone has been reported. It is not clear whether they did n o t detect any significant p r o d u c t i o n o f the metabolite after t h o r o u g h analysis, or overlooked it. As far as we examined steroido-
REFERENCES Coffey J. C. and Johnsonbaugh R. E. (1979) The in vitro metabolism of progesterone, 4-androstene-3,17-dione, testosterone and 17fl-hydroxy-5~-androstan-3-one by fetal rhesus monkey testes. Steroids 33, 427 434. Higashi Y., Takahashi J., Yoshida K., Winters S. J., Oshima H. and Troen P. (1984) Seasonal changes in steroidogenesis in the testis of the rhesus monkey (Macaca mulatta). J. Androl. 5, 70-79. Hoschoian J. C. and Brownie A. C. (1967) Pathways for androgen biosynthesis in monkey testes. Steroids 10, 49-69. Inano H. and Tamaoki B. (1978) In vitro effect of 16~-hydroxyprogesterone on the enzyme activities related to androgen production in human testes. Acta endocr. Copenh. 88, 768 777. Inano H., Egusa M. and Tamaoki B. (1967) Studies on the enzymes related to steroidogenesis in testicular tissue of guinea pig. Biochim. biophys. Acta 144, 165-167. Mizutani S., Tsujimura T., Akashi S. and Matsumoto K. (1977) Lack of metabolism of progesterone, testosterone and pregnenolone to 5~-products in monkey and human testes compared with rodent testes. J. clin. Endocr. Metab. 44, 1023 1031. Mori M., Matsukura S., Kawakura K. and Tamaoki B. (1980) In vitro synthesis of androgen from pregnenolone in the testes of the goat (Capra hircus) and identification of 5-pregnen-3fl, 17ct,20ct-triol as an intermediate in metabolic pathway of pregnenolone. J. Endocr. 84, 381-390. Murota S., Shikita M. and Tamaoki B. (1965) Intracellular distribution of the enzymes related to androgen formation in mouse testes. Steroids 5, 409-413. Oh R. and Tamaoki B. (1970) Steroidogenesis in equine testis. Acta endocr. Copenh. 64, 1 16. Oh R. and Tamaoki B. (1973a) In vitro steroidogenesis in feline testes. Biochim. biophys. Acta 316, 395402. Oh R. and Tamaoki B. (1973b) In vitro biosynthesis of androgen in canine testes. Acta endocr. Copenh. 74, 615-624. Oshima H., Sarada T., Ochi-ai K. and Tamaoki B. (1967) Intracellular distribution and properties of steroid 16ct-hydroxylase in human testes. J. clin. Endocr. Metab. 27, 1249-1254. Preslock J. P. and Steinberger E. (1976) Pathway of testosterone biosynthesis in the testis of the marmoset Sanguinus oedipus. Steroids 28, 775-784. Preslock J. P. and Steinberger E. (1979) Testicular steroido-
16ct-Hydroxylation by monkey testes genesis in the rhesus monkey Macaca mulatta. Steroids 34, 52%537. Sharma D. C., Joshi S. G. and Dorfman R. I. (1967) Biosynthesis of testosterone by monkey testes in vitro. Endocrinology 80, 499-504. Tamaoki B. (1981)Gonadal steroids: their species-specificities and biogenesis. In Advances in Physiological Sciences, Vol. 20, Advances in Animal and Comparative Physiology (Edited by Pethes G. and Frenyo V. L.), pp. 32%336. Pergamon Press, London. Tamaoki B., Inano H. and Nakano H. (1969) In vitro synthesis and conversion of androgens in testicular tissue. In The Gonads (Edited by McKerns K.W.), pp. 547-614. Appleton-Century-Crofts, New York.
57 ADDENDUM
From our personal communication with Dr Hiroyuki Oshima (Yokohama City University School of Medicine), they found that a part of progesterone was converted by testicular preparations of the rhesus monkey (Macaca mulatta) into 16~-hydroxyprogesterone which was tentatively identified, even though unpublished. Higashi et al., (1984) in References [and Yoshida K., Takahashi J., Winters S. J., Oshima H. and Troen P. (1984) Steroidogenesis in the monkey testis: relationship of enzyme organization to endogenous steroids, steroidogenesis and gonadotropin treatment. J. Steroid Biochem. 21, (49-58)].