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Ontogenesis and regulation of steroid sulfatase activity in leydig cells and seminiferous tubules in the long-evans rat
J. swroidEiochem. Vol. 30, No. 1-6, pp. 439-441, Printed in Great Britain
1988
0022-4731/88
$3.00+0.00
Pergamon
Press
plc
ONTOGENESIS AND REGULATION OF STEROID SULFATASE ACTIVITY IN LEYDIG CELLS AND SEMINIFEROUS TUBULES IN THE LONG-EVANS RAT MONIQUE BEDIN*, INSERM
TH~RBSE FOURNIER, NADIA MOUHADJER
and
U. 166, Groupe de Recherches SW I’Endocrinologie de la Reproduction, Bld de Port-Royal, 750 14 Paris, France
GEORGES POINTIS Maternitk Baudelocque,
123,
Summary-Steroid sulfatase (STS) activity was studied in Long-Evans rat testis. The affinity of the enzyme was shown to increase during postnatal development and to be always higher in purified Leydig cells than in seminiferous tubules. STS activity appeared to be higher in the seminiferous tubules at the earlier stages. In vivo injection of 100 IU hCG resulted in a decrease in the affinity and an increase in the activity of the enzyme expressed in Leydig cells with no such modification in seminiferous tubules. This suggests that STS could play a regulatory role in testosterone production by Leydig cells.
INTRODUCTION
In addition to testosterone, large amounts of steroid sulfates have been measured in the testis of some species including man [ 11. In vitro experiments have shown that this tissue is able to both synthesize and hydrolyze these sulfoconjugated steroids and that their secretion is stimulated by hCG-treatment [2,3]. From these observations it has been suggested that these compounds may be used as androgen precursors [4]. Using the rat as an experimental model, previous studies have led us to consider that testicular steroid sulfatase (STS) may act as a regulatory enzyme for the release of free androgens [S, 6, 71. Since testosterone is mainly synthesized in the Leydig cells and no information is available about the possible role of STS in this production, we have investigated the ontogenesis and the effect of hCG on this enzyme in purified Leydig cells derived from Long-Evans rats. EXPERIMENTAL
Animals and treatments Long-Evans male rats, at different stages of postnatal development (from 20 days to adult stage), were used for the ontogenesis study. When the effect of hCG was studied, adult male rats were injected intramuscularly with 100 IU hCG (Organon) diluted in saline containing 0.1% BSA. Control animals were treated with the vehicle alone. Both hCG-treated and control rats were killed 2 h after injection, a lag period resulting into maximal increase in circulating testosterone levels [8]. Isolation of seminiferous tubules and purified Leydig cells After sacrifice, testes were removed and, once
decapsulated, submitted to collagenase digestion in 10 ml medium 199 (Eurobio, Paris) containing 10 mg BSA, 3 mg collagenase type I (ICN Pharmaceuticals Inc., Cleveland, OH) and 4Opg DNAase I (Sigma Chemical Company, St Louis, MO) and processed as previously reported [9]. Seminiferous tubules were allowed to settle and were washed 3 times with 0.9% NaCl so as to eliminate interstitial cells. After filtration through a nylon gauze (pore size 140pm) interstitial cells were collected by centrifugation at 15Og for 15 min and fractionated afterwards by density gradient centrifugation in Percoll as described [lo]. This cell separation allowed us to obtain about 90% of pure Leydig cells identified by staining for 38HSD activity [ 1 I]. Determination of steroid sulfa&se activity Purified Leydig cells and seminiferous tubules free of interstitial cells were homogenized in 0.1 M Tris-HCI, pH 7.2, and sonicated to give complete disruption of the cells. Steroid sulfatase activity was determined as earlier reported [ 121. Aliquots of the suspension were incubated in duplicate at 37°C in the presence of labeled dehydroepiandrosterone-sulfate ([3H]DHA-S, sp. act. 24 Ci/mmol, NEN Chemicals, F.R.G.) at variable final concentrations in a total reaction volume of 250 ~1. The incubations were stopped by addition of 0.1 N NaOH and the yield of liberated unconjugated steroids was measured after direct extraction by the scintillation mixture (Toluen-PPO-POPOP) Results were corrected for methodological losses. The reaction rates were linear within the incubation time and the protein range employed. Protein concentration was determined by the Lowry procedure using BSA as a standard [ 131. The affinity of the enzyme (K,) and the maximum velocity of the reaction (V,) were calculated from Lineweaver-Burk plots.
Proceedings of the 8th International Symposium of The Journal of Steroid Biochemistry “Recent Advances Steroid Biochemistry” (Paris, 24-27 May 1987). *To whom correspondence should be addressed.
in
RESULTS Steroid sulfatase kinetic parameters 439
were calcu-
MONIQUE BEDIN et al.
440 Table 1. Ontogenesis
of STS activity
in purified Leydig cells and seminiferous tubules during postnatal constants are calculated as shown in Fig. 1
Leydig cells
Seminiferous
development.
Kinetic
tubules
Age (days) 20 28 36 42 Adult
K, ( 1O-6
M)
2.30 13.72 9.40 7.70 4.04
V, (nmol/mg protein per 40 min)
K,,,
(lo+
0.35 2.03 1.61 1.56 1.50
lated from double reciprocal plots according to the Lineweaver-Burk representation. The effect of increasing concentrations of DHA-S on the enzymatic activity in homogenates of purified Leydig cells is typically shown in Fig. 1. The mean +-S.E.M. apparent K,,, and V,,,values calculated from three separate experiments were 4.31 ?0.34pM and 1.58-t0.17 nmol/mg protein per 40 min, respectively. The variation of K,,, and V,,, values for homogenates of both Leydig cells and seminiferous tubules as a function of stage of development is reported in Table 1. These results show that the enzyme affinity increased from prepubertal to adult stages in both purified preparations and was always higher in Leydig cells than in seminiferous tubules. Data also indicate that the activity was mainly localized in seminiferous tubule preparation at the earlier stages of development (20-28 days). No marked difference was observed at the later stages. The effect of in vivo hCG treatment (100 II-J) on kinetic constants of testicular steroid sulfatase was subsequently studied. K,,, and V,,, values were simultaneously determined in control and hCGtreated rats. Results from three separate experiments are presented in Fig. 2. This clearly shows that hCG treatment highly reduced the enzyme affinity in Leydig cell homogenates (left panel) as indicated by the 2-3 times increase in the K,,, values: the means -t S.E.M. were 4.16+0.72pM and 9.53+0.47pM in control and hCG-treated rats respectively (PtO.O1). Enhancement of enzymatic activity was concomitantly observed. V, values (mean-t S.E.M.) rose from 1.75 * 0.25 in controls to 3.46~ 0.17 nmol/mg protein per 40 min in hCG-treated males (PtO.O1). Similar determinations performed in seminiferous tubules of control and hCG-treated animals (right panel) indicate that this treatment did not affect the kinetic constants of the enzyme. The means? S.E.M. apparent K,,, @M) and V,,, (nmol/mg protein per 40 min) values were respectively, 9.87 * 0.23 and 0.83 ? 0.13, in controls and 9.95 it_0.62 and 0.83 k 0.15 in hCGtreated rats.
DISCUSSION
The present study clearly demonstrates that Leydig
V, (nmol/mg protein per 40 min)
M)
11.35 22.42 17.67 12.35 9.69
_..-•
f -L
I
I
Q5
0
I/
3.98 4.16 2.19 1.09 1.09
(VDHA-S)
4-n
I
I
1.5
2
I 2.5
x IO6 M
Fig. 1. Typical Lineweaver-Burk plot of the effect of increasing concentrations (0.4-l .6 x 10e6 M) of substrate on the rate of DHA-S hydrolysis in purified adult rat Leydig cells.
Leydig cells
6
Seminiferous tubules
Kl
% '0
(D
=
2
Fig. 2. Influence of hCG treatment (hatched bars) on STS kinetic constants in purified Leydig cells (left panel) and in seminiferous tubules (right panel) as compared with salineinjected males (open bars). *PtO.O 1.
cells isolated from rat testis have the capacity to cleave the sulfate moiety of DHA-S which is known to be a natural substrate for steroid sulfatase in other rat tissues [ 141. Previous works reported that the expression of STS activity in crude interstitial cells prepara-
Steroid sulfatase in rat Leydig cells
tion could be due to the presence of cells from seminiferous tubules, since higher enzymatic activity [lo] was present in this latter testicular compartment [ 151. Our current observation that STS activity is found in purified Leydig cells is the proof that this cell type is able to convert sulfoconjugated into free steroids. As Leydig cells are the main site of testosterone production, the physiological significance of this enzyme in androgen biosynthesis must be reconsidered. In the human the existence of a good correlation between testicular sulfatase, 38-hydroxysteroid dehydrogenase and serum testosterone levels suggest that steroid sulfates may be precursors of testosterone in the testis [4, 161. Although such a relationship might be more questionable in the rat, due to the possible absence of sulfoconjugated steroids in testicular tissue [ 171, our results support the likelihood of steroid sulfatase being implicated in testicular testosterone synthesis in this species. Indeed, we observed a higher affinity of STS towards its substrate in Leydig cells than in seminiferous tubules, together with an increase of this affinity from prepubertal to adult stages. In addition, the kinetic parameters of the enzyme expressed in Leydig cells were modulated by hCG treatment whereas no such modifications occurred in seminiferous tubules. The consistent increase in apparent K,,, values noticed in Leydig cells 2 h after hCG injection could be due to competitive inhibition by free steroids [7], production of which is known to increase after gonadotrophin stimulation. It is unknown at present whether the concomitant increase of V,,, observed after hCG treatment results from induction of new enzyme synthesis or is due to other phenomena. The possibility that hCG could regulate the action of inhibitory or stimulatory effecters on the expression of the enzyme must also be considered since the presence of such effecters has been documented in other tissues [ 181. Although the contribution of steroid sulfates to testosterone biosynthesis is usually reported to be small, our observation that STS kinetic constants change with development and that hCG can modulate these parameters in Leydig cells only, strongly favours the potential regulating role of this enzyme in testicular testosterone production.
Acknowledgements-We
wish to thank Dr Cedard for helpful comments, Dr Ehrhard (Organon) for the gift of hCG and Mrs Verger for careful preparation of the manuscript.
REFERENCES
1. Ruokonen A., Laatikainen T., Laitinen E. A. and Vihko R.: Free and sulfate-conjugated neutral steroids in human. Biochemistry 11 (1972) 141 l-1416.
SB 30:1/6-BB
441
2. Laatikainen T., Laitinen E. A. and Vihko R.: Secretion of free and sulfate-conjugated neutral steroids by the human testis. Effect of administration of human chorionic gonadotropin. J. clin. Endocr. Metab. 32 (1971) 59-64. 3. Bolton N. J., Ruokonen A. 0. and Vihko R. K.: Stimulation of the synthesis of steroids and steroid sulphates in human testicular tissue in vitro by hCG and by &bromo-cyclic AMP. J. steroid Biochem. 22 (1985) 481-485. 4. Ruokonen A.: Steroid metabolism in testis tissue: the metabolism of pregnenolone, pregnenolone sulfate, dehydroepiandrosterone and dehydroepiandrosterone sulfate in human and boar testes in vitro. J. steroid Biochem. 9 (1978) 939-946.
5. Payne A. H., Mason M. and Jaffe R. B.: Testicular steroid sulfatase substrate specificity and inhibition. Steroids 14 (1969) 685-704.
6. Payne A. H. and Jaffe R. B.: Comparative role of dehydroepiandrosterone sulfate and androstenediolsulfate as precursors of testicular androgens. Endocrinology87 (1970) 316-322.
7. Notation A. D. and Ungar F.: Rat testis steroid sulfatase: 2. Kinetic study. Steroids 14 (1969) 15 1- 159. 8. Haour F. and Saez J. M.: hCG-Dependent regulation of gonadotropin receptor sites: negative control in testicular Leydig cells. Molec. Cell Endocr. 7 (1977) 17-24. 9. Rao B., Pointis G. and Cedard L.: Presence of chorionic gonadotrophin-like factor in mouse placental culture during the second half of gestation. J. Reprod. Fert. 66 (1982) 341-348.
10. Browning J. Y., D’Agata R. and Grotjan H. E., Jr: Isolation of purified rat Leydig cells using continuous Percoll gradients. Endocrinology 109 (198 1) 667-669. 11. Pointis G. and Latreille M. T.: Regulation of testosterone production in Leydig cells from fetal mice under dynamic conditions: effect of human chorionic gonadotronhin and 8 bromo-cyclic AMP. J. Endocr. 107 (1985), 409-414. 12. Burstein S. and Dorfman R. I.: Determination of mammalian steroid sulfatase with 7o-[‘H]3/?-hydroxyandrost-5-en-17-one sulfate. J. biol. Chem. 43 (1963) 1656-1660. 13. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193 (195 1) 256-275. 14. Verde A. and Drucker W. D.: Distribution of dehydroepiandrosterone sulfate sulfatase in rat tissue. Endocrinology
(1972) 138-143.
15. Payne A. H., Kawano A., Jaffe R. B.: Formation of dehydro-epiandrosterone and other So-reduced metabolites by isolated seminiferous tubules and suspension of interstitial cells in human testis. J. clin. Endocr. Metab. 37 (1973) 448-453. 16. Payne A. H., Jaffe R. B. and Abel1 M. R.: Gonadal steroid sulfates and sulfatase. III. Correlation of human testicular sulfatase, 3&hydroxysteroid dehydrogenaseisomerase, histologic structure and serum testosterone. J. clin. Endocr. 33 (1971) 582-591.
17. Ruokonen A., Vihko R. and Niemi H.: Steroid metabolism in testis tissue. Concentrations of testosterone, pregnenolone and Sct-androst-l6-en-3~-ol in normal and cryptorchid rat testis, and isolated interstitial and tubular tissue. FEBS Left. 31 (1973) 321-323. 18. Moutaouakkil M. and Adessi G.: Dehydroepiandrosterone sulfatase inhibitor: identification and characterization in soluble proteins of guinea-pig liver. Steroids 47 (1986) 401-411.
1988
0022-4731/88
$3.00+0.00
Pergamon
Press
plc
ONTOGENESIS AND REGULATION OF STEROID SULFATASE ACTIVITY IN LEYDIG CELLS AND SEMINIFEROUS TUBULES IN THE LONG-EVANS RAT MONIQUE BEDIN*, INSERM
TH~RBSE FOURNIER, NADIA MOUHADJER
and
U. 166, Groupe de Recherches SW I’Endocrinologie de la Reproduction, Bld de Port-Royal, 750 14 Paris, France
GEORGES POINTIS Maternitk Baudelocque,
123,
Summary-Steroid sulfatase (STS) activity was studied in Long-Evans rat testis. The affinity of the enzyme was shown to increase during postnatal development and to be always higher in purified Leydig cells than in seminiferous tubules. STS activity appeared to be higher in the seminiferous tubules at the earlier stages. In vivo injection of 100 IU hCG resulted in a decrease in the affinity and an increase in the activity of the enzyme expressed in Leydig cells with no such modification in seminiferous tubules. This suggests that STS could play a regulatory role in testosterone production by Leydig cells.
INTRODUCTION
In addition to testosterone, large amounts of steroid sulfates have been measured in the testis of some species including man [ 11. In vitro experiments have shown that this tissue is able to both synthesize and hydrolyze these sulfoconjugated steroids and that their secretion is stimulated by hCG-treatment [2,3]. From these observations it has been suggested that these compounds may be used as androgen precursors [4]. Using the rat as an experimental model, previous studies have led us to consider that testicular steroid sulfatase (STS) may act as a regulatory enzyme for the release of free androgens [S, 6, 71. Since testosterone is mainly synthesized in the Leydig cells and no information is available about the possible role of STS in this production, we have investigated the ontogenesis and the effect of hCG on this enzyme in purified Leydig cells derived from Long-Evans rats. EXPERIMENTAL
Animals and treatments Long-Evans male rats, at different stages of postnatal development (from 20 days to adult stage), were used for the ontogenesis study. When the effect of hCG was studied, adult male rats were injected intramuscularly with 100 IU hCG (Organon) diluted in saline containing 0.1% BSA. Control animals were treated with the vehicle alone. Both hCG-treated and control rats were killed 2 h after injection, a lag period resulting into maximal increase in circulating testosterone levels [8]. Isolation of seminiferous tubules and purified Leydig cells After sacrifice, testes were removed and, once
decapsulated, submitted to collagenase digestion in 10 ml medium 199 (Eurobio, Paris) containing 10 mg BSA, 3 mg collagenase type I (ICN Pharmaceuticals Inc., Cleveland, OH) and 4Opg DNAase I (Sigma Chemical Company, St Louis, MO) and processed as previously reported [9]. Seminiferous tubules were allowed to settle and were washed 3 times with 0.9% NaCl so as to eliminate interstitial cells. After filtration through a nylon gauze (pore size 140pm) interstitial cells were collected by centrifugation at 15Og for 15 min and fractionated afterwards by density gradient centrifugation in Percoll as described [lo]. This cell separation allowed us to obtain about 90% of pure Leydig cells identified by staining for 38HSD activity [ 1 I]. Determination of steroid sulfa&se activity Purified Leydig cells and seminiferous tubules free of interstitial cells were homogenized in 0.1 M Tris-HCI, pH 7.2, and sonicated to give complete disruption of the cells. Steroid sulfatase activity was determined as earlier reported [ 121. Aliquots of the suspension were incubated in duplicate at 37°C in the presence of labeled dehydroepiandrosterone-sulfate ([3H]DHA-S, sp. act. 24 Ci/mmol, NEN Chemicals, F.R.G.) at variable final concentrations in a total reaction volume of 250 ~1. The incubations were stopped by addition of 0.1 N NaOH and the yield of liberated unconjugated steroids was measured after direct extraction by the scintillation mixture (Toluen-PPO-POPOP) Results were corrected for methodological losses. The reaction rates were linear within the incubation time and the protein range employed. Protein concentration was determined by the Lowry procedure using BSA as a standard [ 131. The affinity of the enzyme (K,) and the maximum velocity of the reaction (V,) were calculated from Lineweaver-Burk plots.
Proceedings of the 8th International Symposium of The Journal of Steroid Biochemistry “Recent Advances Steroid Biochemistry” (Paris, 24-27 May 1987). *To whom correspondence should be addressed.
in
RESULTS Steroid sulfatase kinetic parameters 439
were calcu-
MONIQUE BEDIN et al.
440 Table 1. Ontogenesis
of STS activity
in purified Leydig cells and seminiferous tubules during postnatal constants are calculated as shown in Fig. 1
Leydig cells
Seminiferous
development.
Kinetic
tubules
Age (days) 20 28 36 42 Adult
K, ( 1O-6
M)
2.30 13.72 9.40 7.70 4.04
V, (nmol/mg protein per 40 min)
K,,,
(lo+
0.35 2.03 1.61 1.56 1.50
lated from double reciprocal plots according to the Lineweaver-Burk representation. The effect of increasing concentrations of DHA-S on the enzymatic activity in homogenates of purified Leydig cells is typically shown in Fig. 1. The mean +-S.E.M. apparent K,,, and V,,,values calculated from three separate experiments were 4.31 ?0.34pM and 1.58-t0.17 nmol/mg protein per 40 min, respectively. The variation of K,,, and V,,, values for homogenates of both Leydig cells and seminiferous tubules as a function of stage of development is reported in Table 1. These results show that the enzyme affinity increased from prepubertal to adult stages in both purified preparations and was always higher in Leydig cells than in seminiferous tubules. Data also indicate that the activity was mainly localized in seminiferous tubule preparation at the earlier stages of development (20-28 days). No marked difference was observed at the later stages. The effect of in vivo hCG treatment (100 II-J) on kinetic constants of testicular steroid sulfatase was subsequently studied. K,,, and V,,, values were simultaneously determined in control and hCGtreated rats. Results from three separate experiments are presented in Fig. 2. This clearly shows that hCG treatment highly reduced the enzyme affinity in Leydig cell homogenates (left panel) as indicated by the 2-3 times increase in the K,,, values: the means -t S.E.M. were 4.16+0.72pM and 9.53+0.47pM in control and hCG-treated rats respectively (PtO.O1). Enhancement of enzymatic activity was concomitantly observed. V, values (mean-t S.E.M.) rose from 1.75 * 0.25 in controls to 3.46~ 0.17 nmol/mg protein per 40 min in hCG-treated males (PtO.O1). Similar determinations performed in seminiferous tubules of control and hCG-treated animals (right panel) indicate that this treatment did not affect the kinetic constants of the enzyme. The means? S.E.M. apparent K,,, @M) and V,,, (nmol/mg protein per 40 min) values were respectively, 9.87 * 0.23 and 0.83 ? 0.13, in controls and 9.95 it_0.62 and 0.83 k 0.15 in hCGtreated rats.
DISCUSSION
The present study clearly demonstrates that Leydig
V, (nmol/mg protein per 40 min)
M)
11.35 22.42 17.67 12.35 9.69
_..-•
f -L
I
I
Q5
0
I/
3.98 4.16 2.19 1.09 1.09
(VDHA-S)
4-n
I
I
1.5
2
I 2.5
x IO6 M
Fig. 1. Typical Lineweaver-Burk plot of the effect of increasing concentrations (0.4-l .6 x 10e6 M) of substrate on the rate of DHA-S hydrolysis in purified adult rat Leydig cells.
Leydig cells
6
Seminiferous tubules
Kl
% '0
(D
=
2
Fig. 2. Influence of hCG treatment (hatched bars) on STS kinetic constants in purified Leydig cells (left panel) and in seminiferous tubules (right panel) as compared with salineinjected males (open bars). *PtO.O 1.
cells isolated from rat testis have the capacity to cleave the sulfate moiety of DHA-S which is known to be a natural substrate for steroid sulfatase in other rat tissues [ 141. Previous works reported that the expression of STS activity in crude interstitial cells prepara-
Steroid sulfatase in rat Leydig cells
tion could be due to the presence of cells from seminiferous tubules, since higher enzymatic activity [lo] was present in this latter testicular compartment [ 151. Our current observation that STS activity is found in purified Leydig cells is the proof that this cell type is able to convert sulfoconjugated into free steroids. As Leydig cells are the main site of testosterone production, the physiological significance of this enzyme in androgen biosynthesis must be reconsidered. In the human the existence of a good correlation between testicular sulfatase, 38-hydroxysteroid dehydrogenase and serum testosterone levels suggest that steroid sulfates may be precursors of testosterone in the testis [4, 161. Although such a relationship might be more questionable in the rat, due to the possible absence of sulfoconjugated steroids in testicular tissue [ 171, our results support the likelihood of steroid sulfatase being implicated in testicular testosterone synthesis in this species. Indeed, we observed a higher affinity of STS towards its substrate in Leydig cells than in seminiferous tubules, together with an increase of this affinity from prepubertal to adult stages. In addition, the kinetic parameters of the enzyme expressed in Leydig cells were modulated by hCG treatment whereas no such modifications occurred in seminiferous tubules. The consistent increase in apparent K,,, values noticed in Leydig cells 2 h after hCG injection could be due to competitive inhibition by free steroids [7], production of which is known to increase after gonadotrophin stimulation. It is unknown at present whether the concomitant increase of V,,, observed after hCG treatment results from induction of new enzyme synthesis or is due to other phenomena. The possibility that hCG could regulate the action of inhibitory or stimulatory effecters on the expression of the enzyme must also be considered since the presence of such effecters has been documented in other tissues [ 181. Although the contribution of steroid sulfates to testosterone biosynthesis is usually reported to be small, our observation that STS kinetic constants change with development and that hCG can modulate these parameters in Leydig cells only, strongly favours the potential regulating role of this enzyme in testicular testosterone production.
Acknowledgements-We
wish to thank Dr Cedard for helpful comments, Dr Ehrhard (Organon) for the gift of hCG and Mrs Verger for careful preparation of the manuscript.
REFERENCES
1. Ruokonen A., Laatikainen T., Laitinen E. A. and Vihko R.: Free and sulfate-conjugated neutral steroids in human. Biochemistry 11 (1972) 141 l-1416.
SB 30:1/6-BB
441
2. Laatikainen T., Laitinen E. A. and Vihko R.: Secretion of free and sulfate-conjugated neutral steroids by the human testis. Effect of administration of human chorionic gonadotropin. J. clin. Endocr. Metab. 32 (1971) 59-64. 3. Bolton N. J., Ruokonen A. 0. and Vihko R. K.: Stimulation of the synthesis of steroids and steroid sulphates in human testicular tissue in vitro by hCG and by &bromo-cyclic AMP. J. steroid Biochem. 22 (1985) 481-485. 4. Ruokonen A.: Steroid metabolism in testis tissue: the metabolism of pregnenolone, pregnenolone sulfate, dehydroepiandrosterone and dehydroepiandrosterone sulfate in human and boar testes in vitro. J. steroid Biochem. 9 (1978) 939-946.
5. Payne A. H., Mason M. and Jaffe R. B.: Testicular steroid sulfatase substrate specificity and inhibition. Steroids 14 (1969) 685-704.
6. Payne A. H. and Jaffe R. B.: Comparative role of dehydroepiandrosterone sulfate and androstenediolsulfate as precursors of testicular androgens. Endocrinology87 (1970) 316-322.
7. Notation A. D. and Ungar F.: Rat testis steroid sulfatase: 2. Kinetic study. Steroids 14 (1969) 15 1- 159. 8. Haour F. and Saez J. M.: hCG-Dependent regulation of gonadotropin receptor sites: negative control in testicular Leydig cells. Molec. Cell Endocr. 7 (1977) 17-24. 9. Rao B., Pointis G. and Cedard L.: Presence of chorionic gonadotrophin-like factor in mouse placental culture during the second half of gestation. J. Reprod. Fert. 66 (1982) 341-348.
10. Browning J. Y., D’Agata R. and Grotjan H. E., Jr: Isolation of purified rat Leydig cells using continuous Percoll gradients. Endocrinology 109 (198 1) 667-669. 11. Pointis G. and Latreille M. T.: Regulation of testosterone production in Leydig cells from fetal mice under dynamic conditions: effect of human chorionic gonadotronhin and 8 bromo-cyclic AMP. J. Endocr. 107 (1985), 409-414. 12. Burstein S. and Dorfman R. I.: Determination of mammalian steroid sulfatase with 7o-[‘H]3/?-hydroxyandrost-5-en-17-one sulfate. J. biol. Chem. 43 (1963) 1656-1660. 13. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193 (195 1) 256-275. 14. Verde A. and Drucker W. D.: Distribution of dehydroepiandrosterone sulfate sulfatase in rat tissue. Endocrinology
(1972) 138-143.
15. Payne A. H., Kawano A., Jaffe R. B.: Formation of dehydro-epiandrosterone and other So-reduced metabolites by isolated seminiferous tubules and suspension of interstitial cells in human testis. J. clin. Endocr. Metab. 37 (1973) 448-453. 16. Payne A. H., Jaffe R. B. and Abel1 M. R.: Gonadal steroid sulfates and sulfatase. III. Correlation of human testicular sulfatase, 3&hydroxysteroid dehydrogenaseisomerase, histologic structure and serum testosterone. J. clin. Endocr. 33 (1971) 582-591.
17. Ruokonen A., Vihko R. and Niemi H.: Steroid metabolism in testis tissue. Concentrations of testosterone, pregnenolone and Sct-androst-l6-en-3~-ol in normal and cryptorchid rat testis, and isolated interstitial and tubular tissue. FEBS Left. 31 (1973) 321-323. 18. Moutaouakkil M. and Adessi G.: Dehydroepiandrosterone sulfatase inhibitor: identification and characterization in soluble proteins of guinea-pig liver. Steroids 47 (1986) 401-411.