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Stimulation of ornithine decarboxylase activity by luteinizing hormone in rat testicular interstitial cells in vitro is age dependent
Pergamon Press
Life Sciences, Vol. 33, pp. 353-357 Printed in the U.S.A.
STIMULATION OF ORNITHINE DECARBOXYLASE ACTIVITY BY LUTEINIZING HORMONE IN RAT TESTICULAR INTERSTITIAL CELLS IN VITRO - IS AGE DEPENDENT J. Osterman, D. Barnett, E.P. Murono, T. Lin and H.R. Nankin Medical Service, Wm. Jennings Bryan Dorn Veterans' Hospital; Department of Medicine, University of South Carolina School of Medicine Columbia, South Carolina 29208 (Received in final form May 9, 1983)
Summary The developmental pattern of ornithine decarboxylase (ODC) responsiveness to luteinizing hormone (LH) in isolated rat testicular interstitial cells in vitro was examined and correlated with testosterone production-by the same cells. LH caused a 60100% stimulation of ODC activity in cells from 60-day-old rats but produced no response in cells from 30, 37, 41, 50 and 55-day-old animals. Interstitial cells from 25-day-old rats responded with a moderate (40%) but statistically significant enhancement of ODC activity to the highest LH dose (100.0 ng/ml) only. Testosterone production by control cells was low until day 41 (0.15-0.30 ng/106 cells per 4 h), and then markedly increased to adult levels (2.12 + 0.03 106 cells per 4 h). LH in all concentrations (O.l - 100.0 ng/ml) employed caused a consistent 4 to 7-fold stimulation of testosterone production in interstitial cells at all ages studied. This study shows age-dependent stimulation by LH of ODC activity in rat testicular interstitial cells in vitro and no apparent correlation with testosterone production by the same cells. We have recently demonstrated a specific and dose-dependent stimulation by luteinizing hormone (LH) of adult rat testicular interstitial cell and purified Leydig cell ornithine decarboxylase (ODC) activity in vitro, the ratelimiting enzyme of polyamine biosynthesis (l). This stimulation occurs with physiological doses of LH that also augment testicular steroidogenesis (2). Developmental changes of ODC activity in the whole rat testis in vivo have been described and they follow a characteristic pattern (1,3). The purpose of this study was to examine the developmental pattern of ODC activity and its responsiveness to LH stimulation in isolated testicular interstitial cells in vitro and to determine how it correlates with testosterone production by the-same cells. ~~terials
and Methods
Materials: Ovine LH (NIH-LH-S23) was obtained from the Hormone Distribution Office (NIAMMD, NIH, Bethesda, Maryland). Collagenase (Type I), and 3isobutyl-l-methylxanthine were purchased from Sigma Chemical Company, St. Louis, Missouri. L-[1-14Cl Ornithine monohydrochloride (sp. radioactivity, 58.9 mCi/ mmol) was obtained from New England Nuclear Corporation, Boston, Massachusetts.
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
354
Luteinizing Hormone and ODe Activity
Vol. 33, No.4, 1983
Culture ~Iedium 199 with Hank's salts and 25 mM Hepes [4-(2-hydroxyethyl)-1piperazine-ethanesulphonic acid] buffer (pH 7.4) was from Gibco, Grand Island, New York. Animals and Tissue: Male Sprague-Dawley rats (25-60 days old) were purchased from Zivic-Miller Laboratories, Inc., Allison Park, Pennsylvania. Testes were quickly removed from decapitated rats, decapsulated and kept on ice. Testicular interstitial cells were prepared by collagenase digestion as previously described (4). The incubation with collagenase was carried out for 15 mi n. Culture Conditions: Interstitial cells (2.1-4.2 x 107 viable cells, determined by Trypan-Blue exclusion) were incubated in 10 ml of ~Iedium 199 containing 1.0 mg/ml bovine serum albumin and 25 roM Ilepes buffer, pH 7.4 (culture medium) for 4 h at 34°C in an atmosphere of 02/C02 (95:5, v:v). LH, in concentrations as indicated was addea in the beginning of the incubation. 0.1 mM isobutyl-methylxanthine (dissolved in dimethyl sulfoxide) was added to both control and LH-treated cells in the total volume of 0.5 ul/ml culture medium. Following incubation, cells were recovered by centrifugation (at 600 xg), washed twice in ice-cold 25 mM Tris/HCl buffer, pH 7.4 containing 0.25M sucrose, 0.1 mM EDTA and 5.0 roM dithiothreitol, and then homogenized in 2.5 ml of the same buffer but without sucrose. The homogenates were centrifuged for 3D min at 20,000 xg and supernatants were used for the assay of DOC activity and protein concentration. Enzyme Assay: ODC activity was determined by measuring in duplicate the liberation of 14C0 2 from L-[1-14C] ornithine under conditions as previously described (5). The enzymatic activity was expressed as pmol of CO 2 released/mg cytosolic protein per 30 min. Cytosolic protein concentration was determined by the method of Lowry et al (6) as modified by Munro and Fleck (7), with bovine serum albumin as the standard. Testosterone Radioimmunoassay: Testosterone was measured by radioimmunoassay on ether-extracted aliquots of incubation medium without prior chromatography, following procedures which have been described previously (4). Results are expressed as ng/106 cells per 4 h. Results were analyzed by Student's t test. Results Developmental pattern of LH stimulation of ODC activity in testicular interstitial cells. Because of the rather small number of interstitial cells in rat testes before 25th postnatal day (8), we have limited this study to the age period of 25-60 days. As shown in Table I, the highest dose of LH caused a moderate (40%) but statistically significant stimulation of ODC activity in cells from 25-day-old rats. The hormone caused no stimulation of the enzyme activity in cells from 30-55-day-old rats. All concentrations of LH employed effectively stimulated ODC activity in cells from 60-day-old rats. Developmental pattern of testosterone stimulation by LH in testicular interstitial cells. Testosterone production by control cells was low (0.15 0.31 ng/106 cells per 4 h) before 41st postnatal day and then increased by about 7-fold to adult range (60-day-old). LH caused a consistent 4 to 7-fold stimulaation of testosterone production at all ages studied and with all concentrations of the hormone employed (Table II).
Luteinizing Hormone and ODe Activity
Vol. 33, No.4, 1983
355
TABLE I Developmental Pattern of LH Stimulation of DOC Activity Age (days) 25 30 37 41 50 55 60
ODC Activity (pmol/mg per 30 min) Control LH (ng/ml) 0.1 1.0
100.0
107.63 + 2.90 35.93 + 1.50 11.18+0.10 9.03 + 0.38 12.91 + 0.81 19.42 + 0.65 48.67 ~ 4.59*
122.45 + 4.44* 35.99 + 2.26 10.05 + 0.53 8.55 :;:- 0.49 12.11 + 1.34 21.42 + 1. 57 38.65 + 1.14*
87.39 31.90 10.87 -9. 74 11.15 20.51 23.74
+ 5.25 + 1.22 + 0.59 + 0.32 + 1.06 +0.96 ~ 0.60
98.41 28.64 12.11 9.73 14.39 19.76 41.17
+ 2.67
+ + + + + +
0.43 0.50 0.83 0.44 0.66 0.40*
Testicular interstitial cells were isolated from 5-10 rats of specified age and pooled. They were incubated for 4 h as described in the Methods Section. Each result is a mean ~ SEM of 3 incubations. *p
TABLE II Developmental Pattern of Testosterone Stimulation by LH in Testicular Interstitial Cells Testosterone (ng/l0 6 cells per 4 h) Age (days) 25 30 37 41 50 55 60
Control .1 0.26 0.15 0.21 0.31 0.73 0.98 2.12
+ 0.04
+ + + + + +
0.01 0.01 0.01 0.03 0.02 0.03
0.92 + 0.02 0.76 + 0.02 1.24 + 0.01 2.51+0.10 5.41 + 0.17 4.56+0.06 13.65 ~ 0.35
LH (ng/m1) 1.0 0.93 0.84 1.30 2.71 5.79 5.33 12.86
100.0
+ 0.03
+ + + + + +
0.01 0.05 0.10 0.17 0.10 0.10
1.06 0.86 1.40 2.80 5.97 6.05 14.55
+ + + + + +
0.06 0.01 0.03 0.12 0.25 0.07 ~ 0.20
Testicular interstitial cells were isolated from 5-10 rats at specified ages and pooled. The cells were incubated for 4 h as described in the Method Section. Results are means + SEM of 3 incubation. All experimental cultures differ from respective age controls by p
356
Luteinizing Hormone and ODe Activity
Vol. 33, No.4, 1983
cells in long-term cultures. The role of enhanced polyamine biosynthesis during postnatal development of spermatogenesis and steroidogenesis in mammalian testis remains poorly understood. Several studies examined morphological and biochemical changes in the rat testis and Leydig cells during sexual maturation (8,13-16). Testicular LH receptor concentration progressively increases between 15-38 days of age and plasma testosterone rose rapidly between days 35-55 (16). Isolated interstitial/Leydig cells from rats ranging in age from late fetal to adult responded to human chorionic gonadotropin stimulation in vitro with a 3 to 7-fold increase in testosterone production (8). Our results shown in Table II are consistent with these findings. In contrast, the responsiveness of ODC to LH stimulation in vitro apparently develops at the very end of sexual maturation (55-60 days of agera.s shown in Table 1. It will be of interest to examine the responsiveness of the enzyme to LH stimulation in interstitial cells at even earlier developmental stages « 25 days of age) since we observed moderate stimulation of ODC activity at age 25 days with the highest dose of LH. Recent studies have shown the existence of two populations of Leydig cells in rat testis (obtained during purification procedure) with different responsiveness to LH stimulation (17-19) and the shift in preponderance of Leydig cells from population I to population II during sexual maturation (20). ODC activity in such cell preparations and their hormonal responsiveness remains to be investigated. In conclusion, this study shows age-dependent stimulation by LH of ODC activity in rat testicular interstitial cells in vitro and no apparent correlation with testosterone production by the same cel~ Acknowledgements This research was supported by V.A. Institutional Research funds awarded to the Wm. Jennings Bryan Dorn Veterans' Hospital, and the National Institutes of Health/National Institute of Aging Grant 1 ROl AG 01217-03-REB. We thank Mrs. A. Martin for excellent secretarial assistance. References 1.
2: 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
J. OSTERMAN, E.P. MURONO, 1. LIN and H.R. 11ANKIN, J. Androl. i (1983), in press. . • J. OSTERMAN, E.P. MURONO, T. LIN and H.R. NANKIN, Biochem. Biophys. Res. Commun., 'in press. J.H. t1ACINDOE and R.W. TURKINGTON, Endocrinology 92 595-605 (1973). 1. LIN, E.P. MURONO, J. OSTERr1AN, P. TROEN and H.i['" NArlKIN, Internat. J. Androl. 2 549-558 (1979). J. OSTERMAN and J.M. HAMMOND, Endocrinology 101 1335-1338 (1977). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). H.N. r~UNRO and A. FLECK, r'1ammalian Protein t1etabolism, Vol. 3, P 423, Academic Press, New York (1969). G.F. PAZ, J.S.D. WINTER, F.I. REYES and C. FAlMAN, Steroids 36 675-688 (1980). P.R.K. REDDY and C.A. VILLEE, Biochem. Biophys. Res. Commun. 65 1350-1354 (1975). C.A. VILLEE and J.M. LORING, Structure and Function of Gonadotropins, --p 295, Plenum Press, New York and London-[1978). R. MADHUBALA and P.R.K. REDDY, FEBS Lett, 122 197-198 (1980). G.L. FRANCIS, T.J. TRICHE, T.J. BROWN, H.C~ROWN and B.B. BERCU, J. Androl. 2 312-320 (1981). M. NIEMI-and M. IKONEN, Endocrinology 72 443-448 (1963).
Vol. 33, No.4, 1983
Luteinizing Hormone and ODe Activity
357
14. D.H. LORDING and D.~1. DE KRETSER, J. Reprod. Fert. 29 261-269 (1972). 15. A.H. PAYNE, R.P. KELCH, LP. f·1URONO and J.T. KERLAN~J. Endocr. 72 17-26 (1977). 16. J.-M. KETELSLEGERS, W.D. HETZEL, R.T. SHERINS and K.J. CATT, Endocrinology 103 212-222 (1978). 17 . A.H. PAYNE, J.R. DOWNING and K.-L. WONG, Endocrinology 106 1424-1429 (1980). 18. G.C.C. CHEN, T. LIN, E.P. MURONO, J. OSTERf1AN and H.R. NANKIN, Steroids 37 63-72 (1981). 19. T. LIN, T.M. LINCOLN, N. BROWN, E.P. MURONO, J. OSTERMAN and H.R. NANKIN, Endocrinology III 1391-1393 (1982). 20. D.J. CHASE and A.H . PAYNE, Endocrinology 112 29-34 (1983).
Life Sciences, Vol. 33, pp. 353-357 Printed in the U.S.A.
STIMULATION OF ORNITHINE DECARBOXYLASE ACTIVITY BY LUTEINIZING HORMONE IN RAT TESTICULAR INTERSTITIAL CELLS IN VITRO - IS AGE DEPENDENT J. Osterman, D. Barnett, E.P. Murono, T. Lin and H.R. Nankin Medical Service, Wm. Jennings Bryan Dorn Veterans' Hospital; Department of Medicine, University of South Carolina School of Medicine Columbia, South Carolina 29208 (Received in final form May 9, 1983)
Summary The developmental pattern of ornithine decarboxylase (ODC) responsiveness to luteinizing hormone (LH) in isolated rat testicular interstitial cells in vitro was examined and correlated with testosterone production-by the same cells. LH caused a 60100% stimulation of ODC activity in cells from 60-day-old rats but produced no response in cells from 30, 37, 41, 50 and 55-day-old animals. Interstitial cells from 25-day-old rats responded with a moderate (40%) but statistically significant enhancement of ODC activity to the highest LH dose (100.0 ng/ml) only. Testosterone production by control cells was low until day 41 (0.15-0.30 ng/106 cells per 4 h), and then markedly increased to adult levels (2.12 + 0.03 106 cells per 4 h). LH in all concentrations (O.l - 100.0 ng/ml) employed caused a consistent 4 to 7-fold stimulation of testosterone production in interstitial cells at all ages studied. This study shows age-dependent stimulation by LH of ODC activity in rat testicular interstitial cells in vitro and no apparent correlation with testosterone production by the same cells. We have recently demonstrated a specific and dose-dependent stimulation by luteinizing hormone (LH) of adult rat testicular interstitial cell and purified Leydig cell ornithine decarboxylase (ODC) activity in vitro, the ratelimiting enzyme of polyamine biosynthesis (l). This stimulation occurs with physiological doses of LH that also augment testicular steroidogenesis (2). Developmental changes of ODC activity in the whole rat testis in vivo have been described and they follow a characteristic pattern (1,3). The purpose of this study was to examine the developmental pattern of ODC activity and its responsiveness to LH stimulation in isolated testicular interstitial cells in vitro and to determine how it correlates with testosterone production by the-same cells. ~~terials
and Methods
Materials: Ovine LH (NIH-LH-S23) was obtained from the Hormone Distribution Office (NIAMMD, NIH, Bethesda, Maryland). Collagenase (Type I), and 3isobutyl-l-methylxanthine were purchased from Sigma Chemical Company, St. Louis, Missouri. L-[1-14Cl Ornithine monohydrochloride (sp. radioactivity, 58.9 mCi/ mmol) was obtained from New England Nuclear Corporation, Boston, Massachusetts.
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
354
Luteinizing Hormone and ODe Activity
Vol. 33, No.4, 1983
Culture ~Iedium 199 with Hank's salts and 25 mM Hepes [4-(2-hydroxyethyl)-1piperazine-ethanesulphonic acid] buffer (pH 7.4) was from Gibco, Grand Island, New York. Animals and Tissue: Male Sprague-Dawley rats (25-60 days old) were purchased from Zivic-Miller Laboratories, Inc., Allison Park, Pennsylvania. Testes were quickly removed from decapitated rats, decapsulated and kept on ice. Testicular interstitial cells were prepared by collagenase digestion as previously described (4). The incubation with collagenase was carried out for 15 mi n. Culture Conditions: Interstitial cells (2.1-4.2 x 107 viable cells, determined by Trypan-Blue exclusion) were incubated in 10 ml of ~Iedium 199 containing 1.0 mg/ml bovine serum albumin and 25 roM Ilepes buffer, pH 7.4 (culture medium) for 4 h at 34°C in an atmosphere of 02/C02 (95:5, v:v). LH, in concentrations as indicated was addea in the beginning of the incubation. 0.1 mM isobutyl-methylxanthine (dissolved in dimethyl sulfoxide) was added to both control and LH-treated cells in the total volume of 0.5 ul/ml culture medium. Following incubation, cells were recovered by centrifugation (at 600 xg), washed twice in ice-cold 25 mM Tris/HCl buffer, pH 7.4 containing 0.25M sucrose, 0.1 mM EDTA and 5.0 roM dithiothreitol, and then homogenized in 2.5 ml of the same buffer but without sucrose. The homogenates were centrifuged for 3D min at 20,000 xg and supernatants were used for the assay of DOC activity and protein concentration. Enzyme Assay: ODC activity was determined by measuring in duplicate the liberation of 14C0 2 from L-[1-14C] ornithine under conditions as previously described (5). The enzymatic activity was expressed as pmol of CO 2 released/mg cytosolic protein per 30 min. Cytosolic protein concentration was determined by the method of Lowry et al (6) as modified by Munro and Fleck (7), with bovine serum albumin as the standard. Testosterone Radioimmunoassay: Testosterone was measured by radioimmunoassay on ether-extracted aliquots of incubation medium without prior chromatography, following procedures which have been described previously (4). Results are expressed as ng/106 cells per 4 h. Results were analyzed by Student's t test. Results Developmental pattern of LH stimulation of ODC activity in testicular interstitial cells. Because of the rather small number of interstitial cells in rat testes before 25th postnatal day (8), we have limited this study to the age period of 25-60 days. As shown in Table I, the highest dose of LH caused a moderate (40%) but statistically significant stimulation of ODC activity in cells from 25-day-old rats. The hormone caused no stimulation of the enzyme activity in cells from 30-55-day-old rats. All concentrations of LH employed effectively stimulated ODC activity in cells from 60-day-old rats. Developmental pattern of testosterone stimulation by LH in testicular interstitial cells. Testosterone production by control cells was low (0.15 0.31 ng/106 cells per 4 h) before 41st postnatal day and then increased by about 7-fold to adult range (60-day-old). LH caused a consistent 4 to 7-fold stimulaation of testosterone production at all ages studied and with all concentrations of the hormone employed (Table II).
Luteinizing Hormone and ODe Activity
Vol. 33, No.4, 1983
355
TABLE I Developmental Pattern of LH Stimulation of DOC Activity Age (days) 25 30 37 41 50 55 60
ODC Activity (pmol/mg per 30 min) Control LH (ng/ml) 0.1 1.0
100.0
107.63 + 2.90 35.93 + 1.50 11.18+0.10 9.03 + 0.38 12.91 + 0.81 19.42 + 0.65 48.67 ~ 4.59*
122.45 + 4.44* 35.99 + 2.26 10.05 + 0.53 8.55 :;:- 0.49 12.11 + 1.34 21.42 + 1. 57 38.65 + 1.14*
87.39 31.90 10.87 -9. 74 11.15 20.51 23.74
+ 5.25 + 1.22 + 0.59 + 0.32 + 1.06 +0.96 ~ 0.60
98.41 28.64 12.11 9.73 14.39 19.76 41.17
+ 2.67
+ + + + + +
0.43 0.50 0.83 0.44 0.66 0.40*
Testicular interstitial cells were isolated from 5-10 rats of specified age and pooled. They were incubated for 4 h as described in the Methods Section. Each result is a mean ~ SEM of 3 incubations. *p
TABLE II Developmental Pattern of Testosterone Stimulation by LH in Testicular Interstitial Cells Testosterone (ng/l0 6 cells per 4 h) Age (days) 25 30 37 41 50 55 60
Control .1 0.26 0.15 0.21 0.31 0.73 0.98 2.12
+ 0.04
+ + + + + +
0.01 0.01 0.01 0.03 0.02 0.03
0.92 + 0.02 0.76 + 0.02 1.24 + 0.01 2.51+0.10 5.41 + 0.17 4.56+0.06 13.65 ~ 0.35
LH (ng/m1) 1.0 0.93 0.84 1.30 2.71 5.79 5.33 12.86
100.0
+ 0.03
+ + + + + +
0.01 0.05 0.10 0.17 0.10 0.10
1.06 0.86 1.40 2.80 5.97 6.05 14.55
+ + + + + +
0.06 0.01 0.03 0.12 0.25 0.07 ~ 0.20
Testicular interstitial cells were isolated from 5-10 rats at specified ages and pooled. The cells were incubated for 4 h as described in the Method Section. Results are means + SEM of 3 incubation. All experimental cultures differ from respective age controls by p
356
Luteinizing Hormone and ODe Activity
Vol. 33, No.4, 1983
cells in long-term cultures. The role of enhanced polyamine biosynthesis during postnatal development of spermatogenesis and steroidogenesis in mammalian testis remains poorly understood. Several studies examined morphological and biochemical changes in the rat testis and Leydig cells during sexual maturation (8,13-16). Testicular LH receptor concentration progressively increases between 15-38 days of age and plasma testosterone rose rapidly between days 35-55 (16). Isolated interstitial/Leydig cells from rats ranging in age from late fetal to adult responded to human chorionic gonadotropin stimulation in vitro with a 3 to 7-fold increase in testosterone production (8). Our results shown in Table II are consistent with these findings. In contrast, the responsiveness of ODC to LH stimulation in vitro apparently develops at the very end of sexual maturation (55-60 days of agera.s shown in Table 1. It will be of interest to examine the responsiveness of the enzyme to LH stimulation in interstitial cells at even earlier developmental stages « 25 days of age) since we observed moderate stimulation of ODC activity at age 25 days with the highest dose of LH. Recent studies have shown the existence of two populations of Leydig cells in rat testis (obtained during purification procedure) with different responsiveness to LH stimulation (17-19) and the shift in preponderance of Leydig cells from population I to population II during sexual maturation (20). ODC activity in such cell preparations and their hormonal responsiveness remains to be investigated. In conclusion, this study shows age-dependent stimulation by LH of ODC activity in rat testicular interstitial cells in vitro and no apparent correlation with testosterone production by the same cel~ Acknowledgements This research was supported by V.A. Institutional Research funds awarded to the Wm. Jennings Bryan Dorn Veterans' Hospital, and the National Institutes of Health/National Institute of Aging Grant 1 ROl AG 01217-03-REB. We thank Mrs. A. Martin for excellent secretarial assistance. References 1.
2: 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
J. OSTERMAN, E.P. MURONO, 1. LIN and H.R. 11ANKIN, J. Androl. i (1983), in press. . • J. OSTERMAN, E.P. MURONO, T. LIN and H.R. NANKIN, Biochem. Biophys. Res. Commun., 'in press. J.H. t1ACINDOE and R.W. TURKINGTON, Endocrinology 92 595-605 (1973). 1. LIN, E.P. MURONO, J. OSTERr1AN, P. TROEN and H.i['" NArlKIN, Internat. J. Androl. 2 549-558 (1979). J. OSTERMAN and J.M. HAMMOND, Endocrinology 101 1335-1338 (1977). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). H.N. r~UNRO and A. FLECK, r'1ammalian Protein t1etabolism, Vol. 3, P 423, Academic Press, New York (1969). G.F. PAZ, J.S.D. WINTER, F.I. REYES and C. FAlMAN, Steroids 36 675-688 (1980). P.R.K. REDDY and C.A. VILLEE, Biochem. Biophys. Res. Commun. 65 1350-1354 (1975). C.A. VILLEE and J.M. LORING, Structure and Function of Gonadotropins, --p 295, Plenum Press, New York and London-[1978). R. MADHUBALA and P.R.K. REDDY, FEBS Lett, 122 197-198 (1980). G.L. FRANCIS, T.J. TRICHE, T.J. BROWN, H.C~ROWN and B.B. BERCU, J. Androl. 2 312-320 (1981). M. NIEMI-and M. IKONEN, Endocrinology 72 443-448 (1963).
Vol. 33, No.4, 1983
Luteinizing Hormone and ODe Activity
357
14. D.H. LORDING and D.~1. DE KRETSER, J. Reprod. Fert. 29 261-269 (1972). 15. A.H. PAYNE, R.P. KELCH, LP. f·1URONO and J.T. KERLAN~J. Endocr. 72 17-26 (1977). 16. J.-M. KETELSLEGERS, W.D. HETZEL, R.T. SHERINS and K.J. CATT, Endocrinology 103 212-222 (1978). 17 . A.H. PAYNE, J.R. DOWNING and K.-L. WONG, Endocrinology 106 1424-1429 (1980). 18. G.C.C. CHEN, T. LIN, E.P. MURONO, J. OSTERf1AN and H.R. NANKIN, Steroids 37 63-72 (1981). 19. T. LIN, T.M. LINCOLN, N. BROWN, E.P. MURONO, J. OSTERMAN and H.R. NANKIN, Endocrinology III 1391-1393 (1982). 20. D.J. CHASE and A.H . PAYNE, Endocrinology 112 29-34 (1983).