DOMESTICANIMAL ENDOCRINOLOGY
Vol. 7(2):273-276, 1990
SEXUAL DIMORPHIC PORCINE PITUITARY RESPONSE TO GROWTH HORMONE-RELEASING HORMONE M.L. Heiman, P.L. Surface, D.H. Mowrey and R.D. DiMarchi Lilly Research Laboratories Eli Lilly and Company Indianapolis, IN USA Received August 5, 1989
ABSTRACT The aim of this study was to compare growth hormone (GH) response of barrows and gilts to porcine growth hormone-releasing hormone (pGRH) at the pituitary level. Anterior pituitary cells from barrows and gilts responded to pGRH in a dose-dependent manner. The median effective pGP,H concentration (EC,o) which stimulated GH release from cells of barrows was greater (P < .05) than that for ceils obtained from intact female siblings. Maximal pGRH mediated GH secretion from barrows was not different (P > 0.05) than that from gilt stimulated cells. These data demonstrate that somatotrophs of growing peripubertal gilts are more responsive to pGRH stimulation than are cells from their castrated male siblings. This difference could be caused by castration of the neonatal male. INTRODUCTION A variety of strategies are being pursued to elevate circulating growth hormone (GH) levels in swine. One avenue is to increase endogenous GH levels by administering GH-releasing hormone (GRH). Recently, Dubreuil et al. (1) reported that the acute GH response to a GRH iv stimulus is less in barrows than in gilts. In order to determine w h e t h e r such differential responsiveness to GRH is manifest at the pituitary level, w e examined the GH response to GRH in adenohypophysial cells obtained from both castrated male and intact female pigs. We report that cells obtained from females were more sensitive to GRIt stimulation than were castrated males. MATERIALS AND METHODS Animals. Experimental swine used in this study were from the swine production herd of Eli Lilly (Greenfield, IN) and were a Yorkshire X Hampshire cross. Sixty pigs (from 120 days- to 145 days of age) were used. Swine were fed, a d ltbitum, a diet formulated to meet National Research Council recommendations. All animals were housed under controlled conditions (lights on from 0 6 0 0 hr to 1800 hr) and were allowed free access to water. Thirty males were castrated before two weeks of age. Thirty o f their female siblings were left intact. P i t u i t a r y Cell C u l t u r e . Anterior pituitary glands were removed, dispersed, and cu ltu r ed using an aseptic technique by the following modifications of a previously described m e t hod (2). Each of 5 experiments consisted of cells cultured from a p o ol of six pituitary glands obtained from either barrows or sibling gilts. Plated cells (2x105 cells/well) were maintained at 3"7 C for 96 hr in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) which Copyright © 1990 by DOMENDO, INC.
273
0739-7240/90/$3.00
274
HEIMAN, SURFACE, MOWREY AND DIMARCHI
was supplemented with 2.5% fetal calf serum (Gibco), 3% horse serum (Gibco), 1% MEM non-essential amino acids (Gibco), gentamicin (0.03 mg/ml), and nystatin (11U/ml; Gibco). GRH C h a l l e n g e . pGRH(1-44)OH (pGRH; stucture in ref. 3) was synthesized in our laboratory by standard solid-phase methodology (4) and was administered to triplicate wells at concentrations ranging from 0.1 pM to IIIM in 1 ml medium 199 containing 1% bovine serum albumin (fraction V; Sigma). After 3 hr at 37 C, media was removed and stored at -20 C until assayed for hormone content. Hormone Assay a n d Data Analysis. Media GH was measured by RLA (primary antiserum kindly supplied by Dr. D.N. Marple, Auburn University). Standards used were NIH p522A. [125I]-pGH was prepared by enzymatic iodination using the lactoperoxidase procedure (5). The iodinated pGH was purified by gel chromatography (Sephadex G-100). Specific activity of the tracer ranged between 28.5 ttCi/ug and 54.3 ~tCi/ug. When stored in a phosphate buffered saline at 4 C, the purified [t2~I]-pGH retained binding activity for 4 to 5 weeks. Competition for tracer binding to antiserum with bovine GH, human GH and chicken GH demonstrated no significant cross-reactivity. Displacement of [~25I]pGH binding to antiserum by addition of serial dilutions of either porcine plasma or media from porcine pituitary cell cultures was parallel to that observed by the pGH standard. The intra- and interassay coefficients of variation were 9% and 13%, respectively. Dose-response curves were analyzed by a fourparameter logistic model (6). Efficacy (response to an "infinite" dose) and potency (ECs0; median effective dose) values were generated for each of 5 replicates for both males and females. Data were pooled and analyzed by one way analysis of variance. RESULTS Cells from both sexes responded to pGRI-I in a dose-dependent manner. Mean potency response (ECs0) for GH release from castrate male ceils (8.6 __+ 1.3 nM; mean __+ SEM, 5 different cultures) was greater (P < .05; Table I) than that for intact female siblings (3.6 _.+ 1.3 nM, n----5). Intrinsic activity of pGRH was similar in cells obtained from either sex. Efficacy of maximal pGRH mediated GH secretion was not different for barrows (129.54 - 19.02 ng/ml) (P > 0.05) and gilts (124.92 - 19.02 ng/ml). While there was no difference (P > 0.05) in the varibility of this estimate between the two sexes, the range was 57.3 ng/ml to 188.1 ng/ml and 110.3 ng/ml to 170.0 ng/ml for gilts and barrows, respectively. DISCUSSION Our data demonstrate that somatotrophs of growing peripubertal gilts are more responsive to pGRI-I than are cells from their castrated male siblings. This enhanced responsiveness is a result of increased somatotroph sensitivity to pGRH (smaller ECso value) rather than a result of increased capacity of the somatotroph to secrete GH (similar efficacy values). The data suggest possible interaction of the gonads with the GRH-GH axis. Primary pituitary cells of reproductively mature male rats are more responsive to GRH stimulation than are cells from randomly cycling female rats (7). This sexual dimorphic response is in part a consequence of a greater percent of somatotrophs reported in adult male rats than in females (8). At the cellular
SEX DIFFERENTIATED RESPONSE TO GRH IN SWINE
275
T~eLe 1. MEANEmc.xCV(GH SECRETORYCAPXCITY)ANDPOa'~NCV(EC~0) FOR PITUITARYRESPONSESTO l'GRI'I(1-44)OH IN B~axows (i) XNOGILTS (F) | Sex m f
Mean ECso (ni)
n
Pr>F
Mean GH Secretory CaPacity (ng/ml)
n
Pr>F
8.6 3.6
5 5
0.028
129.5 124.9
5 5
.868
SEM 1.3 19.024 tStandard error of the mean (SEM) values were computed by Statistical Analysis System. level, the m a t u r e male rat s o m a t o t r o p h exhibits a greater c a p a c i t y to secrete G H (8, 9) a n d an e n h a n c e d sensitivity to GRH (8) than the f e m a l e s o m a t o t r o p h . This difference in GRH r e s p o n s i v e n e s s is not o b s e r v e d p r i o r to p u b e r t y (9). W e h r e n b e r g et al. ( 1 0 ) d e m o n s t r a t e d in v f v o that the r e s p o n s e to GRH in a d u l t m a l e rats is greater t h a n that o b s e r v e d in adult f e m a l e rats b u t no a p p a r e n t difference was r e p o r t e d in p r e p u b e r t a l animals. Castration ablates any sexual differentation to GRH ( 1 0 ) . This g o n a d e c t o m i z e d i n d u c e d a t t e n u t a t i o n o f m a l e GRH r e s p o n s i v e n e s s is e v i d e n t at the pituitary level. Ohlsson, Isaksson and Jansson ( 1 1 ) d e m o n s t r a t e d that p e r i f u s e d pituitaries f r o m p r e p u b e r t a l gonad e c t o m i e d m a l e rats release less G H in r e s p o n s e to GRH than do pituitaries f r o m s h a m - o p e r a t e d animals. This effect is m o r e p r o n o u n c e d w h e n the rats w e r e o r c h i e c t o m i z e d as n e o n a t e s t h a n w h e n castrated just p r i o r to p u b e r t y . Similar findings have b e e n r e p o r t e d in pigs. D u b r e v i l et al. ( 1 2 ) d e m o n s t r a t e d that the p l a s m a G H r e s p o n s i v e n e s s to GRH in m a l e piglets w h i c h w e r e castrated at 3 days o f age was significantly less than t h e i r intact c o u n t e r p a r t s . O u r findings are quite r e l e v a n t to these data since barrows u s e d w e r e castrated as neonates. I n d e e d , since these cells a p p e a r to reflect the p h y s i o l o g i c a l state o f the animal at the t i m e that the tissue was obtained, this in v i t r o system c o u l d b e u s e d as a m o d e l to study p h y s i o l o g i c a l r e g u l a t i o n o f pituitary responsiveness to GRH. In c o n c l u s i o n , these data d e m o n s t r a t e that s o m a t o t r o p h s f r o m b a r r o w s are less sensitive to p G R H t h a n cells o b t a i n e d f r o m gilt siblings. It is likely that r e m o v a l o f the testes o f the neonatal m a l e p i g is at least partly r e s p o n s i b l e for t h e s e differences. From a practical p e r s p e c t i v e , neonatal o r c h i e c t o m y in swine is r o u t i n e l y p e r f o r m e d to r e d u c e the o d o r o f c o o k e d b o a r meat.
ACKNOWLEDGEMENTS/FOOTNOT~ We would like to thank Tom Cain for technical assistance, NIADDKfor supply of pGH assay standard, Dr. G. Moore, M. Harris, D. Roland and Dr. D. J. Jones for advice, maintenance and handling of animals and Marlene Carlton for excellent secretarial assistance. Correspondence should be addressed to: M. L. Heiman, Lilly Research Laboratories P.O. Box 708, Building 268 Greenfield, IN 46140, USA
REFERENCES 1. Dubreuil P, Pelletier G, Petitclerc D, Lapierre H, Couture Y, Brazeau P, Gaudreau P, Morisset J. Influence of age and sex on basal secretion of growth hormone (GH) and on GH-induced release by porcine GH-releasing factor pGRH (1-29 NHz) in growing pigs. Domest Anita Endocrinol 4:299-307, 1987. 2. Heiman M L, Nekola M V, Murphy W A, Lance V A, Coy D H. An extremely sensitive in vitro model for elucidating structure-activity relationships of growth hormonereleasing factor analogs. Endocrinology 116:410-415. 1985.
276
HEIMAN, SURFACE, MOWREY AND DIMARCHI
3. Bohlen P, Esch F, Brazeau P, Ling N, Guillemin R. Isolation and characterization of the procine hypothalamic growth hormone releasing factor. Biochem Biophys Res commun 116:726-734, 1983. 4. Merrifield R B, Vizioli L D, Boman H G. Synthesis of the antibacterial peptide Cecropin A (1-33). Biochem 21:5020-5031, 1982. 5. Thorell J I, Johansson B G. Enzymatic iodination of polypeptides with ~2sI to high specific activity. Biochim Biophys Acta 251:363-369, 1971. 6. DeLean A, Munson P J, Rodbard D. Simultaneous analysis of families of sigmoidal curves:application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol 235:E97-E102, 1978. 7. Cronin M J, Rogol A D. Sex differences in the cyclic adenosine 3', 5'-monophosphate and growth hormone response to growth hormone releasing factor in vitro. Biol Repro 31:984-988, 1984. 8. Ho KY, Leong D A, Sinha Y N, Johnson M L, Evans W S, Thorner M O. Sex-related differences in GH secretion in rat using reverse hemolytic plaque assay. Am J Physiol 250 (Endocrinol Metab) 13:E650-E654, 1986. 9. Hoeffler J P, Frawley L S. Capacity of individual somatotropes to release growth hormone varies according to sex:analysis by reverse hemolytic plaque assay. Endocrinology 119:1037-1041, 1986. 10. Wehrenberg W B, Baird A, Ying S-Y, Ling N. The effects of testosterone and estrogen on the pituitary growth hormone response to growth hormone-releasing factor. Biol Repro 32:369-375, 1985. 11. Ohlsson L, Isaksoon O, Jansson J-O. Endogenous testosterone enhances growth hormone (GH)-releasing factor-induced GH secretion in vitro. J Endocr 113:249253, 1987. 12. Dubrevil P, Pelletier G, Couture Y, Lapierre H, Petitclere D, Morisset J, Gaudreau P, Brazeau P. Castration and testosterone effects on endogenous and somatocrinininduced growth hormone release in intact and castrated male pits. Domest Anita Endocrinol 6:15-24, 1989.
Vol. 7(2):273-276, 1990
SEXUAL DIMORPHIC PORCINE PITUITARY RESPONSE TO GROWTH HORMONE-RELEASING HORMONE M.L. Heiman, P.L. Surface, D.H. Mowrey and R.D. DiMarchi Lilly Research Laboratories Eli Lilly and Company Indianapolis, IN USA Received August 5, 1989
ABSTRACT The aim of this study was to compare growth hormone (GH) response of barrows and gilts to porcine growth hormone-releasing hormone (pGRH) at the pituitary level. Anterior pituitary cells from barrows and gilts responded to pGRH in a dose-dependent manner. The median effective pGP,H concentration (EC,o) which stimulated GH release from cells of barrows was greater (P < .05) than that for ceils obtained from intact female siblings. Maximal pGRH mediated GH secretion from barrows was not different (P > 0.05) than that from gilt stimulated cells. These data demonstrate that somatotrophs of growing peripubertal gilts are more responsive to pGRH stimulation than are cells from their castrated male siblings. This difference could be caused by castration of the neonatal male. INTRODUCTION A variety of strategies are being pursued to elevate circulating growth hormone (GH) levels in swine. One avenue is to increase endogenous GH levels by administering GH-releasing hormone (GRH). Recently, Dubreuil et al. (1) reported that the acute GH response to a GRH iv stimulus is less in barrows than in gilts. In order to determine w h e t h e r such differential responsiveness to GRH is manifest at the pituitary level, w e examined the GH response to GRH in adenohypophysial cells obtained from both castrated male and intact female pigs. We report that cells obtained from females were more sensitive to GRIt stimulation than were castrated males. MATERIALS AND METHODS Animals. Experimental swine used in this study were from the swine production herd of Eli Lilly (Greenfield, IN) and were a Yorkshire X Hampshire cross. Sixty pigs (from 120 days- to 145 days of age) were used. Swine were fed, a d ltbitum, a diet formulated to meet National Research Council recommendations. All animals were housed under controlled conditions (lights on from 0 6 0 0 hr to 1800 hr) and were allowed free access to water. Thirty males were castrated before two weeks of age. Thirty o f their female siblings were left intact. P i t u i t a r y Cell C u l t u r e . Anterior pituitary glands were removed, dispersed, and cu ltu r ed using an aseptic technique by the following modifications of a previously described m e t hod (2). Each of 5 experiments consisted of cells cultured from a p o ol of six pituitary glands obtained from either barrows or sibling gilts. Plated cells (2x105 cells/well) were maintained at 3"7 C for 96 hr in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) which Copyright © 1990 by DOMENDO, INC.
273
0739-7240/90/$3.00
274
HEIMAN, SURFACE, MOWREY AND DIMARCHI
was supplemented with 2.5% fetal calf serum (Gibco), 3% horse serum (Gibco), 1% MEM non-essential amino acids (Gibco), gentamicin (0.03 mg/ml), and nystatin (11U/ml; Gibco). GRH C h a l l e n g e . pGRH(1-44)OH (pGRH; stucture in ref. 3) was synthesized in our laboratory by standard solid-phase methodology (4) and was administered to triplicate wells at concentrations ranging from 0.1 pM to IIIM in 1 ml medium 199 containing 1% bovine serum albumin (fraction V; Sigma). After 3 hr at 37 C, media was removed and stored at -20 C until assayed for hormone content. Hormone Assay a n d Data Analysis. Media GH was measured by RLA (primary antiserum kindly supplied by Dr. D.N. Marple, Auburn University). Standards used were NIH p522A. [125I]-pGH was prepared by enzymatic iodination using the lactoperoxidase procedure (5). The iodinated pGH was purified by gel chromatography (Sephadex G-100). Specific activity of the tracer ranged between 28.5 ttCi/ug and 54.3 ~tCi/ug. When stored in a phosphate buffered saline at 4 C, the purified [t2~I]-pGH retained binding activity for 4 to 5 weeks. Competition for tracer binding to antiserum with bovine GH, human GH and chicken GH demonstrated no significant cross-reactivity. Displacement of [~25I]pGH binding to antiserum by addition of serial dilutions of either porcine plasma or media from porcine pituitary cell cultures was parallel to that observed by the pGH standard. The intra- and interassay coefficients of variation were 9% and 13%, respectively. Dose-response curves were analyzed by a fourparameter logistic model (6). Efficacy (response to an "infinite" dose) and potency (ECs0; median effective dose) values were generated for each of 5 replicates for both males and females. Data were pooled and analyzed by one way analysis of variance. RESULTS Cells from both sexes responded to pGRI-I in a dose-dependent manner. Mean potency response (ECs0) for GH release from castrate male ceils (8.6 __+ 1.3 nM; mean __+ SEM, 5 different cultures) was greater (P < .05; Table I) than that for intact female siblings (3.6 _.+ 1.3 nM, n----5). Intrinsic activity of pGRH was similar in cells obtained from either sex. Efficacy of maximal pGRH mediated GH secretion was not different for barrows (129.54 - 19.02 ng/ml) (P > 0.05) and gilts (124.92 - 19.02 ng/ml). While there was no difference (P > 0.05) in the varibility of this estimate between the two sexes, the range was 57.3 ng/ml to 188.1 ng/ml and 110.3 ng/ml to 170.0 ng/ml for gilts and barrows, respectively. DISCUSSION Our data demonstrate that somatotrophs of growing peripubertal gilts are more responsive to pGRI-I than are cells from their castrated male siblings. This enhanced responsiveness is a result of increased somatotroph sensitivity to pGRH (smaller ECso value) rather than a result of increased capacity of the somatotroph to secrete GH (similar efficacy values). The data suggest possible interaction of the gonads with the GRH-GH axis. Primary pituitary cells of reproductively mature male rats are more responsive to GRH stimulation than are cells from randomly cycling female rats (7). This sexual dimorphic response is in part a consequence of a greater percent of somatotrophs reported in adult male rats than in females (8). At the cellular
SEX DIFFERENTIATED RESPONSE TO GRH IN SWINE
275
T~eLe 1. MEANEmc.xCV(GH SECRETORYCAPXCITY)ANDPOa'~NCV(EC~0) FOR PITUITARYRESPONSESTO l'GRI'I(1-44)OH IN B~axows (i) XNOGILTS (F) | Sex m f
Mean ECso (ni)
n
Pr>F
Mean GH Secretory CaPacity (ng/ml)
n
Pr>F
8.6 3.6
5 5
0.028
129.5 124.9
5 5
.868
SEM 1.3 19.024 tStandard error of the mean (SEM) values were computed by Statistical Analysis System. level, the m a t u r e male rat s o m a t o t r o p h exhibits a greater c a p a c i t y to secrete G H (8, 9) a n d an e n h a n c e d sensitivity to GRH (8) than the f e m a l e s o m a t o t r o p h . This difference in GRH r e s p o n s i v e n e s s is not o b s e r v e d p r i o r to p u b e r t y (9). W e h r e n b e r g et al. ( 1 0 ) d e m o n s t r a t e d in v f v o that the r e s p o n s e to GRH in a d u l t m a l e rats is greater t h a n that o b s e r v e d in adult f e m a l e rats b u t no a p p a r e n t difference was r e p o r t e d in p r e p u b e r t a l animals. Castration ablates any sexual differentation to GRH ( 1 0 ) . This g o n a d e c t o m i z e d i n d u c e d a t t e n u t a t i o n o f m a l e GRH r e s p o n s i v e n e s s is e v i d e n t at the pituitary level. Ohlsson, Isaksson and Jansson ( 1 1 ) d e m o n s t r a t e d that p e r i f u s e d pituitaries f r o m p r e p u b e r t a l gonad e c t o m i e d m a l e rats release less G H in r e s p o n s e to GRH than do pituitaries f r o m s h a m - o p e r a t e d animals. This effect is m o r e p r o n o u n c e d w h e n the rats w e r e o r c h i e c t o m i z e d as n e o n a t e s t h a n w h e n castrated just p r i o r to p u b e r t y . Similar findings have b e e n r e p o r t e d in pigs. D u b r e v i l et al. ( 1 2 ) d e m o n s t r a t e d that the p l a s m a G H r e s p o n s i v e n e s s to GRH in m a l e piglets w h i c h w e r e castrated at 3 days o f age was significantly less than t h e i r intact c o u n t e r p a r t s . O u r findings are quite r e l e v a n t to these data since barrows u s e d w e r e castrated as neonates. I n d e e d , since these cells a p p e a r to reflect the p h y s i o l o g i c a l state o f the animal at the t i m e that the tissue was obtained, this in v i t r o system c o u l d b e u s e d as a m o d e l to study p h y s i o l o g i c a l r e g u l a t i o n o f pituitary responsiveness to GRH. In c o n c l u s i o n , these data d e m o n s t r a t e that s o m a t o t r o p h s f r o m b a r r o w s are less sensitive to p G R H t h a n cells o b t a i n e d f r o m gilt siblings. It is likely that r e m o v a l o f the testes o f the neonatal m a l e p i g is at least partly r e s p o n s i b l e for t h e s e differences. From a practical p e r s p e c t i v e , neonatal o r c h i e c t o m y in swine is r o u t i n e l y p e r f o r m e d to r e d u c e the o d o r o f c o o k e d b o a r meat.
ACKNOWLEDGEMENTS/FOOTNOT~ We would like to thank Tom Cain for technical assistance, NIADDKfor supply of pGH assay standard, Dr. G. Moore, M. Harris, D. Roland and Dr. D. J. Jones for advice, maintenance and handling of animals and Marlene Carlton for excellent secretarial assistance. Correspondence should be addressed to: M. L. Heiman, Lilly Research Laboratories P.O. Box 708, Building 268 Greenfield, IN 46140, USA
REFERENCES 1. Dubreuil P, Pelletier G, Petitclerc D, Lapierre H, Couture Y, Brazeau P, Gaudreau P, Morisset J. Influence of age and sex on basal secretion of growth hormone (GH) and on GH-induced release by porcine GH-releasing factor pGRH (1-29 NHz) in growing pigs. Domest Anita Endocrinol 4:299-307, 1987. 2. Heiman M L, Nekola M V, Murphy W A, Lance V A, Coy D H. An extremely sensitive in vitro model for elucidating structure-activity relationships of growth hormonereleasing factor analogs. Endocrinology 116:410-415. 1985.
276
HEIMAN, SURFACE, MOWREY AND DIMARCHI
3. Bohlen P, Esch F, Brazeau P, Ling N, Guillemin R. Isolation and characterization of the procine hypothalamic growth hormone releasing factor. Biochem Biophys Res commun 116:726-734, 1983. 4. Merrifield R B, Vizioli L D, Boman H G. Synthesis of the antibacterial peptide Cecropin A (1-33). Biochem 21:5020-5031, 1982. 5. Thorell J I, Johansson B G. Enzymatic iodination of polypeptides with ~2sI to high specific activity. Biochim Biophys Acta 251:363-369, 1971. 6. DeLean A, Munson P J, Rodbard D. Simultaneous analysis of families of sigmoidal curves:application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol 235:E97-E102, 1978. 7. Cronin M J, Rogol A D. Sex differences in the cyclic adenosine 3', 5'-monophosphate and growth hormone response to growth hormone releasing factor in vitro. Biol Repro 31:984-988, 1984. 8. Ho KY, Leong D A, Sinha Y N, Johnson M L, Evans W S, Thorner M O. Sex-related differences in GH secretion in rat using reverse hemolytic plaque assay. Am J Physiol 250 (Endocrinol Metab) 13:E650-E654, 1986. 9. Hoeffler J P, Frawley L S. Capacity of individual somatotropes to release growth hormone varies according to sex:analysis by reverse hemolytic plaque assay. Endocrinology 119:1037-1041, 1986. 10. Wehrenberg W B, Baird A, Ying S-Y, Ling N. The effects of testosterone and estrogen on the pituitary growth hormone response to growth hormone-releasing factor. Biol Repro 32:369-375, 1985. 11. Ohlsson L, Isaksoon O, Jansson J-O. Endogenous testosterone enhances growth hormone (GH)-releasing factor-induced GH secretion in vitro. J Endocr 113:249253, 1987. 12. Dubrevil P, Pelletier G, Couture Y, Lapierre H, Petitclere D, Morisset J, Gaudreau P, Brazeau P. Castration and testosterone effects on endogenous and somatocrinininduced growth hormone release in intact and castrated male pits. Domest Anita Endocrinol 6:15-24, 1989.
