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Temporal relationships among peripheral blood concentrations of corticosteroids, luteinizing hormone and testosterone in bulls
THERIOGENOLOCY TEMPORAL RELATIONSHIPS AKR'K PERIPHERALBLOOD CONCFXl!RATIONSOF CORTICOSTEROIDS, LUTEINIZING HORMONEAND TFSTOSTERONE INBULLS T. I-I. Welsh', R. D. Rand=12 and B. H. Johnson' 1 Reproductive Physiology Research Laboratory Department of Animal Science North Carolina State University Raleigh, North Carolina 27650
2Texas A
& M Agricultural Research and Extension Center Texas A & M University Overton, Texas 75684
Received for Publication:
June 4, 1979
ABSTRACT Interrelationships among peripheral blood concentrations of corticosteroids (CS), luteinizing hormone (LH) and testosterone (T) were evaluated over a 24-hr period in four Angus bulls (18 months of age and 450 kg in body weight). Concentrations of LH and T were determined by radioiarsunoassay and concentrations of CS by competitive protein binding assay of blood samples collected via jugular cannula at hourly intervals for 24 consecutive hr. A positive temporal relationship was observed between LH and T as significant positive correlations were obtained between concentrations of LH at one hour and concentrations of T at the subsequent hour in 3 of 4 bulls. Although LH peaks preceded T peaks by 1 hr, variation in this temporal relationship was observed as LH peaks occurred which were not accompanied by T peaks in some bulls. LH peaks were usually preceded by basal or declining concentrations of CS and prolonged elevations in concentrations of CS were often coincident with basal concentrations of LH and T. Negative correlations were obtained between concentrations of CS at one hour and concentrations of LH and T at the subsequent hour. These data describe the positive regulatory role of LH in testicular T production in the bull and suggest that alterations in endogenous concentrations of CS may influence peripheral concentrations of LH and T in the bull.
The authors are grateful to: Dr. Harold Hafs (Michigan State University, East Lansing, MI) for testosterone antiserum (MSU f/74); Dr. Leo Reichert, Jr. (Emory University, Atlanta, GA) for purified bovine LH (LER-1716-2); Dr. Gordon Niswender (Colorado State University, Ft. Collins, CO) for bovine LH antiserum (B-225); and NIAMD for the bovine LH (NIH-LH-B9). The skillful technical assistance of Ms. Z Turner, Mr. B. Severt and Ms. J. Schmidt is gratefully acknowledged.
SEPTEMBER
1979 VOL. 12 NO. 3
THERIOGENOLOGY INTRODUCTION The concept of a possible endocrine relationship between the adrenal cortex and testis has recently received attention in males of several manmmlian species (1, 2, 3, 4, 5). Administration of dexamethasone, a synthetic corticosteroid (CS), has been reported to inhibit the episodic secretion of luteinizing hormone (LH) and testosterone (T) (6) and to suppress the release of LH following a luteinizing hormone-releasing hormone (LHRH) challenge in bulls (7). However, relationships of endogenous concentrations of CS, LH and T in peripheral blood have not been reported for the bull. The purpose of this study was to examine the temporal relationships among peripheral blood concentrations of CS, LH and T during a 24-hr period in puberal bulls. MAlERIALS AND METHODS Animals and Blood Collection Four Angus bulls 18 months of age and 450 kg in body weight were housed in individual stalls (2.3m x 1.3m) with room temperature maintained at 23 C. Bulls were halter-broken at 9 months of age and were placed in their stalls one week prior to being fitted with an indwelling jugular cannula. Serial blood samples (10 ml) were obtained via cannula at hourly intervals for 24 hr, allowed to clot for 24 hr at 5 C, centrifuged at 5 C and sera stored at -20 C until hormone assays were performed. Hormone Assays Concentrations of LH were determined in duplicate 200~1 aliquots of serum by a double-antibody radioinnmn p$3say (8, 9). Purified bovine LH (LER-1716-a was labelled with I by the chloramine T method (10) and separated on a 1 x 30 cm Sephadex G-100 column. The LH antibody (antibovine LH serum B225) was used at a dilution of l:lOO,OOO and the sheep anti-rabbit ganana globulin was used at a dilution of 1:30. Intra-assay and inter-assay coefficients of variation were 9.9 and 13.1%, respectively. Concentrations of T were determined in duplicate 100~1 aliquots of serum via radioimmunoassay (9, 11, 12). .The antiserum was used at a dilution of 1:25,000 to obtain 50% binding of radiolabelled T to the antitestosterone serum. Of the major androgens, progestogens, estrogens and corticosteroids, the antiserum crossreacted significantly only with dihydrotestosterone (50%). Serum levels of dihydrotestosterone have been reported to be quite low (0.10 ng/ml) in the bull (13) therefore chromatographic techniques were not utilized to separate T and dihydrotestosterone. Intra-assay and
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inter-assay coefficients of variation were 6.8 and 10.5%, respectively. Concentrations of total CS were determined in duplicate lOOu1 aliquots of serum by competitive protein binding assay (9, 14, 15). Serum samples were first extracted with benzene:hexane (1:2) to remove androgens, progestogens and estrogens prior to extraction with ethyl acetate to remove CS. Dog plasma was the source of binding protein and tritiated cortisol in 2.5% flourisil-washed dog plasma was the competitor. Intra-assay and inter-assay coefficients of variation were 9.5 and 15.0%, respectively. Statistical Analyses Time series analysis (16) identified basal and peak concentrations of hormones and partial correlation coefficients (17) described temporal relationships among concentrations of hormones (9). Partial correlations between concentrations of hormones in blood samples of the same hour were estimated for concentrations of LH and T (LHTCORR), CS and T (CSTCORR), and CS and LH (CSLHCORR). Lagged hormone correlations also were estimated for each bull. A lagged hormone correlation was defined to be the partial correlation between the concentration of a hormone at one hour and the concentration of another hormone the subsequent hour for all such combinations over the 24-hr period. Lagged hormone correlations were determined between concentrations of LH and T (LAG-LHTCORR),CS and T (LAG-CSTCORR) and CS and LH (LAG-CSLH CORR). RESULTS AND DISCUSSION Twenty-four hour profiles of concentrations of CS, LH and T in peripheral blood of four bulls (A, B, C and D) are illustrated in Fig.1 and 2. In bulls A and B, LH peaks consistently preceded T peaks, whereas, only half of the LH peaks in bulls C and D were followed by T peaks. The failure of some LH peaks to induce elevations in T suggests a possible inconsistency in the classical concept of the temporal relationship between LH and T. Biosynthesis of androgens within Leydig cells and the dependence of testicular steroidogenesis upon LH stimulation are well-documented (18, 19). However, the steroidogenic function of Leydig cells has recently been demonstrated to be regulated by the presence and functionality of LH receptors on plasma membranes of rat Leydig cells (20, 21, 22). Furthermore, receptor population and function are also influenced indirectly and directly by other hormones such as androgens, corticosteroids, estrogens, follicle stimulating hormone, progestogens, and prolactin which suggest that Leydig cell function may not be static but dynamic and subject to numerous endocrine interrelationships (23). For
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1979 VOL. 12 NO. 3
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THERIOGENOLOGY
example, an unexpecteddiminished steroidogenicresponse of rat Leydig cells to exogenousLH has been attributed to either a decreasedpopulation of LH receptors or a desensitizationof the receptormechanism (23, 24). Existence of an analogous endocrine mechanism in the bovine testis has not been assessed; however, in this study endogenousLH peaks were observed which were not temporallyassociatedwith T peaks. Temporal relationshipsbetween concentrationsof CS, LH and T in the four bulls can be assessed from the correlationcoefficients in Table I. Low and nonsignificantvalues were obtained for LHTCOEE, whereas, values of greater magnitude were obtained for LAG-LHTCOBB. Occurrence of T peaks within 1 hr after LH peaks (Fig. 1 and 2), and a significantpositive relationshipbetween concentrationsof LH and T described by LAG-LHTCOBE (Table I), indicate that an elevation in endogenousconcentrationof LH normally exerts a stiumlatoryeffect upon T synthesisand secretion after a timelag of 1 hr. Investigations which utilized an exogenous supply of LH suggesteda timelag of 60-120 min for maximal T response to iv injection of LH in the bull (25, 26). Analysis of the relationshipsbetween concentrationsof CS and concentrationsof LH and T revealed that 93% of al.1LH peaks occurred at the time of basal or declining concentrationsof CS. Each LH peak in bull A was preceded by basal concentrationsof CS and followed within 1 hr by a T peak. Two instancesof prolonged maintenanceof elevated concentrationsof CS were coincidentwith basal concentrations of LH and T in bull A. Each LH peak in bull B was preceded by the decline of peak concentrationsof CS and two instances of elevated concentrationsof CS were coincidentwith basal concentrationsof LH and T. Each LH peak in bull C and four of five LH peaks in bull D were preceded by basal or declining concentrationsof CS. However, each LH peak was not accompaniedby a T peak. The first LH peak observed in bull C was not followed by a T peak but was closely followedby a CS peak. In bull D the first LH peak was preceded by a CS peak and was not followedby a T peak. A prolonged elevation in concentration of LH was later observed in bull D which was associatedwith a delayed increase in concentrationof T. Coincidentwith the delayed T response was an elevation in concentrationof CS. Overall, these observationssuggest that concentrationsof LH and T in peripheral blood are inversely related to concentrationsof CS. Correlationcoefficientsdescribing the temporal relationships between endogenousconcentrationsof CS, LH and T further suggest a negative influence of CS upon the regulationof LH and T production in the bull (Table I). Negative values were obtained for both CSLHCOBR and LAG-CSLHCOBEin bulls A, B and C. In contrast,positive
172
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THERIOGENOLOGY values were obtained for these two temporal relationships between concentration of CS and LH in bull D. Also, bull D was the only bull without a positive LAG-LHTCORR. A negative relationship was also observed between concentrations of CS and T as negative values were obtained for both CSTCORR and LAG-CSTCORR. Analogous to the trend of LAG-LHTCORR being more positive relative to LHTCORR, LAGCSTCORR was more negative relative to CSTCORR. A negative influence of exogenous CS on testicular steroidogenesis has been reported for several maranalian species. A decrease in T production has been reported in men following administration of either exogenous cortisol (27) or dexamethasone (28). Similarly, dexamethasone inhibited episodic secretion of LH and T in the bull (6). The cellular site and mechanism of action whereby CS may influence testicular steroidogenesis is unknown. However, a glucocorticoid receptor has been identified in the prepuberal rat Leydig cell (29) and a direct effect of CS on Leydig cells has been suggested by the observation that dexamethasone decreased binding of 1251-HCG to LH/HCG receptors on rat Leydig cells (30). The observation that dexamethasone suppressed LH response to LHRH challenge in bulls (7) suggests a possible direct effect of CS on the hypothalamic-pituitary axis. In all of these studies, exogenous chemical messengers were used to study the influence of adrenal CS on testicular T production. The results of this study provide further evidence of a functional relationship between adrenal and testicular steroidogenic processes in the bull. This evidence is based upon the observation that endogenous concentrations of adrenal CS in peripheral blood were negatively associated with concentrations of T and LH. This suggests that adrenal dysfunction could significantly influence testicular function in the bull.
Paper No. 6016 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned.
SEPTEMBER 1979 VOL. 12 NO. 3
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REFERENCES 1.
Liptrap, R. M. and Raeside, J. I. Effect of corticotrophin and corticosteroids on plasma ICSH and urinary steroids in the boar. J. Endocr. 42:33-43. (1968)
2.
Pasley, J. N. and Christian, J. J. The effect of ACTH, group caging, and adrenalectomy in Peromyscus leucopus with emphasis on suppression of reproductive function. Proc. Sot. Exp. Biol. Med. 139:921-925. (1972)
3.
Schaison, G., Durand, F. and Mowszowicz, I. Effect of glucocorticoids on plasma testosterone in men. Acta End&r. 89:126-131. (1978)
4.
Beitins, I. Z., Bayard, F., Kowarski, A. and Migeon, C. J. The effect of ACTH administration on plasma testosterone, dihydrotestosterone and serum LH concentrations in normal men. Steroids 21:553-563. (1973)
5.
Wassermann, G. F. and Eik-Nes, K. B. Interrelation between adrenal function and formation of testosterone -in vivo in the testis of the dog. -Acta Endocr. 61:33-47. (1969)
6.
Thibier, M. and Rolland, 0. The effect of dexamethasone (DXM) on circulating testosterone (T) and luteinizing hormone (LH) in young postpubertal bulls. Theriogenology 5:53-60. (1976)
7.
Chantaraprateep, P. and Thibier, M. Effects of dexamethasone on the responses of luteinizing hormone and testosterone to two injections of luteinizing hormone releasing hormone in young postpubertal bulls. J. Endocr. 77:389-395. (1978)
8.
Niswender, G. D., Reichert, L. E., Midgley, A. R. and Nalbandov, A. V. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84:1166-1173. (1969)
9.
Welsh, T. H., McGraw, R. L. and Johnson, B. H. Influence of corticosteroids on testosterone production in the bull. Biol. Reprod. In press.
10.
Greenwood, F. D Hunter, W. M. and Glover, J. S. The preparation of Is1I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89:114-123. (1963)
11.
Smith, 0. W. and Hafs, H. D. Competitive protein binding and radioinznunoassay for testosterone in bulls and rabbits; blood serum testosterone after injection of LH or prolactin in rabbits. Proc. Sot. Exp. Biol. Med. 142:804-810. (1973).
174
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12.
Johnson, B. H. Effects of hem&castration on testicular functions in adult and young puberal bulls. Theriogenology 10:257-264. (1978)
13.
Schanbacher, B. D. Rapid chromatography for quantitation of radioimmunoassayable 5%androstone-178-ol-3-one and testosterone in ram, bull and boar serum. Endocr. Res. Courn. 3:71-82. (1976)
14.
Murphy, B. E. P. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurements of various steroids in body fluids by competitive protein-binding radioassay. J. Clin. Endocr. Metab. 27:973990. (1967)
15.
Smith, V. G., Convey, E. M. and Edgerton, L. A. Bovine serum corticoid response to milking and exteroceptive stimuli. J. Dairy Sci. 55:1170-1173. (1972)
16.
Christian, L. E., Everson, D. 0. and Davis, S. L. A statistical method for detection of hormone secretory spikes. J. Anim. $c& 46:699-706. (1978)
17.
Barr, A. J., Goodnight, J. H *, Sall, J. P. and Helwig, J. T. A Users Guide to SAS 76. Sparks Press, Raleigh, N. C. pp. 92256. (1976)
18.
Eik-Nes, K. B. Biosynthesis and secretion of testicular steroids. 2: Handbook of Physiology. Section 7: Endocrinology, Vol. V. Male reproductive system (D. W. Hamilton and R. 0. Greep, eds.), American Physiological Society, Washington, D. C. pp. 95-115. (1975)
19.
Bartke, A., Hafiez, A. A., Bex, F. J. and Dalterio, S. Hormonal interactions in regulation of androgen secretion. Biol. Reprod. 18:44-54. (1978)
20.
Catt, K. J., Tsuruhara, T., Mendelson, C., Ketelslegers, J. M. and Dufau, M. L. Gonadotropin binding and activation of the intestitial cells of the testis. In: Hormone binding and target cell activation in the test= (M. L. Dufau and A. R. Means, eds.), Plenum Press, New York, pp. l-30. (1974)
21.
Hsueh, A. J., Dufau, M. L. and Catt, K. J. Regulation of luteinizing hormone receptors in testicular interstitial cells by gonadotropin. Biochem. Biophys. Res. Conan. 72:1145-1152. (1976)
22.
Hsueh, A. J., Dufau, M. L. and Catt, K. J. Gonadotropin-induced regulation of luteinizing hormone receptors and desensitization of testicular 3':5'-cyclic AMP and testosterone responses. Proc. Natl. Acad. Sci. 74:592-595. (1977)
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THERIOGENOLOGY 23. Purvis, K. and Hansson, V. Hormonal regulation of Leydig cell function.Mol. Cell. Endocr. 12:123-138. (1978) 24. Catt, K. J., Baukal, A. J., Davies, T. F. and Dufau, M. L. Luteinizing hormone-releasinghormone-inducedregulationof gonadotropinand prolactin receptors in the rat testis. Endocrinologv104:17-25. (1979) 25. Smith, 0. W., Mongkonpunya,K., Hafs, H. D., Convey, E. M. and Oxender, W. D. Blood serum testosteroneafter sexual preparation or ejaculation,or after injections of LH or prolactin in bulls. J. Anim. Sci. 37:979-984.(1973) 26. Kiser, T. E., Milvae, R. A., Hafs, H. D., Oxender, W. E. and Louis, T. M. Comparisonof testosteroneand androstenedione or luteinizing secretion in bulls given prostaglandinF hormone. J. Anim. Sci. 46:436-442. (1978 27. Doerr, P. and Pirke, K. M. Cortisol-inducedsuppressionof plasma testosteronein normal adult males. J. Clin. Endocr. Metab. 43:622-629. (1976) 28. Doerr, P. and Pirke, K. M. Dexamethasone-inducedsuppression of the circadian rhythm of plasma testosteronein normal adult males. J. Steroid Biochem. 10:81-86. (1979) 29. Evain, D., Morera, A. M. and Saez, J. M. Glucocorticoidreceptors in interstitialcells of the rat testis. J. Steroid uochem, 7:1135-1139.(1976) 30. Saez, J. M., Morera, A. M., Haour, F. and Evain, D. Effects of in viva administrationof dexamethasone,corticotropinand -human chorionic gonadotropinon steroidogenesisand protein and DNA synthesisof testicularinterstitialcells in prepuberal rats. Endocrinology101:1256-1263.(1977)
176
SEPTEMBER 1979 VOL. 12 NO. 3
.12
-.06
.oo
-.18
A
B
C
D
-.06
.40c
,50b
.7ga
LAG-LHTCORR
.37c
-.16
-.29
-.21
CSLHCORR
.27
-.04
-.28
-.27
LAG-CSLHCORR
-.05
.07
-.3ac
-.18
CSTCORR
.
-.21
-.12
-.4gb
-.35c
LAG-CSTCORR
cP ( .05
bP < .Ol
aP < .OOOl
*See Materials and Methods for definition of column headings. Correlations and lagged correlations were based upon 25 and 24 observations per bull, respectively.
LHTCORR
Bull
Temporal Relationships*
Correlation coefficients between concentrations of corticosteroids, luteinizing hormone and testosterone over a 24-hr period within individual bulls
TABLE I
THERIOGENOLOGY
i 16
BULL
A
! \ i \
160
14
I40
12
120
10
lOi
8
80
6
60
7
zo-
$2
"-
23
SEi! I -I .
"
101
-4
m
E
2
-I 9am
9pm
g
I I :
5 I-
16
I
I4 12
10
8
80
6
4
2
cl”““““:“““““’ 9am
9(
9pm TIME IN HOURS
Fig. 1.
178
Peripheral blood concentrations of corticosteroids, luteinizing hormone and testosterone over a 24-hr period in bulls A and B.
SEPTEMBER
1979 VOL. 12 NO. 3
e
f-5
h)
8 8
m
f3
m
g
G
g
g
-TESTOSTERONE FL ii
(ng/ml)
&.-.-.-‘A CORTICOSTEROIDS
g
6
l---¤LH L
(ng/ml)
88sagg
?.!
(ng/ml)
0
0
b
is
E
z
s
F
2Texas A
& M Agricultural Research and Extension Center Texas A & M University Overton, Texas 75684
Received for Publication:
June 4, 1979
ABSTRACT Interrelationships among peripheral blood concentrations of corticosteroids (CS), luteinizing hormone (LH) and testosterone (T) were evaluated over a 24-hr period in four Angus bulls (18 months of age and 450 kg in body weight). Concentrations of LH and T were determined by radioiarsunoassay and concentrations of CS by competitive protein binding assay of blood samples collected via jugular cannula at hourly intervals for 24 consecutive hr. A positive temporal relationship was observed between LH and T as significant positive correlations were obtained between concentrations of LH at one hour and concentrations of T at the subsequent hour in 3 of 4 bulls. Although LH peaks preceded T peaks by 1 hr, variation in this temporal relationship was observed as LH peaks occurred which were not accompanied by T peaks in some bulls. LH peaks were usually preceded by basal or declining concentrations of CS and prolonged elevations in concentrations of CS were often coincident with basal concentrations of LH and T. Negative correlations were obtained between concentrations of CS at one hour and concentrations of LH and T at the subsequent hour. These data describe the positive regulatory role of LH in testicular T production in the bull and suggest that alterations in endogenous concentrations of CS may influence peripheral concentrations of LH and T in the bull.
The authors are grateful to: Dr. Harold Hafs (Michigan State University, East Lansing, MI) for testosterone antiserum (MSU f/74); Dr. Leo Reichert, Jr. (Emory University, Atlanta, GA) for purified bovine LH (LER-1716-2); Dr. Gordon Niswender (Colorado State University, Ft. Collins, CO) for bovine LH antiserum (B-225); and NIAMD for the bovine LH (NIH-LH-B9). The skillful technical assistance of Ms. Z Turner, Mr. B. Severt and Ms. J. Schmidt is gratefully acknowledged.
SEPTEMBER
1979 VOL. 12 NO. 3
THERIOGENOLOGY INTRODUCTION The concept of a possible endocrine relationship between the adrenal cortex and testis has recently received attention in males of several manmmlian species (1, 2, 3, 4, 5). Administration of dexamethasone, a synthetic corticosteroid (CS), has been reported to inhibit the episodic secretion of luteinizing hormone (LH) and testosterone (T) (6) and to suppress the release of LH following a luteinizing hormone-releasing hormone (LHRH) challenge in bulls (7). However, relationships of endogenous concentrations of CS, LH and T in peripheral blood have not been reported for the bull. The purpose of this study was to examine the temporal relationships among peripheral blood concentrations of CS, LH and T during a 24-hr period in puberal bulls. MAlERIALS AND METHODS Animals and Blood Collection Four Angus bulls 18 months of age and 450 kg in body weight were housed in individual stalls (2.3m x 1.3m) with room temperature maintained at 23 C. Bulls were halter-broken at 9 months of age and were placed in their stalls one week prior to being fitted with an indwelling jugular cannula. Serial blood samples (10 ml) were obtained via cannula at hourly intervals for 24 hr, allowed to clot for 24 hr at 5 C, centrifuged at 5 C and sera stored at -20 C until hormone assays were performed. Hormone Assays Concentrations of LH were determined in duplicate 200~1 aliquots of serum by a double-antibody radioinnmn p$3say (8, 9). Purified bovine LH (LER-1716-a was labelled with I by the chloramine T method (10) and separated on a 1 x 30 cm Sephadex G-100 column. The LH antibody (antibovine LH serum B225) was used at a dilution of l:lOO,OOO and the sheep anti-rabbit ganana globulin was used at a dilution of 1:30. Intra-assay and inter-assay coefficients of variation were 9.9 and 13.1%, respectively. Concentrations of T were determined in duplicate 100~1 aliquots of serum via radioimmunoassay (9, 11, 12). .The antiserum was used at a dilution of 1:25,000 to obtain 50% binding of radiolabelled T to the antitestosterone serum. Of the major androgens, progestogens, estrogens and corticosteroids, the antiserum crossreacted significantly only with dihydrotestosterone (50%). Serum levels of dihydrotestosterone have been reported to be quite low (0.10 ng/ml) in the bull (13) therefore chromatographic techniques were not utilized to separate T and dihydrotestosterone. Intra-assay and
170
SEPTEMBER
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THERIOCENOLOGY
inter-assay coefficients of variation were 6.8 and 10.5%, respectively. Concentrations of total CS were determined in duplicate lOOu1 aliquots of serum by competitive protein binding assay (9, 14, 15). Serum samples were first extracted with benzene:hexane (1:2) to remove androgens, progestogens and estrogens prior to extraction with ethyl acetate to remove CS. Dog plasma was the source of binding protein and tritiated cortisol in 2.5% flourisil-washed dog plasma was the competitor. Intra-assay and inter-assay coefficients of variation were 9.5 and 15.0%, respectively. Statistical Analyses Time series analysis (16) identified basal and peak concentrations of hormones and partial correlation coefficients (17) described temporal relationships among concentrations of hormones (9). Partial correlations between concentrations of hormones in blood samples of the same hour were estimated for concentrations of LH and T (LHTCORR), CS and T (CSTCORR), and CS and LH (CSLHCORR). Lagged hormone correlations also were estimated for each bull. A lagged hormone correlation was defined to be the partial correlation between the concentration of a hormone at one hour and the concentration of another hormone the subsequent hour for all such combinations over the 24-hr period. Lagged hormone correlations were determined between concentrations of LH and T (LAG-LHTCORR),CS and T (LAG-CSTCORR) and CS and LH (LAG-CSLH CORR). RESULTS AND DISCUSSION Twenty-four hour profiles of concentrations of CS, LH and T in peripheral blood of four bulls (A, B, C and D) are illustrated in Fig.1 and 2. In bulls A and B, LH peaks consistently preceded T peaks, whereas, only half of the LH peaks in bulls C and D were followed by T peaks. The failure of some LH peaks to induce elevations in T suggests a possible inconsistency in the classical concept of the temporal relationship between LH and T. Biosynthesis of androgens within Leydig cells and the dependence of testicular steroidogenesis upon LH stimulation are well-documented (18, 19). However, the steroidogenic function of Leydig cells has recently been demonstrated to be regulated by the presence and functionality of LH receptors on plasma membranes of rat Leydig cells (20, 21, 22). Furthermore, receptor population and function are also influenced indirectly and directly by other hormones such as androgens, corticosteroids, estrogens, follicle stimulating hormone, progestogens, and prolactin which suggest that Leydig cell function may not be static but dynamic and subject to numerous endocrine interrelationships (23). For
SEPTEMBER
1979 VOL. 12 NO. 3
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THERIOGENOLOGY
example, an unexpecteddiminished steroidogenicresponse of rat Leydig cells to exogenousLH has been attributed to either a decreasedpopulation of LH receptors or a desensitizationof the receptormechanism (23, 24). Existence of an analogous endocrine mechanism in the bovine testis has not been assessed; however, in this study endogenousLH peaks were observed which were not temporallyassociatedwith T peaks. Temporal relationshipsbetween concentrationsof CS, LH and T in the four bulls can be assessed from the correlationcoefficients in Table I. Low and nonsignificantvalues were obtained for LHTCOEE, whereas, values of greater magnitude were obtained for LAG-LHTCOBB. Occurrence of T peaks within 1 hr after LH peaks (Fig. 1 and 2), and a significantpositive relationshipbetween concentrationsof LH and T described by LAG-LHTCOBE (Table I), indicate that an elevation in endogenousconcentrationof LH normally exerts a stiumlatoryeffect upon T synthesisand secretion after a timelag of 1 hr. Investigations which utilized an exogenous supply of LH suggesteda timelag of 60-120 min for maximal T response to iv injection of LH in the bull (25, 26). Analysis of the relationshipsbetween concentrationsof CS and concentrationsof LH and T revealed that 93% of al.1LH peaks occurred at the time of basal or declining concentrationsof CS. Each LH peak in bull A was preceded by basal concentrationsof CS and followed within 1 hr by a T peak. Two instancesof prolonged maintenanceof elevated concentrationsof CS were coincidentwith basal concentrations of LH and T in bull A. Each LH peak in bull B was preceded by the decline of peak concentrationsof CS and two instances of elevated concentrationsof CS were coincidentwith basal concentrationsof LH and T. Each LH peak in bull C and four of five LH peaks in bull D were preceded by basal or declining concentrationsof CS. However, each LH peak was not accompaniedby a T peak. The first LH peak observed in bull C was not followed by a T peak but was closely followedby a CS peak. In bull D the first LH peak was preceded by a CS peak and was not followedby a T peak. A prolonged elevation in concentration of LH was later observed in bull D which was associatedwith a delayed increase in concentrationof T. Coincidentwith the delayed T response was an elevation in concentrationof CS. Overall, these observationssuggest that concentrationsof LH and T in peripheral blood are inversely related to concentrationsof CS. Correlationcoefficientsdescribing the temporal relationships between endogenousconcentrationsof CS, LH and T further suggest a negative influence of CS upon the regulationof LH and T production in the bull (Table I). Negative values were obtained for both CSLHCOBR and LAG-CSLHCOBEin bulls A, B and C. In contrast,positive
172
SEPTEMBER
1979 VOL. 12 NO. 3
THERIOGENOLOGY values were obtained for these two temporal relationships between concentration of CS and LH in bull D. Also, bull D was the only bull without a positive LAG-LHTCORR. A negative relationship was also observed between concentrations of CS and T as negative values were obtained for both CSTCORR and LAG-CSTCORR. Analogous to the trend of LAG-LHTCORR being more positive relative to LHTCORR, LAGCSTCORR was more negative relative to CSTCORR. A negative influence of exogenous CS on testicular steroidogenesis has been reported for several maranalian species. A decrease in T production has been reported in men following administration of either exogenous cortisol (27) or dexamethasone (28). Similarly, dexamethasone inhibited episodic secretion of LH and T in the bull (6). The cellular site and mechanism of action whereby CS may influence testicular steroidogenesis is unknown. However, a glucocorticoid receptor has been identified in the prepuberal rat Leydig cell (29) and a direct effect of CS on Leydig cells has been suggested by the observation that dexamethasone decreased binding of 1251-HCG to LH/HCG receptors on rat Leydig cells (30). The observation that dexamethasone suppressed LH response to LHRH challenge in bulls (7) suggests a possible direct effect of CS on the hypothalamic-pituitary axis. In all of these studies, exogenous chemical messengers were used to study the influence of adrenal CS on testicular T production. The results of this study provide further evidence of a functional relationship between adrenal and testicular steroidogenic processes in the bull. This evidence is based upon the observation that endogenous concentrations of adrenal CS in peripheral blood were negatively associated with concentrations of T and LH. This suggests that adrenal dysfunction could significantly influence testicular function in the bull.
Paper No. 6016 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned.
SEPTEMBER 1979 VOL. 12 NO. 3
173
THERIOCENOLOCY
REFERENCES 1.
Liptrap, R. M. and Raeside, J. I. Effect of corticotrophin and corticosteroids on plasma ICSH and urinary steroids in the boar. J. Endocr. 42:33-43. (1968)
2.
Pasley, J. N. and Christian, J. J. The effect of ACTH, group caging, and adrenalectomy in Peromyscus leucopus with emphasis on suppression of reproductive function. Proc. Sot. Exp. Biol. Med. 139:921-925. (1972)
3.
Schaison, G., Durand, F. and Mowszowicz, I. Effect of glucocorticoids on plasma testosterone in men. Acta End&r. 89:126-131. (1978)
4.
Beitins, I. Z., Bayard, F., Kowarski, A. and Migeon, C. J. The effect of ACTH administration on plasma testosterone, dihydrotestosterone and serum LH concentrations in normal men. Steroids 21:553-563. (1973)
5.
Wassermann, G. F. and Eik-Nes, K. B. Interrelation between adrenal function and formation of testosterone -in vivo in the testis of the dog. -Acta Endocr. 61:33-47. (1969)
6.
Thibier, M. and Rolland, 0. The effect of dexamethasone (DXM) on circulating testosterone (T) and luteinizing hormone (LH) in young postpubertal bulls. Theriogenology 5:53-60. (1976)
7.
Chantaraprateep, P. and Thibier, M. Effects of dexamethasone on the responses of luteinizing hormone and testosterone to two injections of luteinizing hormone releasing hormone in young postpubertal bulls. J. Endocr. 77:389-395. (1978)
8.
Niswender, G. D., Reichert, L. E., Midgley, A. R. and Nalbandov, A. V. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84:1166-1173. (1969)
9.
Welsh, T. H., McGraw, R. L. and Johnson, B. H. Influence of corticosteroids on testosterone production in the bull. Biol. Reprod. In press.
10.
Greenwood, F. D Hunter, W. M. and Glover, J. S. The preparation of Is1I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89:114-123. (1963)
11.
Smith, 0. W. and Hafs, H. D. Competitive protein binding and radioinznunoassay for testosterone in bulls and rabbits; blood serum testosterone after injection of LH or prolactin in rabbits. Proc. Sot. Exp. Biol. Med. 142:804-810. (1973).
174
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1979 VOL. 12 NO. 3
THERIOGENOLOGY
12.
Johnson, B. H. Effects of hem&castration on testicular functions in adult and young puberal bulls. Theriogenology 10:257-264. (1978)
13.
Schanbacher, B. D. Rapid chromatography for quantitation of radioimmunoassayable 5%androstone-178-ol-3-one and testosterone in ram, bull and boar serum. Endocr. Res. Courn. 3:71-82. (1976)
14.
Murphy, B. E. P. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurements of various steroids in body fluids by competitive protein-binding radioassay. J. Clin. Endocr. Metab. 27:973990. (1967)
15.
Smith, V. G., Convey, E. M. and Edgerton, L. A. Bovine serum corticoid response to milking and exteroceptive stimuli. J. Dairy Sci. 55:1170-1173. (1972)
16.
Christian, L. E., Everson, D. 0. and Davis, S. L. A statistical method for detection of hormone secretory spikes. J. Anim. $c& 46:699-706. (1978)
17.
Barr, A. J., Goodnight, J. H *, Sall, J. P. and Helwig, J. T. A Users Guide to SAS 76. Sparks Press, Raleigh, N. C. pp. 92256. (1976)
18.
Eik-Nes, K. B. Biosynthesis and secretion of testicular steroids. 2: Handbook of Physiology. Section 7: Endocrinology, Vol. V. Male reproductive system (D. W. Hamilton and R. 0. Greep, eds.), American Physiological Society, Washington, D. C. pp. 95-115. (1975)
19.
Bartke, A., Hafiez, A. A., Bex, F. J. and Dalterio, S. Hormonal interactions in regulation of androgen secretion. Biol. Reprod. 18:44-54. (1978)
20.
Catt, K. J., Tsuruhara, T., Mendelson, C., Ketelslegers, J. M. and Dufau, M. L. Gonadotropin binding and activation of the intestitial cells of the testis. In: Hormone binding and target cell activation in the test= (M. L. Dufau and A. R. Means, eds.), Plenum Press, New York, pp. l-30. (1974)
21.
Hsueh, A. J., Dufau, M. L. and Catt, K. J. Regulation of luteinizing hormone receptors in testicular interstitial cells by gonadotropin. Biochem. Biophys. Res. Conan. 72:1145-1152. (1976)
22.
Hsueh, A. J., Dufau, M. L. and Catt, K. J. Gonadotropin-induced regulation of luteinizing hormone receptors and desensitization of testicular 3':5'-cyclic AMP and testosterone responses. Proc. Natl. Acad. Sci. 74:592-595. (1977)
SEPTEMBER
1979 VOL. 12 NO. 3
175
THERIOGENOLOGY 23. Purvis, K. and Hansson, V. Hormonal regulation of Leydig cell function.Mol. Cell. Endocr. 12:123-138. (1978) 24. Catt, K. J., Baukal, A. J., Davies, T. F. and Dufau, M. L. Luteinizing hormone-releasinghormone-inducedregulationof gonadotropinand prolactin receptors in the rat testis. Endocrinologv104:17-25. (1979) 25. Smith, 0. W., Mongkonpunya,K., Hafs, H. D., Convey, E. M. and Oxender, W. D. Blood serum testosteroneafter sexual preparation or ejaculation,or after injections of LH or prolactin in bulls. J. Anim. Sci. 37:979-984.(1973) 26. Kiser, T. E., Milvae, R. A., Hafs, H. D., Oxender, W. E. and Louis, T. M. Comparisonof testosteroneand androstenedione or luteinizing secretion in bulls given prostaglandinF hormone. J. Anim. Sci. 46:436-442. (1978 27. Doerr, P. and Pirke, K. M. Cortisol-inducedsuppressionof plasma testosteronein normal adult males. J. Clin. Endocr. Metab. 43:622-629. (1976) 28. Doerr, P. and Pirke, K. M. Dexamethasone-inducedsuppression of the circadian rhythm of plasma testosteronein normal adult males. J. Steroid Biochem. 10:81-86. (1979) 29. Evain, D., Morera, A. M. and Saez, J. M. Glucocorticoidreceptors in interstitialcells of the rat testis. J. Steroid uochem, 7:1135-1139.(1976) 30. Saez, J. M., Morera, A. M., Haour, F. and Evain, D. Effects of in viva administrationof dexamethasone,corticotropinand -human chorionic gonadotropinon steroidogenesisand protein and DNA synthesisof testicularinterstitialcells in prepuberal rats. Endocrinology101:1256-1263.(1977)
176
SEPTEMBER 1979 VOL. 12 NO. 3
.12
-.06
.oo
-.18
A
B
C
D
-.06
.40c
,50b
.7ga
LAG-LHTCORR
.37c
-.16
-.29
-.21
CSLHCORR
.27
-.04
-.28
-.27
LAG-CSLHCORR
-.05
.07
-.3ac
-.18
CSTCORR
.
-.21
-.12
-.4gb
-.35c
LAG-CSTCORR
cP ( .05
bP < .Ol
aP < .OOOl
*See Materials and Methods for definition of column headings. Correlations and lagged correlations were based upon 25 and 24 observations per bull, respectively.
LHTCORR
Bull
Temporal Relationships*
Correlation coefficients between concentrations of corticosteroids, luteinizing hormone and testosterone over a 24-hr period within individual bulls
TABLE I
THERIOGENOLOGY
i 16
BULL
A
! \ i \
160
14
I40
12
120
10
lOi
8
80
6
60
7
zo-
$2
"-
23
SEi! I -I .
"
101
-4
m
E
2
-I 9am
9pm
g
I I :
5 I-
16
I
I4 12
10
8
80
6
4
2
cl”““““:“““““’ 9am
9(
9pm TIME IN HOURS
Fig. 1.
178
Peripheral blood concentrations of corticosteroids, luteinizing hormone and testosterone over a 24-hr period in bulls A and B.
SEPTEMBER
1979 VOL. 12 NO. 3
e
f-5
h)
8 8
m
f3
m
g
G
g
g
-TESTOSTERONE FL ii
(ng/ml)
&.-.-.-‘A CORTICOSTEROIDS
g
6
l---¤LH L
(ng/ml)
88sagg
?.!
(ng/ml)
0
0
b
is
E
z
s
F