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Regular Article |
a Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97201
b Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331
c United States Sheep Experiment Station, Dubois, Idaho 83423
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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behavior, male sexual function, neuroendocrinology, stress, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testosterone and its component androgenic and estrogenic signaling pathways in the nervous system are critical for the expression of masculine sexual behaviors in vertebrates [3]. Minimal threshold concentrations of testosterone are required for expression of heterosexual mating behavior in male sheep [4]. MORs appear to have at least the minimum basal concentrations of testosterone required for exhibiting heterosexual courtship and copulatory behaviors [5, 6]. However, in contrast to FORs, MORs do not respond with an increment in systemic concentrations of testosterone when allowed to interact with estrous ewes for several hours [5, 6].
In a previous study [7] we found that aromatase activity in the medial preoptic area was lower in MORs than in FORs, suggesting that the capacity for androgen signaling mediated through the aromatase pathway and estrogen receptor differs between FORs and MORs, and may relate to the expression of sexual partner preferences. In addition, MORs exhibited significantly lower systemic concentrations of testosterone than FORs, as well as a lower capacity for testicular testosterone synthesis. These latter observations raised the possibility that basal levels of circulating testosterone may also be causative or predictive of sexual partner preferences in rams. However, subsequent studies by other investigators [8, 9] did not find differences in mean circulating testosterone concentrations between FORs and MORs when multiple blood samples from the jugular vein were analyzed. Moreover, Perkins et al. [6] reported that some MORs had greater mean plasma concentrations of testosterone following physical contact and copulation with male-stimulus animals. Thus, it is not clear whether MORs have inherently lower concentrations of testosterone than FORs.
The purpose of the present experiment was to reexamine this issue by measuring systemic concentrations of testosterone under 3 separate conditions; namely, in conscious rams during the nonbreeding season (June) and breeding season (November), and in anesthetized rams during the breeding season. The latter condition mimics that of our previous study [7]. In addition, a group of asexual rams with indeterminate sexual orientation (NORs) was included in the study for comparison of basal systemic concentrations of testosterone between all three behaviorally classified groups.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirty adult rams (3.3 ± 0.9 yr old) of different Western breeds (Columbia, n = 5; Polypay, n = 11; Rambouillet, n = 10; and Targhee, n = 4) were used in this study. The number of rams in each experimental group was as follows: FOR (n = 8), MOR (n = 6), and NOR (n = 16). Sexual performance and partner preference tests were given to each subject to determine its level of libido and sexual orientation. These tests were performed over a 2-yr period at the United States Sheep Experimental Station in Dubois, ID. The details of these procedures have been published previously [7]. Briefly, at approximately 18 mo of age, the rams were tested 18 separate times with mature ovariectomized ewes in which estrus was induced artificially with exogenous progestin and estrogen treatments. Repeated testing revealed 2 groups of rams: those that mated vigorously and repeatedly with estrous ewes (FORs) and those that did not mate with estrous ewes (i.e., potentially MORs or NORs). Rams that did not mount estrous ewes were further given three 30-min sexual partner preference tests. For these tests, rams were exposed simultaneously to 2 females in estrus and 2 randomly chosen males. The stimulus animals were restrained in a 4-way stanchion so that they were all readily accessible to the test ram. In all behavioral tests the numbers of genital sniffs, foreleg kicks, vocalizations, Flehmens (a behavior characterized by a raised head and curled upper lip that presumably allows odors to reach the vomeronasal organ in rams), mounts, and ejaculations were recorded. Approximately 1 yr later, the rams were given a second set of 18 performance tests with ewes in estrus followed by a sexual partner preference test to confirm all behavioral classifications. In the spring, the rams were trucked from Dubois, ID, to Corvallis, OR, where they were housed together in a large, fenced pasture with free access to water. Blood sampling was initiated in the nonbreeding season after the rams had adapted to their surroundings for approximately 2 mo.
Procedures for Obtaining Blood
Daily conscious blood samples were drawn between 0900 and 1100 h from the jugular veins of all 30 rams during the last 2 wk of June (nonbreeding season) and then again for the same period of time in late October (breeding season). This was accomplished by jugular venipuncture after the rams were placed into a 30 ;ts 30 foot pen adjacent to their pasture. Three weeks after the final bleeding of the breeding season, 12 rams, 4 from each behavioral group, were transported to Portland, OR (
90 miles). The rams were tranquilized with Telazole (tiletamine HCl & zolazepam HCl, 2 mg/kg, i.m.; Fort Dodge Laboratories, Cherry Hill, NJ) and approximately 15–30 min later anesthetized with 15 mg/kg of sodium pentobarbital. Blood samples were then collected from both the jugular and the spermatic veins. Sera, harvested from blood clotted overnight at 4°C and centrifuged at 1000 ;ts g for 15 min, were stored frozen (-20°C) until assayed for steroids.
Hormone Measurements
Testosterone was quantified in 100 µl of serum by radioimmunoassay after extraction with ethyl ether and chromatography on Sephadex LH-20 (Sigma, St. Louis, MO) columns [10]. The assay extraction efficiency for testosterone was 66.3% ± 1.0%. Interassay and intraassay coefficients of variation were 9.6% and 6.5%, respectively. The lower limit of detectability, defined as the first point on the standard curve lying outside of 3 standard deviations from the counts per minute in tubes containing unlabeled hormone, was 3.2 pg/tube. Cortisol was measured in serum after extraction with ethyl ether by a radioimmunoassay using antibody R2P8 (ICN Biochemicals, Inc., Costa Mesa, CA) raised against cortisol-3-O-carboxymethyl ether:BSA. The antibody cross-reacts 11.4% with 21-desoxycortisol, 8.9% with desoxycorticosterone, 1.6% with corticosterone, and 0.6% with cortisone; but less than 0.1% with estradiol, testosterone, progesterone, and aldosterone. Incubations of multiple dilutions of serum extract were parallel with the standard curve. All samples were measured in duplicate in the same assay. Assay extraction efficiency for cortisol was 83.6% ± 3.6%. The intraassay coefficient of variation was 5.2% and the limit of detectability was 5.3 pg/tube. Concentrations of LH were determined in duplicate using NIH-anti-ovine-LH as primary antibody and NIH-ovine-LH-13 as the standard according to previously published methods [11]. The sensitivity of the assay ranged from 0.25 to 7 ng. The intraassay and interassay coefficients of variation were 13% and 15%, respectively.
Statistical Analysis
Hormone data were analyzed by use of one-way ANOVA. Significant overall effects were noted among groups of anesthetized rams, and differences among means were evaluated by the Newman-Keuls multiple range test. Behavior data were analyzed by the Wilcoxon signed rank test. Simple linear correlation coefficients were calculated to examine within-ram interrelationships between serum hormone concentrations. Probability values of P < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Each ram was given at least 36 performance tests over the course of 2 breeding seasons to identify FORs and potential MORs and NORs. In none of the performance tests did any ram, which was eventually classified as a MOR or a NOR, attempt to mount the stimulus ewe. The FORs used in this study were all high-libido males with an average mount frequency of 15.1 ± 2.0 and an ejaculation frequency of 3.2 ± 0.2 per 30-min test. Behavioral data from the last sexual preference test of males classified as MOR and NOR are presented in Table 1. Based on the results of the partner preference tests, males were classified as MOR if they consistently courted and mounted other males in preference to females. In this study, MORs directed significantly more behavior toward males than toward females in all behavioral categories except Flehmens and ejaculations, which were rarely displayed (Table 1). Males were classified as NOR if they demonstrated consistently low levels of courtship with no clear evidence of a preference for either a male or female stimulus animal and no mounting or ejaculatory behavior. All 16 NORs identified for this study met these criteria.
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Hormone Concentrations
Mean daily testosterone concentrations in serum samples obtained from conscious rams during the nonbreeding season are shown in Figure 1A. Serum concentrations of testosterone were erratic, showing variations in magnitude from 2-fold to 35-fold in individual animals over time, but there were no apparent differences among groups. Mean daily serum concentrations of testosterone collected over a 2-wk period for each of the 3 behaviorally characterized groups are shown in Figure 1B. Average concentrations did not differ significantly among the treatment groups. During the breeding season, concentrations of testosterone were generally increased 2-fold compared with concentrations in rams during the nonbreeding season, and still exhibited an erratic secretory pattern in individual rams (Fig. 2A). Average systemic serum concentrations of testosterone during the 14-day sampling period of the breeding season were equivalent in all behavioral groups (Figure 2B).
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Testosterone concentrations in serum from the jugular vein of rams that were tranquilized with Telazole and anesthetized with pentobarbital before the blood samples were collected are shown in Figure 3A. Mean concentrations of testosterone in the systemic circulation of anesthetized FORs differed significantly from NORs, but not from MORs. However, systemic concentrations of testosterone did not differ between NORs and MORs. Concentrations of testosterone in serum from spermatic veins of anesthetized FORs differed significantly from those in both MORs and NORs, but concentrations in spermatic veins were not significantly different between MORs and NORs (Figure 3B). Concentrations of testosterone in the spermatic vein were 10-fold to 20-fold higher than concentrations in the jugular vein. Concentrations of testosterone in the jugular vein and spermatic vein of the same anesthetized ram were positively correlated (r = 0.9928, P < 0.0001). However, concentrations of testosterone in the jugular vein of the same ram during conscious and unconscious states were not correlated (r = 0.1703, P > 0.05).
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Concentrations of LH in anesthetized rams did not differ significantly among groups (1.90 ± 0.17 ng/ml, mean ± SEM for all groups; n = 12).
Systemic cortisol was significantly higher in anesthetized MORs (25.1 ± 4.2 ng/ml) and NORs (27.2 ± 4.4 ng/ml) than in FORs (10.9 ± 1.8 ng/ml) (Fig. 4). Concentrations of cortisol and testosterone in the jugular vein of anesthetized rams were negatively correlated (r = -0.6695, P < 0.05). For comparison, cortisol concentrations were measured on the eighth day of sampling during the breeding season in the serum of the same rams while they were conscious. Systemic concentrations of cortisol in conscious rams did not differ significantly among groups (8.66 ± 0.99 ng/ml, mean ± SEM for all groups; n = 12). Systemic serum concentrations of cortisol and testosterone in conscious rams were not correlated (r = 0.3336, P > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In agreement with a study by Perkins et al. [6], we found that asexual rams had concentrations of testosterone that were well within the range of the high-libido FORs used in the current study. Most previous studies in other species have found no differences in circulating testosterone concentrations between sexually active and inactive males [12, 13]. Moreover, sexually unresponsive males are not induced to mate following treatment with high doses of exogenous testosterone [13–16]. These results taken together suggest that hypogonadism is not the primary cause for the failure of asexual rams to breed.
In contrast to the results in conscious rams, we confirmed our previous observation showing that systemic concentrations of testosterone after anesthetization are lower in MORs than in FORs. Furthermore, the present study provides new data showing that systemic testosterone concentrations are also lower in asexual rams after anesthesia. The differences in systemic concentrations of testosterone were accompanied by nearly identical group differences in the concentrations of testosterone in spermatic venous blood. These differences appear to result from an anesthesia-induced suppression of testosterone concentrations in MORs and NORs, which does not occur in FORs. Despite the low concentrations of testosterone in MORs and NORs compared with FORs, LH concentrations were not different among the groups. Previous studies using repeated sampling procedures did not observe differences in basal LH concentrations among similar behaviorally defined groups of rams [6, 17]. These results indicate that variations in testosterone concentrations may arise as a result of a difference in the functional responsiveness of the testis to LH rather than in the sensitivity of the hypothalamic-pituitary axis or in the peripheral metabolism and clearance of testosterone. The observation that the anterior pituitary of FORs, MORs, and NORs exhibit equivalent responses to a GnRH challenge provides additional evidence to suggest that the sensitivity of the anterior pituitary is not involved [5, 6]. However, testosterone concentrations before and after GnRH challenge tests were not correlated in asexual rams, although this association existed in high-performing FORs [6]. The less-predictable response in testosterone secretion by asexual rams to elevated LH suggests that in comparison to FORs, the testes of NORs may be less sensitive to the stimulatory effects of LH on androgen synthesis and secretion. Whether similar differences in testicular responsiveness exist between FORs and MORs has not yet been analyzed and would be an interesting avenue for further study.
The difference in hormone concentrations between conscious and anesthetized rams may relate to either a direct effect of anesthesia on the central nervous system, or to the acute stress induced by the physiologic reaction to anesthesia and immobilization. Various anesthetics are known to influence neuronal activity and thereby can directly modulate hypothalamic function. Although little is known specifically about Telazole, the dissociative anesthetics as a class do not appear to effect gonadal or adrenal hormone secretion in primates and sheep [18, 19]. In contrast, barbiturate anesthetics, such as pentobarbital, generally suppress endocrine functions mediated through the hypothalamus as well as systemic testosterone and cortisol concentrations in males [20, 21]. Anesthesia has been used as a controlled stressor to test the effect of stress on the pituitary-adrenal-testis axis in many species [22–26]. The significantly higher cortisol concentrations measured in anesthetized MORs and NORs compared with FORs suggest that the former two types of rams were relatively more stressed than the latter. In contrast, we found no group differences in serum concentrations of testosterone or cortisol after repeated daily blood sampling during the breeding season in conscious rams. Whether anesthesia is acting directly at the level of the neuron or as a stressor cannot be answered definitively from the present study alone and further studies will be needed to clarify this point. However, because in either case the endocrine response to anesthesia is most likely mediated through the central nervous system, the present results indicate that functional differences exist between the brains of rams that differ in sexual behavior expression and partner preference. This conclusion is supported by previous studies that demonstrated differences in aromatase activity [7], estrogen receptors [17], behaviorally induced neuronal fos responses [27], and neuronal soma sizes [28] among ram types.
The differences in anesthesia-induced concentrations of systemic corticosteroids could be in part responsible for the differential suppression of systemic androgens among the 3 types of rams. This idea is supported indirectly by the observation that serum concentrations of testosterone and cortisol concentrations are inversely correlated in anesthetized rams. In bulls and rats, plasma concentrations of cortisol are inversely related with the amount of testosterone secreted during stressful situations [29, 30]. It is well documented that corticosteroids have direct actions on the testis to suppress testosterone synthesis [31, 32]. Furthermore, acute dexamethasone treatment suppresses serum testosterone in rams and nonhuman primates, but it does not alter basal or GnRH-stimulated LH release [26, 33]. Similar to what we observed in rams, acute immobilization stress in rats is accompanied by decreases in systemic serum concentrations of testosterone and increases in systemic concentrations of corticosterone, but is not associated with concomitant decreases in serum LH concentrations [34]. Neither acute stress nor exogenous glucocorticoid administration to rats reduces the capacity for LH binding in Leydig cells [35]. Instead, the decline of systemic androgen appears to be mediated by increased concentrations of corticosterone acting through glucocorticoid receptors in testes to interfere with LH signaling and, in turn, to inhibit androgen synthesis [30]. Both acute stress and exogenous glucocorticoids have been shown to suppress androgen synthesis by inhibiting the activities of 3ß-hydroxysteroid dehydrogenase and 17
-hydroxylase/C17,20 lyase (P450c17) [35]. In our previous study [7], we noted that the capacity of testicular homogenates to biosynthesize 17-hydroxyprogesterone and testosterone from progesterone was significantly lower in MORs than in FORs. This conversion is dependent on the activity of P450c17, and it is interesting to speculate that this could reflect a further manifestation of the difference in adrenal response to anesthesia between MORs and FORs.
Individual differences in social behavior and stress responses have been described previously. In particular, Sapolsky [36] has reported that high-ranking males, by the criterion of reproductive activity, exhibit significantly lower cortisol concentrations than those of subordinates, when measured immediately after immobilization with anesthetic. These males appear to be more sensitive to negative feedback by glucocorticoids because they exhibit a faster and more extreme suppression of systemic cortisol in response to exogenous dexamethasone. Similarly, the present study suggests that ram social behaviors, as defined by sexual partner preference and sexual drive, seem to be associated with distinct endocrine responses under specific testing conditions (i.e., anesthesia or stress). Future studies are needed to further characterize the endocrine and neuroendocrine mechanisms underlying these associations.
Some anesthetics may have direct suppressive effects on the testis in addition to actions mediated through the adrenal axis. Thiopentane, a short-acting barbiturate, inhibits LH-stimulated testosterone output from mouse Leydig cell cultures [37]. Diazepam, like zolazepam in the Telazole preparation, is a benzodiazepine tranquilizer. Although chronic administration of diazepam blocks in vitro testicular androgen synthesis and reduces the concentration of peripheral benzodiazepine receptor in testis, acute administration is without effect [38]. Although available data are sparse, they do not support the possibility that the anesthesia used is acting directly at the level of the testis.
In summary, this study, together with our previously published research, demonstrate that circulating concentrations of testosterone are related to sexual orientation in anesthetized rams, but not in conscious rams. These results make it seem highly unlikely that differences in basal androgen concentrations in adulthood are responsible for the expression of male-oriented preferences or low libido in sheep. Instead, because the endocrine response to anesthesia is most likely coordinated through the central nervous system, we hypothesize that the neural substrates mediating these responses differ among FORs, MORs, and NORs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Institutes of Health grant RR14270 to C.E.R. 
2 Correspondence: Charles E. Roselli, Department of Physiology and Pharmacology L334, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098. FAX: 503 494 4352; rosellic{at}ohsu.edu
Accepted: February 7, 2002.
Received: November 3, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  J. N. Stellflug Comparison of cortisol, luteinizing hormone, and testosterone responses to a defined stressor in sexually inactive rams and sexually active female-oriented and male-oriented rams J Anim Sci, June 1, 2006; 84(6): 1520 - 1525. [Abstract] [Full Text] [PDF]
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 J. A. Morris, K. L. Gobrogge, C. L. Jordan, and S. M. Breedlove Brain Aromatase: Dyed-in-the-Wool Homosexuality Endocrinology, February 1, 2004; 145(2): 475 - 477. [Full Text] [PDF]
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C. E. Roselli, K. Larkin, J. A. Resko, J. N. Stellflug, and F. Stormshak The Volume of a Sexually Dimorphic Nucleus in the Ovine Medial Preoptic Area/Anterior Hypothalamus Varies with Sexual Partner Preference Endocrinology, February 1, 2004; 145(2): 478 - 483. [Abstract] [Full Text] [PDF]
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