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Testis |
a Population Council
b Rockefeller University, New York, New York 10021
c Institute of Anatomy II, Heinrich Heine University, D-40001 Duesseldorf, Germany
d Departments of Psychology and
e Anatomy and Reproductive Biology, University of Hawaii, Honolulu, Hawaii 96822
f Department of Psychiatry, University of Cincinnati, Cincinnati, Ohio 45267
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corticosterone, Leydig cells, luteinizing hormone, stress, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leydig cells are able to resist the inhibitory action of CORT through oxidative inactivation of the steroid by 11ß-hydroxysteroid dehydrogenase (11ßHSD) [7]. The type I form of 11ßHSD known to be present in Leydig cells [8, 9] is predominantly reductive in many other cell types when the enzyme's oxidative and reductive activities are assayed using intact cells [10, 11]. However, intact, freshly isolated, adult Sprague Dawley rat Leydig cells possess a net oxidative activity [12]. This implies that type I 11ßHSD activities are either dependent on context, such as by cell type-specific posttranslational modification of the protein, or that another isoform of 11ßHSD is present in Leydig cells in addition to type I.
The VBS provides a means of studying naturally occurring psychosocial stress in the laboratory setting (see [13] for review). Experiments employing the VBS involve a highly aggressive subline of the Long-Evans rat. Within the closed environment of the VBS, male rats establish a dominance hierarchy topped by a single dominant animal that repeatedly attacks other males, especially during the first few days of group housing. Subordinates show a number of behavioral changes, including reductions in aggression, activity, and sexual and social behaviors, and an increase in a range of defensive responses to the dominant male [13, 14]. Relative to dominants and to singly housed control, basal serum CORT values in subordinates have been shown to be significantly higher, 100 ng/ml in subordinates versus 38 ng/ml in dominants and 21 ng/ml in controls [15], and T to be lower, at the end of 14 days in the VBS [16]. Moreover, Spencer et al. [15] documented that of the three groups, subordinates have the lowest levels of plasma CORT binding globulin, suggesting that the unbound, physiologically active CORT in subordinates is markedly elevated compared to dominant and control animals. Since Leydig cell T production is directly inhibited by CORT in vitro [17], oxidative inactivation of CORT intracellularly by 11ßHSD in Leydig cells could determine the extent of glucocorticoid action. Lower levels of 11ßHSD oxidative activity in homogenates from testes of the subordinates led to the hypothesis that these animals have lower T because their Leydig cells are more susceptible to glucocorticoid-mediated inhibition compared to controls and dominants.
The goal of the present study was to assess the impact of psychosocial stress on Leydig cell function by measuring serum LH as well as T production and 11ßHSD activities in Leydig cells of rats after housing in the VBS. The amount of 11ßHSD reductive activity in Leydig cells of VBS rats is currently unknown. If the reductive activity were to predominate, the 11ßHSD in VBS Leydig cells would amplify glucocorticoid-mediated stress, which is inconsistent with a protective role. Therefore, the present study determined the predominant direction of catalysis by 11ßHSD in VBS rat Leydig cells. The activity was measured in intact cells because 11ßHSD reductive activity is rapidly lost after homogenization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The VBS apparatus and experimental procedure has been described in detail elsewhere [13, 18]. Briefly, a "colony" of four male and two female adult rats of the Long-Evans strain (maintained by the University of Hawaii Laboratory Animal Services) were housed in a VBS. Female rats served to potentiate male territoriality and the formation of dominance hierarchies. Food and water were available ad libitum in the surface area and the two side chambers. The VBS consisted of an 85 x 65-cm surface area connected to two smaller chambers fitted with clear Plexiglas tops, all connected by a series of clear Plexiglas tunnels. A 15-W incandescent bulb in the surface area provided the only light on a 12L:12D cycle, whereas the chambers and tunnels remained shielded from the light throughout the experiment. Activity within the VBS was recorded using a video camera and infrared light source mounted above the apparatus. Thus activity could be monitored in complete darkness during the lights-out period. Digital photos were taken to document each rat's unique coat pattern. This allowed us to identify every animal in videotapes of colony activity at night and facilitated behavioral analysis and determination of dominance. From the videotapes it was possible to score offensive and defensive behaviors and time spent on the VBS surface area for each animal. By using this information in conjunction with subject body weight, the dominant/subordinate status of each animal was determined (for further details see [13]). Control animals were age- and weight-matched single males that were housed with a single female in conventional cages. After 4, 7, or 14 days of group or pair housing, VBS and control males were decapitated and trunk blood and organs were collected for later use. In separate animals, blood was also taken through a small tail nick from VBS and control animals on Days 4, 7, and 14 of VBS housing. All animal protocols were approved by the University of Hawaii Institutional Animal Care and Use Committee, and the health of the animals was carefully monitored by veterinary staff throughout the experiment.
Leydig Cell Isolation
Leydig cells were isolated from individual male Long-Evans rats by centrifugal elutriation followed by Percoll density gradient centrifugation using the technique of Klinefelter et al. [19]. This procedure was used immediately following killing of the animals without modification to obtain Leydig cells from adult Long-Evans rats between 60 and 90 days of age. The purity of the isolated cell fraction was evaluated by histochemical staining for 3ß-hydroxysteroid dehydrogenase enzyme activity, with 0.4 mM etiocholanolone as the steroid substrate [20]. Enrichment of Leydig cells was typically 95%, regardless of age.
Serum Testosterone, LH, and Corticosterone Concentrations
Blood samples were centrifuged at 500 x g, and the sera were stored at -20°C until RIA of T, LH, and CORT. Serum T concentrations were measured with a tritium-based RIA as previously described [21]. Interassay variation of the T, LH, and CORT RIA was, respectively, between 7% and 8%, 4% and 5% and 7% and 8%. Serum LH concentrations were measured using the method from Chandrashekar et al. [22]. Rat LH standards NIDDK-r-LH-I9 and rat LH antibody NIDDK-anti-rLH-S-11 were obtained through the National Hormone and Pituitary Program. Radioactive 125I-rat LH was obtained through Covance Laboratories (Vienna, VA), and IgG antiserum was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Serum CORT was measured using the method from Spencer et al. [15]. The CORT antiserum B3-163 was obtained from Endocrine Sciences (Calabasas, CA).
Analysis of 11ßHSD Activity
The levels of 11ßHSD activity in Leydig cell samples from individual control and VBS rats were evaluated as described previously [23]. In brief, intact Leydig cells were incubated for 10 min in the presence of radiolabeled substrates for the oxidative CORT to dehydrocorticosterone (11DHC) or reductive dehydrocorticosterone to CORT reactions. The reactions were terminated by the addition of ice-chilled ethyl acetate. After extraction of the organic phase, each sample was dried under nitrogen gas, resuspended in ethyl acetate, and subjected to thin-layer chromatography to resolve bands corresponding to CORT and 11DHC. The amount of steroid in each band was quantified by radiometric scanning (System 200/AC3000, Bioscan, Inc., Washington, DC). Steady-state mRNA levels for type I 11ßHSD were estimated after amplification by RT-PCR as previously published [23]; ribosomal S16 mRNA was used as an internal control.
Immunohistochemical Localization of Type I 11ßHSD
At the end of 14 days of VBS housing, testes from dominants (n = 5), subordinates (n = 5), and controls (n = 4) were removed, quickly frozen, and subsequently processed for type 1 11ßHSD immunohistochemistry. Testes were paraffin embedded and 6-µm-thick sections were cut, mounted, and dewaxed just prior to immunohistochemical staining. The sections were incubated in a humidified chamber as follows: 3% H2O2 for 5 min; 0.05 M Tris buffer (pH 7.6) washed for 5 min; incubated in donkey serum diluted in Tris buffer 1:10 for 30 min; and incubated in primary antibody sheep anti-human type I 11ßHSD (from Binding Site, Birmingham, U.K.; dilution 1:2000 in Tris buffer) at 4°C overnight [24]. The following morning the sections were washed in buffer for 5 min; incubated in secondary antibody rabbit anti-goat 1:100 diluted in Tris buffer supplemented with 100 µl rat serum for 30 min; washed in buffer for 5 min; and followed by a goat peroxidase-antiperoxidase diluted 1:100 in Tris buffer incubation for 30 min. The sections were then washed in buffer for 5 min, followed by a 10-min incubation in DAB/H2O2 solution for 10 min. They were then washed for 5 min in distilled H2O and subsequently dehydrated in serial isopropyl alcohol baths and cover-slipped. Negative controls were run in parallel with Tris buffer in place of primary antibody.
11ßHSD Enzyme Histochemical Activity
On Days 4, 7, and 14, testes were removed and quickly frozen in liquid nitrogen at -190°C and then kept frozen at -80°C. Ten-micrometer-thick cryostat sections were prepared at -20°C and prefixed in acetone at 4°C for 3 min. The incubation solution contained 2.5 mM CORT as substrate and 2 mM NADP as a cofactor. In addition, the incubation medium contained tetranitroblue tetrazolium as a H+ acceptor, 0.1 M potassium cyanide, 0.05 M magnesium chloride, 20% polyvinyl alcohol, and 0.1 phosphate buffer at pH 7.5. The substrate was dissolved in dimethylformamide. Just prior to use, 0.2 mM phenazine methosulfate was added to the incubation medium as an intermediate electron acceptor. The sections were incubated for 75 min at 37°C in a humidified chamber in darkness. Additional sections were incubated without substrate as negative controls in order to determine nonspecific dehydrogenase effect. The sample included 25 rats on Day 4, 33 rats on Day 7, and 32 rats on Day 14 of VBS housing. The enzyme reaction was independently evaluated in the light microscope by two different observers who did not know the dominance status of the animals. The staining intensities of the reaction products were graded on the following scale: + (weak), ++ (moderate), +++ (strong), and ++++ (very strong).
Statistics
The data were analyzed by one-way analysis of variance (ANOVA) followed by planned comparisons with the Sidak adjustment of the P value, estimating the experiment-wise error rate, to identify significant differences between dominant and control or between subordinate and control [25]. All data are expressed as means ± SEM. Differences were regarded as significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Luteinizing hormone Serum LH concentrations in dominant animals exceeded control values on Day 4, but did not differ on Days 7 and 14 (Fig. 1). The time of sampling on Day 4 corresponded to a period of more frequent aggressive encounters between the dominant and subordinate males, with the dominants emerging as the victors. In contrast, LH declined in subordinates to approximately 0.1 ng/ml on Day 7, but did not differ from control on Days 4 and 14.
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Testosterone Serum T concentrations in dominant males were consistent with the trends observed for LH (Fig. 2). On Day 4, T levels were higher in the dominants relative to control males, whereas the two groups were equivalent on Days 7 and 14. These results indicated a relationship between elevated T in dominant males during the first few days of housing in the VBS and their victories in aggressive encounters. Serum T levels were lower in subordinate males relative to controls during Days 7 and 14, and the reproductive suppression may be associated with the establishment of social subordination.
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Corticosterone Serum CORT concentrations in dominant animals exceeded control values on Day 4 (P = 0.05) but did not differ on Days 7 and 14 (Fig. 3). In subordinate males, CORT levels were higher than control on Days 4 and 7. Neither dominants nor subordinates differed from control on Day 14, although this may have resulted from an apparent upward variation in control values. These results were consistent with an endocrine stress response in the subordinate males.
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11ßHSD Enzyme and Immunohistochemical Activity
Immunohistochemical reaction for type I 11ßHSD showed strongest intensity in the Leydig cells of the dominant animals (Fig. 4C) compared with control (Fig. 4A) and subordinates (Fig. 4B) in the case of VBS housing after 14 days. No specific staining was observed in the negative control sections (data not shown). High levels of 11ßHSD enzyme activity were observed in the cytoplasm of the Leydig cells of the control rats (Fig. 4D). The intensity in subordinate rats was lower after 4 days of VBS housing relative to control (Fig. 4E), but did not differ on Days 7 and 14. Dominant rats had a stronger 11ßHSD staining intensity compared with control and subordinates, which was maintained on Days 4, 7, and 14 (Fig. 4F, Table 1). The negative control sections, incubated without substrate, only had nonspecific precipitates within seminiferous tubules, particularly in the sperm flagellum.
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11ß-Hydroxysteroid Dehydrogenase Enzyme Activity
11ßHSD oxidative and reductive activities were measured in purified Leydig cells on Days 4, 7, and 14 of VBS housing (Fig. 5). Oxidative activity exceeded reductive activity in all groups. This corroborated earlier data showing that 11ßHSD is a predominant dehydrogenase in adult rat Leydig cells, which enables these cells to oxidatively inactivate CORT [26]. Oxidative activity in Leydig cells of dominant animals was consistently higher than control at all three time points. In contrast, oxidative activities in Leydig cells from subordinate males were equivalent to control on Days 4, 7, and 14. Reductive activities were unchanged except in subordinate males on Day 7 when the enzyme activity was elevated. These results suggested that, relative to control and subordinates, Leydig cells from dominant males were more resistant to direct inhibition of T production due to increases in serum CORT concentrations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two phases in the endocrine response to VBS housing were observed. In the initial housing period tested on Day 4, serum LH, T, and CORT were higher in dominant males relative to control. We attribute the increases in androgen levels to aggressive encounters in which the dominants emerged as victors. During the second phase, changes in endocrine parameters shifted to the subordinates: T levels were lower on Days 7 and 14, which was consistent with reproductive suppression resulting from social stress. The trends in T concentrations during VBS housing appeared to follow the trends for LH, with the exception of Day 14. On Day 14, subordinate males had lower serum T values, whereas their LH values did not differ from control. The lower T values on Day 14 would normally be expected to elicit increases in LH because of alleviation of the negative feedback effects of androgen. LH concentrations were not increased in subordinates on Day 14, and these results were therefore consistent with inhibition of the reproductive axis in subordinate males at the level of the hypothalamus and pituitary. The condition of the subordinate rats is analogous to primary hypogonadism with secondary hypopituitarism. Reminiscent of may endocrinopathies. It is likely that the elevated CORT levels suppress LH secondarily, thus preventing the rebound in secretion when T is decreased. The data are consistent with the inhibition of the reproductive axis being primarily due to a Leydig cell effect.
In addition to a decline in gonadotropic stimulation, several stress-induced factors with receptor-mediated effects on Leydig cells have been postulated to cause reduced T production. These include catecholamines, CORT, arginine vasopressin, and proopiomelanocortin-derived (POMC-derived) peptides (see [27, 28] for reviews). However, catecholamines and POMC-derived peptides such as endorphin have been found to stimulate T production by Leydig cells in vitro. Arginine vasopressin, which consistently inhibits T levels, is not always increased by stress. The suppressive effect of CORT on Leydig cell steroidogenesis is well-established, and Monder et al. [16] demonstrated an inverse relationship between serum T and CORT in a previous study of rats housed in the VBS. Therefore it is likely that increased levels of CORT action are involved in the suppression of T in subordinates on Days 7 and 14. The magnitude of CORT activity is determined by the circulating levels of the steroid, the relative degree of CBG, and by the degree of inactivation catalyzed by 11ßHSD.
The measurement of 11ßHSD enzyme activities in intact Leydig cells showed that oxidation exceeded reduction in all three groups of rats. This agreed with earlier data using intact Sprague Dawley rat Leydig cells [26] and corroborated the hypothesis that 11ßHSD in Leydig cells is a net oxidase. Other investigators have proposed that 11ßHSD reduction predominates in rat Leydig cells [29, 30], and it is now clear that the two activities of the enzyme can be influenced by assay conditions. Predominantly oxidative activity occurs in freshly isolated, intact adult Leydig cells in the presence of physiological concentrations of substrate 25 nM. If 11ßHSD is studied in immature Leydig cells, after prolonged culture in vitro, or in the presence of high substrate concentrations greater than 50 nM, the ratio of oxidative to reductive activity varies, typically favoring the reductase.
The rates of oxidative 11ßHSD activity were higher in Leydig cells from dominant males at all three time points during VBS housing. The enzyme histochemical observations on the oxidative enzyme reaction in situ support this finding and indicate that the difference did not arise from in vitro conditions. The immunohistochemical data provide confirmation of increased type I 11ßHSD protein in Leydig cells of dominant rats. It is notable that serum CORT levels in dominant rats are significantly elevated with respect to control [18]. Studies of the hormonal regulation of 11ßHSD in Leydig cells have shown that glucocorticoid treatment in vitro increases 11ßHSD oxidation [26]. In contrast, despite higher serum CORT levels in subordinates [31], 11ßHSD oxidative activities were unchanged and reductive activity was elevated on Day 7. In the presence of an increased glucocorticoid load, Leydig cells in the subordinates did not compensate through increased levels of 11ßHSD oxidative inactivation. Indeed, on Day 7 the 11ßHSD activity became relatively more reductive, which would regenerate additional CORT from inactive 11-dehydro-CORT. The enzyme histochemistry can detect only the oxidative component of 11ßHSD, but the results clearly confirmed the trends that were obtained with isolated Leydig cells in vitro. These results differed from but were not inconsistent with the findings of Monder et al. [16] who performed measurements on homogenates, rather than intact cells. Our observation that 11ßHSD oxidative activity was higher in dominant than in control Leydig cells could explain how rats maintain T levels in the presence of elevated serum CORT. The levels of 11ßHSD oxidative activity in homogenates prepared from the thymus gland were unchanged in VBS-housed rats despite significant reductions in thymus gland weight in both dominants and subordinates relative to control [32]. However, it will be necessary to reinvestigate the trends for thymus given the instability of 11ßHSD reductive activity in homogenized tissues.
In conclusion, two types of behaviorally induced changes occur during housing in the VBS. Initially on Day 4, dominant males had elevated T levels associated with their victorious encounters with subordinates. Subsequently, on Days 7 and 14 T levels were lower in the subordinate males due in part to suppressed LH secretion. Direct effects of glucocorticoid on Leydig cells were implicated by the observation that dominant males had consistently higher capacities to inactivate this steroid through 11ßHSD oxidation. Conversely, insufficient 11ßHSD oxidative activity in subordinate males is postulated to increase the sensitivity of their Leydig cells to glucocorticoid inhibition.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Matthew P. Hardy, Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 7678; mhardy{at}popcbr.rockefeller.edu
Received: 10 April 2002.
First decision: 9 May 2002.
Accepted: 25 June 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxylase and 17,20-lyase without affecting the binding of LH/hCG receptors. J Androl 1994 15:302-308-dihydrotestosterone, 5-androstane-3,17ß-diol, 5-androstane-3ß,17ß-diol and 17ß-estradiol from male beagles with spontaneous or induced benign prostatic hyperplasia. Invest Urol 1981 19:142-147[Medline]This article has been cited by other articles:
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  L. J Martin and J. J Tremblay Glucocorticoids antagonize cAMP-induced Star transcription in Leydig cells through the orphan nuclear receptor NR4A1 J. Mol. Endocrinol., September 1, 2008; 41(3): 165 - 175. [Abstract] [Full Text] [PDF]
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 IN MEMORIAM Matthew P. Hardy, Ph.D. 1957-2007 Biol Reprod, March 1, 2008; 78(3): 563 - 564. [Full Text] [PDF]
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 M. M. N. Nguyen, K. L. K. Tamashiro, S. J. Melhorn, L. Y. Ma, S. R. Gardner, and R. R. Sakai Androgenic Influences on Behavior, Body Weight, and Body Composition in a Model of Chronic Social Stress Endocrinology, December 1, 2007; 148(12): 6145 - 6156. [Abstract] [Full Text] [PDF]
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 K. L. K. Tamashiro, M. M. N. Nguyen, M. M. Ostrander, S. R. Gardner, L. Y. Ma, S. C. Woods, and R. R. Sakai Social stress and recovery: implications for body weight and body composition Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2007; 293(5): R1864 - R1874. [Abstract] [Full Text] [PDF]
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 J. W. Tomlinson, E. A. Walker, I. J. Bujalska, N. Draper, G. G. Lavery, M. S. Cooper, M. Hewison, and P. M. Stewart 11{beta}-Hydroxysteroid Dehydrogenase Type 1: A Tissue-Specific Regulator of Glucocorticoid Response Endocr. Rev., October 1, 2004; 25(5): 831 - 866. [Abstract] [Full Text] [PDF]
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 C. E. Wood, J. M. Cline, M. S. Anthony, T. C. Register, and J. R. Kaplan Adrenocortical Effects of Oral Estrogens and Soy Isoflavones in Female Monkeys J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2319 - 2325. [Abstract] [Full Text] [PDF]
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