Effects of Repeated Ether Stress on the Hypothalamic-Pituitary-Testes Axis in Adult Rats with Special Reference to Inhibin Secretion

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1 Effects of Repeated Ether Stress on the Hypothalamic-Pituitary-Testes Axis in Adult Rats with Special Reference to Inhibin Secretion Atsushi TOHEI, Taeko TOMABECHI, Masayuki MAMADA, Makoto AKAI, Gen WATANABE, and Kazuyoshi TAYA Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan (Received 3 October 1996/Accepted 17 December 1996) ABSTRACT. Effects of ether stress on the hypothalamo-hypophysial-gonadal axis in adult male rats were examined. To clarify the role of adrenal glucocorticoids in gonadal function, the effects of adrenalectomy and Dexamethasone treatment were also investigated. Ether stress increased the plasma concentrations of ACTH and corticosterone, but decreased the plasma concentrations of LH, FSH, inhibin and testosterone. The pituitary responsiveness to LH-RH for LH release and testicular responsiveness to the endogenous LH for testosterone release were maintained in stressed rats. Adrenalectomy caused an increase in the plasma concentrations of ACTH, but decreased the plasma concentrations of LH, FSH and testosterone. Dexamethasone treatment in adrenalectomized rats recovered the levels of plasma gonadotropins to control levels. The concentration of plasma inhibin did not change in adrenalectomized rats, but it was decreased compared to control rats by Dexamethasone treatment. Treatments of Dexamethasone in intact male rats resulted in a decline in plasma levels of testosterone and inhibin without a decrease in the levels of LH and FSH, indicating the direct effect of Dexamethasone on the testes. These results indicate that increased ACTH secretion in stressed rats is probably due to hypersecretion of CRH from the hypothalamus, which suppresses gonadotropin secretion via the inhibition of LH-RH. The decreased levels of testosterone may be caused by a stress-induced decrease in plasma LH concentrations and increased secretion of corticosterone in the ether stressed rats. The low levels of plasma inhibin in stressed rats was also probably due to the direct effect of corticosterone on the Sertoli cells. KEY WORDS: corticoid, CRH, gonad, inhibin, stress. J. Vet. Med. Sci. 59(5): , 1997 One of the consequences of stress is disruption to the reproductive functions of mammals [26]. Stress is accompanied by both an increase in the activity of the hypothalamic-pituitary-adrenal (HPA) axis and a decrease in the activity of the hypothalamic-pituitary-gonadal (HPG) axis [33]. These observations have suggested a possible relationship between hormonal regulation of the HPA axis and those of HPG axis. Stress-related hormones, for example, corticotropin releasing hormone (CRH), pro-opiomelanocortin (POMC)- derived peptides and adrenal corticosteroids can influence the reproductive function at all three levels of the HPG axis [26]. It has been reported that a high concentration of serum corticosterone increases sensitivity to the negative feedback effects of testosterone and inhibits the testicular function in rats [41]. In humans, the inhibition of sexual functions has often been observed during pathological circumstances accompanied by elevated levels of circulating corticosteroids (such as Cushing s syndrome), and usually disappears after a return to the normal adrenal function [16, 19, 34]. Arianavarreta et al. [3] reported that not only the adrenal cortex, but also the medulla may be involved in gonadotropin inhibition during chronic stress. It has also been reported that β-endorphin, a major product of the maturation process of POMC and CRH, participate either directly or indirectly in the inhibition of luteinizing hormone releasing hormone (LH-RH) neuronal activity during stress [26]. Inhibin is a glycoprotein hormone secreted from Sertoli cells and is now known to be one of the important gonadal hormones which regulate the secretion of follicle-stimulating hormone (FSH) [13]. Many kinds of stress, such as immobilization, psychological stress and electro-shocks have been reported to increase activity of the HPA axis and inhibit reproductive functions [26, 27, 30]. But the effects of stress on inhibin secretions are not clearly understood. In the present study, we investigated the effects of repeated ether stress on HPA and HPG axes, especially inhibin secretions in adult male rats. MATERIALS AND METHODS Animals: One hundred sixty five adult male rats ( g) of Wistar strain were used throughout the present study. The animals were maintained on a 14 hr light and 10 hr dark lighting schedule (light on at 05:00), and the room was kept at C. They received a standard laboratory diet and water ad libitum. One group of 5 animals was used in each experiment. Effects of repeated ether anesthesia combined with blood withdrawal: Two groups of animals were used in this experiment. One group of animals was anesthetized 6 times at 1-hr intervals with ether. Blood samples (1 ml) were taken from the jugular vein under ether anesthesia and centrifuged for the determination of plasma concentrations of adrenocorticotropic hormone (ACTH), corticosterone, luteinizing hormone (LH), FSH, inhibin and testosterone. The red blood cells were not returned to the animal. The other group of animals was sacrificed by decapitation without anesthesia as a control group and trunk blood was collected. To investigate the effects of ether anesthesia on the pituitary responsiveness to LH-RH, LH-RH challenges were performed. LH-RH (250, 500 or 1,000 ng) dissolved in 0.2

2 330 A. TOHEI, ET AL. ml 50% polyvinylpyrrolidone were injected subcutaneously. Blood samples (1 ml) were taken 6 times at 1-hr intervals from the jugular vein under ether anesthesia. Roles of adrenal corticosteroids in HPG axis: To investigate the roles of adrenal glands in gonadal functions, the animals were adrenalectomized or sham operated. They were sacrificed by decapitation on days 0, 1 and 2 after the operation. Adrenalectomized rats were treated intraperitoneally with Dexamethasone (1,320 µg, Decadron, Banyu Pharmaceutical Co., Ltd, Tokyo, Japan) on days 1 and 2 after the operation. To investigate the effects of glucocorticoid on HPG axis, intact animals were injected intravenously with two doses of Dexamethasone (500 µg, 2,000 µg) dissolved in saline. Blood was collected by decapitation at 0, 1, 2, 6, 12 and 24 hr after the injection. Radioimmunoassay (RIA): Concentrations of LH and FSH were measured using NIDDK rat RIA kits for rat LH and FSH. The hormones for iodination were rat LH-I-7 and rat FSH-I-7. The antisera used were anti-rat LH-S-10 and antirat FSH-S-11. The results were expressed in terms of NIDDK rat LH-RP-2 and FSH-RP-2, and the intra- and inter-assay coefficients of variation were 5.5 and 13.9 for LH and 7.9 and 17.5 for FSH, respectively. ACTH [38], inhibin [11], testosterone [39] and corticosterone [14] were measured by double-antibody RIAs using 125 I-labeled radioligands as described previously. Antisera to testosterone and corticosterone were kindly provided by Dr. G. D. Niswender (Colorado State University, Fort Collins, CO, U.S.A.). The intra- and interassay coefficients of variation were 11.3 and 11.9 for ACTH, 9.5 and 16.4 for testosterone, and 9.8 and 17.5 for corticosterone, respectively. Statistical analyses: All results are expressed as the mean ± SEM. The data was analyzed by Student s t-test but when more than two means were compared, analysis of variance was carried out and the significance of the difference between the means was determined by Duncan s multiple range test [33]. RESULTS Effects of repeated ether anesthesia combined with blood withdrawal: Compared with the control rats, plasma concentrations of ACTH and corticosterone were increased throughout the experimental period in the ether anesthetized rats (Fig. 1). On the other hand, the plasma concentrations of LH, FSH, inhibin and testosterone in the ether stressed rats significantly decreased compared to the decapitated rats by the second administrations of ether, and continued to decrease in a manner dependant on the number of times of ether administration (Fig. 1). Plasma concentrations of LH and testosterone significantly increased in anesthetized rats after the administration of 3 different doses of LH-RH in a dose related manner (Fig. 2). Plasma concentrations of FSH also increased in anesthetized rats after LH-RH challenges Fig. 1. Plasma levels of (a) ACTH, (b) LH, (c) FSH, (d) corticosterone, (e) testosterone and (f) inhibin in ether anesthetized ( ) and decapitated ( ) rats. *P<0.05 compared to control group (Student s t-test). though the plasma concentrations of FSH were not dose dependant (Fig. 2). Roles of adrenal corticoids in HPG axis: Adrenalectomy caused an increase in plasma concentrations of ACTH, but Dexamethasone treatment decreased the plasma concentrations of ACTH to control levels. Plasma concentrations of LH and FSH had decreased 1 day after adrenalectomy, and Dexamethasone treatment recovered them to control levels (Fig. 3). Plasma levels of testosterone also decreased in adrenalectomized rats, and decreased further as compared to that of adrenalectomized rat by Dexamethasone treatment. The concentration of plasma inhibin did not change in adrenalectomized rats, but decreased significantly, compared to the control rats, by Dexamethasone treatment in adrenalectomized rats. Both doses (500 µg and 2,000 µg) of Dexamethasone administrated to intact rats caused a decrease in the plasma concentrations of inhibin and testosterone by 2 hr after the treatment, and these low levels were maintained until 12 hr after the treatment (Fig. 4). Plasma concentrations of testosterone recovered to control levels by 24 hr, but the levels of inhibin were not recovered by 24 hr after the treatment. Plasma concentrations of LH were unchanged until 12 hr after treatment with Dexamethasone, but significantly increased by 24 hr after the treatment. The concentration of plasma FSH was not altered in Dexamethasone treated rats.

3 EFFECTS OF STRESS ON GONADAL AXIS 331 Fig. 3. Plasma concentrations of (a) ACTH, (b) LH, (c) FSH, (d) testosterone and (e) inhibin in adrenalectomized rats (solid bars), adrenalectomized rats treated with Dexamethasone (hatched bars) and control rats (open bars). *P<0.05 compared to control group (Student s t-test). Fig. 2. Plasma levels of (a) LH, (b) FSH and (c) testosterone in ether anesthetized rats after a single sc administration of 0 ( ), 250 ( ), 500 ( ), or 1,000 ( ) ng LH-RH dissolved in Polyvinylpyrrolidone (PVP). Fig. 4. Plasma concentrations of (a) LH, (b) testosterone, (c) FSH and (d) inhibin after a single i.v. injection of 0 ( ), 500 ( ) or 2,000 ( ) µg Dexamethasone in adult male rats. *P<0.05 compared to control group (Duncan s multiple range test).

4 332 A. TOHEI, ET AL. DISCUSSION Repeated ether anesthesia caused a decrease in the plasma concentrations of LH, FSH, inhibin and testosterone, however, the pituitary responsiveness to LH-RH for LH release and the testicular responsiveness to the endogenous LH for testosterone release were still maintained, since plasma concentrations of both hormones were increased after administration of LH-RH in a dose dependent manner. These results suggest that pituitary and testicular functions were not affected in ether stressed rats, and the decreased levels of plasma LH in stressed rats may be due to a reduction of LH-RH release from the hypothalamus. Compared to control rats, plasma concentrations of ACTH and corticosterone increased in anesthetized rats. It has been reported that CRH is an important factor in ACTH release induced by ether stress, and blockade of its effects by antiserum eliminated the ACTH response to ether [5, 21]. Vasopressin and oxytocin have also been implicated in ether stressed rats using either the antagonist or immunoneutralizing antibodies, either of which can reduce the ACTH response [5, 22]. Adrenalectomy also increased the plasma levels of ACTH, and these increases were restored to control levels by administration of Dexamethasone. Plasma concentrations of LH, FSH and testosterone decreased in adrenalectomized rats. Plasma LH and FSH recovered to control levels after administration of Dexamethasone, although plasma levels of testosterone did not recover. Plasma concentrations of testosterone and inhibin decreased in intact rats treated with Dexamethasone, while plasma concentrations of LH and FSH were unchanged with the exception of the plasma concentration of LH 24 hr after the administration. The high level of plasma LH at 24 hr after Dexamethasone administration probably depends on the low levels of testosterone in the treated rats. These results suggest that Dexamethasone affects the testes directly and causes a decrease in the plasma concentrations of testosterone and inhibin, whereas adrenalectomy suppresses the gonadal function at hypothalamic-pituitary levels. It has been reported that adrenalectomy, as a model of adrenal dysfunction, inhibits the pituitary LH release [32, 40], markedly enhances the staining for CRH [1 2, 24] and increases the amounts of mrna transcripts of CRH gene [2] in the hypothalamic paraventricular nucleus (PVN) which contains glucocorticoid receptors [25]. In addition, an increase in the concentrations of CRH in hypophysial portal blood [15, 20] and the median eminence (ME) [12, 36] after adrenalectomy has also been reported. One of the main factors known to exert a potent inhibitory influence on LH-RH secretion is CRH [26 29]. It has been shown that CRH inhibits the in vitro release of LH-RH at the level of neurosecretory terminals in the ME [10], which have high concentrations of both CRH [37] and CRH receptors [7]. There is also immunocytochemical evidence for direct synaptic connections between CRH and LH-RH containing neurons [18]. In the present study, repeated ether anesthesia of rats decreased their plasma concentrations of inhibin and Dexamethasone treatment in intact rats also decreased plasma levels of inhibin. The decrease in plasma levels of inhibin was probably due to the inhibitory effects of increased corticosterone in ether stressed rats. Glucocorticoids are known to inhibit testicular function, both in vivo and in vitro [4, 8, 31], and the presence of glucocorticoid receptors in Leydig cells [9] and Sertoli cells [17] has been demonstrated. It has been reported that immobilization caused a suppression of testicular steroidogenesis [23] by inhibiting the activities of steroidogenic enzymes [23] or by a stress-induced decrease in plasma concentrations of LH [6], while inhibin remained unaffected [6]. Repeated ether stress may be effective at inducing high levels of plasma corticosterone, and the increased corticosterone probably suppresses the secretion of inhibin from Sertoli cells. Recently, receptors and mrnas for CRH are found in rodent testes [26, and CRH secreted from the testes may also suppress the functions of Leydig cells by autocrine or paracrine reaction. In summary, repeated ether stress increased the plasma concentrations of ACTH and corticosterone. The increased ACTH secretion is probably due to hypersecretion of CRH from hypothalamus, which suppresses gonadotropin secretion via the inhibition of LH-RH. Decreased levels of testosterone may be caused by a stress-induced decrease in plasma LH concentrations and the increased secretion of corticosterone. Low levels of plasma inhibin are also probably due to the direct effect of corticosterone on the Sertoli cells. ACKNOWLEDGEMENTS. We are grateful to the Rat Pituitary Hormone Distribution Program, NIDDK, NIH, Bethesda, MD, U.S.A. for providing RIA materials; Dr. G. D. Niswender, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Co, U.S.A. for providing antisera to corticosterone (GDN B377) and testosterone (GDN 250). This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education of Japan (No ). A. Tohei received a fellowship from the Japan Society for the Promotion of Science (JSPS). REFERENCES 1. Antoni, F. A., Parcovits, M., Makara, G. B., Linton, E. A., Lowry, P. J., and Kiss, J. Z Immunoreactive corticotropin releasing hormone (CRF) in the hypothalamo-infundibular tract. Neuroendocrinology 36: Antoni, F. A Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor. Endoc. Rev. 7: Arianavarreta, C., Calderon, M. D., Tresguerres, J. A. F., and Lopez-Calderon, A Effect of adrenomedullectomy and propranolol treatment on the response of gonadotropins to chronic stress in male rats. J. Endocrinol. 120:

5 EFFECTS OF STRESS ON GONADAL AXIS Bambino, T. H. and Hsueh, A. J. W Direct inhibitory effect of glucocorticoids upon testicular luteinizing hormone receptor and steroidogenesis in vivo and in vitro. Endocrinology 108: Bruhn, T. O., Plotsky, P. M., and Vale, W. W Effect of paraventricular lesions on corticotropin-releasing factor-like immunoreactivity into the stalk-median eminence: studies on the adrenocorticotropin response to ether stress and exogenous CRF. Endocrinology 114: Demura, R., Suzuki, T., Nakamura, S., Komatsu, H., Odagiri, E., and Demura, H Effect of immobilization stress on testosterone and inhibin in male rats. J. Androl. 10: DeSouza, E. B., Perrin, M. H., Insel, T. R., Riviel, J., Vale, W., and Kuhar, M. J Corticotropin-releasing factor receptors in rat forebrain : autoradiografic identification. Science 224: Doerr, P. and Pirke, K. M Cortisol-induced suppression of plasma testosterone in normal adult males. J. Clin. Endocrinol. Metab. 43: Evain, D., Morera, A. M., and Saez, J. M Glucocorticoid receptors in interstitial cells of the rat testes. J. Steroid. Biochem 7: Gambacciani, M., Yen, S. S. C., and Rasmussen, D. D GnRH release from the mediobasal hypothalamus: in vitro inhibition by corticotropin-releasing factor. Neuroendocrinology 43: Hamada, T., Watanabe, G., Kokuho, T., Taya, K., Sasamoto, S., Hasegawa,Y., Miyamoto, K., and Igarashi, M Radioimmunoassay of inhibin in various mammals. J. Endocrinol. 122: Holemes, M. C., Antoni, F. A., Catt, K. J., and Aguilera, G Predominant release of vasopressin vs corticotropin releasing factor from the isolated median eminence after adrenalectomy. Neuroendocrinology 43: dejong, F. H Inhibin. Physiol. Rev. 68: Kanesaka, T., Taya, K., and Sasamoto, S Radioimmunoassay of corticosterone using 125 I-labeled radioligand. J. Reprod. Dev. 38: Kooy, A., de Greef, W. J., Vreeburg, J. T. M., Hackeng, W. H. L., Ooms, M. P., Lamberts, S. W. J., and Weber, R. F. A Evidence for the involvement of corticotropin-releasing factor in the inhibition of gonadotropin release induced by hyperprolactinemia. Neuroendocrinology 51: Luton, J. P., Thieblot, P., Valcke, J. C., Mahoudeau, J. A., and Bricaire, H Reversible gonadotropin deficiency in male Cushing s disease. J. Clin. Endocrinol. Metab. 45: Levy, F. O., Ree, A. H., Eikvar, L., Govindan, V. M., Jahnsen, T., and Hansson, V Glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells. Endocrinology 124: MacLusky, N. J., Naftolin, F., and Leranth, C Immunocytochemical evidence for direct synaptic connections between corticotropin-releasing factor (CRF) and gonadotropin-releasing factor (GnRH)-containing neurons in the preoptic area of the rat. Brain Res. 439: MacKenna, T. J., Lorber, D., Lacroix, A., and Rabin, D Testicular activity in Cuhing s disease. Acta Endocrinol. (Copenh.) 91: Oliver, C., Conte-Devolx B., Rey, M., Boudouresque, F., Giraud, P., Castanas, E., and Porter, J. C Immunoreactive 41-CRF in hypophysial portal blood of intact and adrenalectomized rats. Acta Endocrinol. (Copenh) 103 (Suppl. 256): Ono, N., Samson, W. K., McDonald, J. K., Lumpkin, M. D., Berdan de Castro, J., and McCann, S. M Effects of intravenous and intraventricular injection of antisera directed against corticotropin-releasing factor on the secretion of anterior pituitary hormones. Proc. Natl. Acad. Sci. U.S.A. 82: Ono, N., Berdan de Castro, J., Khorram, O., and McCann, S. M Role of arginine vasopressin in control of ACTH and LH release during stress. Life Sci. 36: Orr, T. E., Taylor, M. F., Bhattacharyya, A. K., Collins, D. C., and Mann, D. R Acute immobilization stress disrupts testicular steroidogenesis in adult male rats by inhibiting the activities of 17α-hydroxylase and 17, 20-lyase without affecting the binding of LH/hCG receptors. J. Androl 15: Paull, W. K. and Gibbs, F. P The corticotropin releasing factor (CRF) neurosecretory system in intact, adrenalectomized and adrenalectomized-dexamethasone treated rats. Histochemistry 78: Reul, J. M. H. M. and De Kloet, E. R Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117: Rivier, C. and Riviest, S Effect of stress on the activity of the hypothalamic- pituitary-gonadal axis: peripheral and central mechanisms. Biol. Reprod. 45: Rivier, C., Rivier, J., and Vale, W Stress-induced inhibition of reproductive function: role of endogenous corticotropin-releasing factor. Science 231: Rivier, C. and Vale, W Influence of corticotropinreleasing factor (CRF) on reproductive functions in the rats. Endocrinology 114: Rivier, C. and Vale, W Effect of long-term administration of corticotropin releasing factor on the pituitary-adrenal and pituitary-gonadal axis in the male rat. J. Clin. Invest. 75: Romero, L. M. and Sapolsky, R. M Patterns of ACTH secretagog secretion in response to psychological stimuli. J. Neuroendocrinol. 8: Saez, J. M., Morera, A. M., Haour, F., and Evain, D Effects of in vivo administration of dexamethasone, corticotropin and human chorionic gonadotropin on steroidogenesis and protein and DNA synthesis of testicular interstitial cells in prepubertal rats. Endocrinology 101: Schwartz, N. B. and Justo, S. N Acute changes in serum gonadotrophins and steroids following orchidectomy: role of the adrenal gland. Endocrinology 100: Selye, H Effect of adaptation to various damaging agents on the female sex organs in the rats. Endocrinology 25: Smal, A. G. H., Kloppenborg, P. W. C., and Benraad, T. J Plasma testosterone profiles in Cushing s syndrome. J. Clin. Endocrinol. Metab. 45: Steel, R. G. D. and Torrie, J. H pp Principles and Procedures of Statistics, New York, McGraw-Hill. 36. Suda, T., Tomori, N., Tozawa, F., Mouri, T., Demura, H., and Shizume, K Effects of bilateral adrenalectomy on immunoreactive corticotropin-releasing factor in the rat median eminence and intermediate-posterior pituitary. Endocrinology 113: Swanson, L. W., Sawchenko, P. E., Rivier, J., and Vale, W. W Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rats brain: an immunohistochemical study. Neuroendocrinology 36:

6 334 A. TOHEI, ET AL. 38. Taya, K. and Sasamoto, S Involvement of the adrenal gland in the suckling-induced decrease in LH and FSH secretion and concomitant increase in prolactin secretion in the rat. J. Endocrinol. 125: Taya, K., Watanabe, G., and Sasamoto, S Radioimmunoassay for progesterone, testosterone and estradiol-17β using 125 I-iodohistamine radioligands. Jpn. J. Anim. Reprod. 31: Tohei, A., Akai, M., Tomabechi, T., and Taya, K Gonadal and adrenal functions in the hypothyroid adult male rat. Biol. Reprod. (Suppl. 52): Vreeburg, J. T. M., DeGreef, W. J., Ooms, M. P., VanWouw, P., and Weber, R. F. A Effects of adrenocorticotropin and corticosterone on the negative feedback action of testosterone in the adult male rat. Endocrinology 115: