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Asian Pacific Journal of Reproduction
journal homepage: www.apjr.net
Original research http://dx.doi.org/10.1016/j.apjr.2016.07.005
Testicular germ cells apoptosis following exposure to chronic stress in rats
Maryam Karami Kheirabad1, Zahra Khodabandeh2, Farhad Rahmanifar3, Amin Tamadon2*, Bahia Namavar Jahromi4*, Maryam Owjfard2, Omid Koohi-Hosseinabadi5
1 Department of Basic Sciences, Azad University, Gachsaran Branch, Gachsaran, Iran 2Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran 3Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
4Infertility Research Center, Department of OB-GYN, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 5Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
ABSTRACT
Objective: To evaluate the effects of chronic stress on testosterone hormone level and germ cells apoptosis in testes and the inhibitory role of glucocorticoid receptor antagonist, RU486.
Methods: Adult male Sprague-Dawley rats were randomly assigned to four groups (n = 6): control; stress; RU486; stress/RU486. Animals in RU486 and stress/RU486 subjected to subcutaneous injections of 2.5 mg RU486/kg 1 h before stress session. Rats were submitted to chronic restraint stress (1 h daily for 12 consecutive days) whereas control animals were not subjected to stress. Serum testosterone assay was performed and the occurrence of DNA fragmentation in the testis sections was examined using TUNEL staining.
Results: Chronic restraint stress significantly induced a decrease in serum testosterone level with increase in apoptosis in spermatogonia. Systemic administration of RU486 significantly restored serum testosterone levels and attenuates stress-induced apoptosis in spermatogonia.
Conclusion: The restraint stress-induced change in serum testosterone levels and seminiferous tubules apoptosis closely associated with the glucocorticoid receptor.
ARTICLE INFO
Article history: Received 12 Apr 2016 Received in revised form 6 Jul 2016 Accepted 12 Jul 2016 Available online 6 Aug 2016
Keywords:
Apoptosis
Restraint stress
Spermatogenesis
Testosterone
1. Introduction
Exposure to psychophysical stressors has adverse effects on reproductive system. Testis performs steroidogenesis and sper-matogenesis that are functionally relevant. All species respond to stress by a decrease in the levels of gonadal steroids [1,2]. Psychophysical stress experienced by adolescents can shift the onset of puberty [3]. Chronic environmental stressors are
*Corresponding authors. Amin Tamadon, Transgenic Technology Research Center, Shiraz University of Medical Sciences, Neshat Ave., Near Sina & Sadra Halls, Shiraz 71348-74478, Iran.
Tel/Fax: +98 71 32341025 E-mail: amintamaddon@yahoo.com
Bahia Namavar Jahromi, Infertility Research Center, Ghadir Mother & Child Hospital, Quran Gate, Shiraz, Iran. Tel/Fax: +98 71 32279715 E-mail: namavarb@sums.ac.ir
Peer review under responsibility of Hainan Medical College. Foundation Project: This work was supported by the grant from Infertility Research Centre, Shiraz University of Medical Sciences, Iran.
associated with low plasma testosterone in mammals [4]. Testicular germ cell apoptosis has been demonstrated in response to high temperature stress and testosterone reduction [5].
The neuroendocrine mechanisms responsible for testosterone reduction under stressful situations are not clear. A variety of factors could be involved including increase glucocorticoids, catecholamine, or decreased gonadotropins. There is evidence that stress hormones and testosterone are work against each other. Glucocorticoids appear to exert much of their effects through a member of the nuclear steroid receptor superfamily, glucocorticoid receptor, that functions as a ligand dependent transcription factor to regulate the expression of glucocorticoid receptor target genes either positively or negatively [6]. In mammals, the interstitial cells of Leydig which produce steroids, notably testosterone and dihydrotestosterone, contain glucocorticoid receptors [7]. In the presence of luteinizing hormone (LH), Leydig cells produce and secrete testosterone for spermatogenesis through cytochromes P450 enzymes [8].
2305-0500/Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Systemic injection of exogenous glucocorticoids causes apoptosis of testicular germ cell [9]. Administration of adrenocorticotropine hormone has been shown to reduce testosterone level [10]. Sensitivity of Leydig cells to gonadotropins decrease by stress [11]. Moreover, cortisol induces reduction in plasma testosterone level, presumably by inhibiting testicular 17a-hydroxylase or 17, 20-lyase activity [12]. Although an inhibitory role of acute stress on testosterone level is well established [13,14], the effect of prolonged stress on testicular steroidogenesis has not been fully examined.
The restraint stress has been used in modeling physical and psychological stress in animal studies [15]. The current study intended to assess the role of glucocorticoids during chronic restraint stress on spermatogenesis. For this purpose, we have examined the effect of glucocorticoid receptor antagonist, RU486, on apoptosis using the terminal deoxynucleotidyl transferase mediated dUDP nick end labeling (TUNEL) method in parallel with serum testosterone concentration changes.
2. Material and methods
2.1. Animals
Adult male Sprague-Dawley rats bred in Laboratory Animal Center, Shiraz University of Medical Sciences, Iran and raised under controlled environmental conditions (temperature (23 ± 1) °C; 12 h light/12 h dark) with food and water ad libitum were used for experiments.
The rats were randomly divided into four groups (n = 6); stress, RU486, stress/RU486, and control groups: control group consisted of unstressed animals; stress group were exposed to restraint (1 h for 12 consecutive days); RU486 group, the rats were injected subcutaneously with RU486 (2.5 mg/kg, 20 mL/ rat; ab120356, Abcam Ltd, Cambridge, UK) 60 min before restraint stress was applied; stress/RU486 group, the rats were injected subcutaneously with the same dose of RU486, 60 min before the stress session. Restraint stress was performed daily for 12 d. Briefly, rats were handled before experiments. After that, animals were individually restraint for 1 h through wrapping of their upper and lower limbs by plastic cylinders (20.5 cm x 8 cm x 6 cm) which with holes for ventilation and their extended tails. The cylinders were just large enough to allow rats of the size used to turn around easily. All experiments were conducted during the light phase between 8 and 12 a.m. The experimental investigation was approved by Shiraz University of Medical Sciences ethics committee. Twenty four hours after the last session of stress, the animals were killed by cervical dislocation and trunk blood was collected. Serum samples were stored at -70 °C until assayed for testosterone by testosterone kit (ELISA, Diagnostics Biochem Canada Inc., Ontario, Canada) and the kit sensitivity was 0.022 ng/mL.
2.2. TUNEL assay
Twenty four hours after the last session of stress, the rats were weighted and anaesthetized by inhalation of ether and euthanized by cervical dislocation. Testes were removed and fixed in 4% (v:v) paraformaldehyde for 16 h at 4 ° C, then an additional 8 h at 25 °C, prior to dehydration and paraffin embedding. Six-micron-thick testis sections were cut and
mounted on Supersoft Plus slides (Fisher Scientific, Pittsburgh, PA). The specimens were subjected to TUNEL staining using a TACS 2 TdT-DAB in situ Apoptosis Detection Kit (Catalogue No. 4810-30-K; Trevigen, Inc., Gaithersburg, MD) according to the manufacturer's protocol. The TUNEL assay was carried out on paraffin-embedded sections of testis. Briefly, sections were deparaffinized and dehydrated in graded concentrations of xylene and ethanol. The sections were digested with 20 mg/mL proteinase K for 15 min at room temperature. The sections were then washed and incubated with the TUNEL reaction mixture (enzyme solution and labeling solution) for 60 min at 37 °C in a humidified atmosphere. Negative controls were processed according to the same protocol, except for the incubation with TdT. TUNEL-positive cell detection was based on dark labeling as intense as, or more intense than, that of apoptotic cells observed in the positive control slide labeled simultaneously. All labeled sections were viewed at 400x magnification with an Olympus BX50 microscope, Tokyo, Japan.
TUNEL analyses were expressed by two indices. First, a histogram of the number of total spermatogonia or spermato-cytes per an individual seminiferous tubule was established. Secondly, the number of TUNEL-positive cells in at least five cross sections of the tubules with apoptotic cells was counted. Then the percentages of the number of TUNEL-positive cells were calculated by dividing positive cells to total spermatogonia or spermatocytes.
2.3. Statistical analysis
Data of serum testosterone concentration and total and percentage of TUNEL-positive cells were analyzed by one-way analysis of variance (ANOVA) followed by LSD post hoc test (SPSS for Windows, version 20, SPSS Inc., Chicago, Illinois). All data are expressed as means and standard errors of means. Differences were regarded as significant at P < 0.05.
3. Results
Restraint stress (1 h/d for 12 consecutive days) significantly decreased serum testosterone levels (P < 0.05), and subcutaneous (sc) administration of RU486 (2.5 mg/kg in 20 mL per rat)
Figure 1. The mean and standard error of serum testosterone concentration after chronic restraint stress-induced rats (n = 6).
a Different superscript letters indicate significant differences between groups. RU486, glucocorticoid receptor antagonist.
Figure 2. TUNEL detection of apoptotic germ cells in testes.
A, TUNEL-positive germ cells are rare in control testes (A), RU486, glucocorticoid receptor antagonist (B) and chronic stress pretreated with RU486 (D). C, high TUNEL-positive spermatogonia and spermatocytes in testes of rats which exposed to chronic stress.
Figure 3. The mean and standard error of percentage of TUNEL-positive spermatogonia per seminiferous tubules cross sections after chronic restraint stress-induced rats (n = 6).
a,b Different superscript letters indicate significant differences between groups. RU486, glucocorticoid receptor antagonist.
Figure 4. The mean and standard error of percentage of TUNEL-positive spermatocytes per seminiferous tubules cross sections after chronic restraint stress-induced rats (n = 6).
a Different superscript letters indicate significant differences between groups. RU486, glucocorticoid receptor antagonist.
blunted the inhibitory effects of chronic stress on serum testosterone concentration (Figure 1).
TUNEL analysis of testicular tissues showed that apoptosis was confined to the basal germ cells, indicating suppression of spermatogenesis. The number of TUNEL-positive cells in the testis markedly increased in stress group (Figure 2). TUNEL-positive Leydig cells were detected in the sections prepared from the stressed animals, consistent with the low testosterone concentration in stress group. Apoptotic cells were not detected in spermatids located close to the lumen.
In the stress group, enlarged tubular lumen was seen due to the decrease in the number of germ cells. The numbers of TUNEL-positive spermatogonia per seminiferous tubule of the stress group increased relative to those of the control groups (P < 0.05, Figure 3). However, the stress/RU486 group showed a reduction in apoptotic cells when compared with stress group (P < 0.05) and to levels equivalent to those observed in control testes. The number of TUNEL-positive spermatocytes per seminiferous tubule was not increased significantly in the stress group, but an increasing trend of apoptotic spermatocyte in this group was observed (Figure 4).
4. Discussion
The findings of testicular apoptosis following chronic stress suggest possible role of the androgen changes in germ cell apoptosis. Various types of stress can have adverse effect on fertility and reproductive system. Heat stress causes apoptosis of spermatogonia in mice [16]. Consistent with our findings, exposure of rat to immobilization stress led to increase in testicular germ cell DNA fragmentation [17]. In addition, chronic stress by immobilization causes Leydig cell apoptosis through glucocorticoid receptor while adrenalectomised rats have increased frequencies of apoptosis in Leydig cells [18]. Repeated immobilization stress for 10 d negatively affects testicular steroidogenesis related to cAMP signaling [19]. Glucocorticoid receptor are expressed in various cell types of testis and highly conserved across species [20,21]. Increased glucocorticoids (stress-induced or exogenous administration) exert effects on reproductive system. Glucocorticoids directly inhibit enzymes involve in testosterone biosynthesis and also number of LH receptor in testis [22]. Psychophysical stress causes testicular defects by acting on the testis and decreased enzyme content [19] and testosterone production [23] or whether due to effect on the higher level in hypothalamic-pituitary axis [24], it is something all publications deal with to some extent. Results presented in this study agree with other work in animal investigations [19,25] which showed a significant decrease in serum testosterone level following chronic stress. Results of this study showed that serum testosterone levels were decreased in rats exposed to restraint and increased apoptotic spermatogonia and Leydig cells from these animals. Production of testosterone in the Leydig cells is vital for the spermatogenesis in the seminiferous tubules [26]. Corticosterone suppresses the 3b-hydroxysteroid dehydrogenase type 1 (HSD3B1) and 17b-hydroxysteroid dehydrogenase type 3 (HSD17B3) enzymes mRNA expression in Leydig cells and through that decreased steroidogenic activity of testis [27]. Therefore, in the testis, the enzymes catalyze testosterone synthesis are linked to spermatogenesis and sperm maturation. RU486 pretreatment attenuated the stress-induced apoptosis. Such anti-apoptotic effects of RU486
on the seminiferous tubules appear to closely parallel increases in the serum level of testosterone. The anti-glucocorticoid effect of RU486 is related to masking DNA domain of the glucocor-ticoid receptor [28], however it appears RU486 to impair nuclear translocation of glucocorticoid receptors maybe due to inefficient release of heat-shock proteins [29].
The present study indicates that administration of RU486 prevent stress induced apoptosis of spermatogonia. Glucocorti-coid receptor blockade by intra testicular administration of RU486, prevented the immobilization-induced decline in plasma testosterone levels, so the rapid changes in testosterone suggest a suppression of androgen biosynthesis directly by glucocorticoid through a non-genomic mechanism [30]. Our results are not appropriate to reveal how restraint stress decreased serum testosterone level. Based on other animal studies that serum LH levels are either unchanged or decline during stress [31,32]. This study confirms findings available in literature on the increased in germ cell apoptosis related to glucocorticoid receptor during stress. In view of these glucocorticoids could account for the apoptosis of damaged germ cells, but not spermatocyte, in order to prevent genetic abnormalities to offspring.
In conclusion, our data demonstrate that chronic restraint stress impaired testicular steroidogenesis and causes apoptosis in spermatogonia related to increase in glucocorticoid action on testes.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgments
This work was supported by the grant from Infertility Research Centre, Shiraz University of Medical Sciences, Iran.
References
[1] Schmid B, Tam-Dafond L, Jenni-Eiermann S, Arlettaz R, Schaub M, Jenni L. Modulation of the adrenocortical response to acute stress with respect to brood value, reproductive success and survival in the Eurasian hoopoe. Oecologia 2013; 173(1): 33-44.
[2] Lucas LD, French SS. Stress-induced tradeoffs in a free-living lizard across a variable landscape: consequences for individuals and populations. PLoS One 2012; 7(11): e49895.
[3] Genazzani AD, Ricchieri F, Lanzoni C, Strucchi C, Jasonni VM. Diagnostic and therapeutic approach to hypothalamic amenorrhea. Ann N YAcad Sci 2006; 1092(1): 103-113.
[4] Malan NT, Stalder T, Schlaich MP, Lambert GW, Hamer M, Schutte AE, et al. Chronic distress and acute vascular stress responses associated with ambulatory blood pressure in low-testosterone African men: the SABPA Study. J Hum Hypertens 2014; 28(6): 393-398.
[5] Kanter M, Aktas C, Erboga M. Heat stress decreases testicular germ cell proliferation and increases apoptosis in short term: an immunohistochemical and ultrastructural study. Toxicol Ind Health 2013; 29(2): 99-113.
[6] Nicolaides NC, Galata Z, Kino T, Chrousos GP, Charmandari E. The human glucocorticoid receptor: molecular basis of biologic function. Steroids 2010; 75(1): 1-12.
[7] Hazra R, Upton D, Jimenez M, Desai R, Handelsman DJ, Allan CM. In vivo actions of the Sertoli cell glucocorticoid receptor. Endocrinology 2014; 155(3): 1120-1130.
[8] Weinbauer GF, Luetjens CM, Simoni M, Nieschlag E. Physiology of testicular function. Andrology. Berlin, Heidelberg: SpringerVerlag; 2010, p. 11-59.
[9] Mukherjee A, Haldar C, Vishwas DK. Melatonin prevents dexamethasone-induced testicular oxidative stress and germ cell apoptosis in golden hamster, Mesocricetus auratus. Andrologia 2014; 47(8): 920-931.
[10] Romeo RD, Minhas S, Svirsky SE, Hall BS, Savenkova M, Karatsoreos IN. Pubertal shifts in adrenal responsiveness to stress and adrenocorticotropic hormone in male rats. Psychoneur-oendocrinology 2014; 42: 146-152.
[11] Stojkov NJ, Janjic MM, Baburski AZ, Mihajlovic AI, Drljaca DM, Sokanovic SJ, et al. Sustained in vivo blockade of a-adrenergic receptors prevented some of stress-triggered effects on steroido-genic machinery in Leydig cells. Am J Physiol Endocrinol Metab 2013; 305(2): E194-E204.
[12] Abbott DH, Eisner JR, Bird IM, Rafferty SW, Moore WR, Schotzinger RJ. Plasma steroid concentrations in male rhesus monkeys following treatment with the P450c17 (CYP17) inhibitors VT-464 and abiraterone acetate: a comparison to human 17,20-lyase (lyase) and combined lyase/17a-hydroxylase (hydroxylase) deficiencies. J Clin Endocrinol Metab 2012; 95: 994-999.
[13] Fernandez-Guasti A, Fiedler J, Herrera L, Handa R. Sex, stress, and mood disorders: at the intersection of adrenal and gonadal hormones. Horm Metab Res 2012; 44(8): 607.
[14] Damian JP, Bausero M, Bielli A. Acute stress, hypothalamic-hypophyseal-gonadal axis and testicular function. Ann Anim Sci 2015; 15(1): 31-50.
[15] Olesen MV, Wortwein G, Pakkenberg B. Electroconvulsive stimulation, but not chronic restraint stress, causes structural alterations in adult rat hippocampus - stereological study. Hippocampus 2015; 25(1): 72-80.
[16] Zhang M, Jiang M, Bi Y, Zhu H, Zhou Z, Sha J. Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice. PLoS One 2012; 7(7): e41412.
[17] Xu M, Wei Q, Zheng K, Mao D, Zheng Y, Li Y, et al. Protective effects of Big-leaf mulberry and physiological roles of nitric oxide synthases in the testis of mice following water immersion and restraint stress. Acta Histochem 2014; 116(8): 1323-1330.
[18] Chen Y, Wang Q, Wang F-F, Gao H-B, Zhang P. Stress induces glucocorticoid-mediated apoptosis of rat Leydig cells in vivo. Stress 2012; 15(1): 74-84.
[19] Stojkov NJ, Janjic MM, Bjelic MM, Mihajlovic AI, Kostic TS, Andric SA. Repeated immobilization stress disturbed steroidogenic machinery and stimulated the expression of cAMP signaling elements and adrenergic receptors in Leydig cells. Am J Physiol Endocrinol Metab 2012; 302(10): E1239-E1251.
[20] Herrera-Luna C, Budik S, Aurich C. Gene expression of ACTH, glucocorticoid receptors, 11bHSD enzymes, LH-, FSH-, GH
receptors and aromatase in equine epididymal and testicular tissue. Reprod DomestAnim 2012; 47(6): 928-935.
[21] Nirupama M, Devaki M, Nirupama R, Yajurvedi H. Chronic intermittent stress-induced alterations in the spermatogenesis and antioxidant status of the testis are irreversible in albino rat. J Physiol Biochem 2013; 69(1): 59-68.
[22] Xiao YC, Huang YD, Hardy DO, Li XK, Ge RS. Glucocorticoid suppresses steroidogenesis in rat progenitor Leydig cells. J Androl 2010; 31(4): 365-371.
[23] Turner TT, Lysiak JJ. Oxidative stress: a common factor in testicular dysfunction. J Androl 2008; 29(5): 488-498.
[24] Kirby ED, Geraghty AC, Ubuka T, Bentley GE, Kaufer D. Stress increases putative gonadotropin inhibitory hormone and decreases luteinizing hormone in male rats. Proc Natl Acad Sci USA 2009; 106(27): 11324-11329.
[25] Wang F-F, Wang Q, Chen Y, Lin Q, Gao H-B, Zhang P. Chronic stress induces ageing-associated degeneration in rat Leydig cells. Asian J Androl 2012; 14(4): 643.
[26] O'Hara L, Smith LB. Androgen receptor roles in spermatogenesis and infertility. Best Pract Res Clin Endocrinol Metab 2015; 29(4): 595-605.
[27] Badrinarayanan R, Rengarajan S, Nithya P, Balasubramanian K. Corticosterone impairs the mRNA expression and activity of 3 band 17 b-hydroxysteroid dehydrogenases in adult rat Leydig cells. Biochem Cell Biol 2006; 84(5): 745-754.
[28] Groeneweg FL, van Royen ME, Fenz S, Keizer VI, Geverts B, Prins J, et al. Quantitation of glucocorticoid receptor DNA-binding dynamics by single-molecule microscopy and FRAP. PLoS One 2013; 9(3): e90532.
[29] Czar MJ, Lyons RH, Welsh MJ, Renoir J-M, Pratt WB. Evidence that the FK506-binding immunophilin heat shock protein 56 is required for trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus. Mol Endocrinol 1995; 9(11): 1549-1560.
[30] Dong Q, Salva A, Sottas CM, Niu E, Holmes M, Hardy MP. Rapid glucocorticoid mediation of suppressed testosterone biosynthesis in male mice subjected to immobilization stress. J Androl 2004; 25(6): 973-981.
[31] Orr TE, Taylor MF, Bhattacharyya AK, Collins DC, Mann DR. Acute immobilization stress disrupts testicular steroidogenesis in adult male rats by inhibiting the activities of 17 alpha-hydroxylase and 17,20-lyase without affecting the binding of LH/hCG receptors. J Androl 1994; 15(4): 302-308.
[32] Maric D, Kostic T, Kovacevic R. Effects of acute and chronic immobilization stress on rat Leydig cell steroidogenesis. J Steroid Biochem Mol Biol 1996; 58(3): 351-355.
