Life Sciences 146 (2016) 184–191

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Testosterone production by a Leydig tumor cell line is suppressed by hyperthermia-induced endoplasmic reticulum stress in mice Jung-Hak Kim a,1, Sun-Ji Park a,1, Tae-Shin Kim b, Jin-Man Kim c, Dong-Seok Lee a,⁎ a b c

School of Life Sciences and Biotechnology, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu 702-701, Republic of Korea Embryology Laboratory, Neway Fertility, 115 East 57th Street Suite 500, New York, NY 10022, USA Cancer Research Institute and Department of Pathology, School of Medicine, Chungnam National University, Daejeon 305-732, Republic of Korea

a r t i c l e

i n f o

Article history: Received 7 April 2015 Received in revised form 21 December 2015 Accepted 22 December 2015 Available online 29 December 2015 Keywords: Leydig cells Heat stress Testosterone Endoplasmic reticulum stress Apoptosis

a b s t r a c t Aims: Leydig cells are characterized by their ability to produce testosterone. When the Leydig cells are unable to produce enough testosterone, spermatogenesis fails completely. Considering this, it is of great interest to investigate whether the expressions of steroidogenic enzymes are affected by testicular heat stress. This study aimed to demonstrate that heat induced ER-stress significantly influences steroidogenic enzyme expression and testosterone production in the Leydig cells. Main methods: C57BL/6 mice were subjected to repetitive testicular heat-treatment at 42 °C for 15 min per day, and heat-treated mLTC-1 cells following hCG treatment for 1 h. The protein and RNA expressions were measured by Western blot, RT-PCR. The testosterone and progesterone levels were detected by EIA. The histological and pathological characteristics using hematoxylin and eosin (H&E) and antibody stains. Key findings: The 3β-HSD expression was decreased by heat-stress and hCG treatment. While the GRP78/BiP and CHOP levels were increased by ER-stress inducers, those of the steroidogenic enzyme and progesterone were decreased. In contrast, an ER-stress inhibitor rescued the testosterone levels, even under heat-stress conditions. Moreover, the Leydig cells were randomly scattered, and severely damaged upon repetitive testicular heattreatment. Additionally, immunohistochemical analyses revealed that cleaved caspase-3 was elevated in the testicular Leydig cells, and rescued by TUDCA. Thus, repetitive testicular heat-treatment in mice promotes excessive ER-stress, thereby leading to apoptosis of the Leydig cells and thus, decreased testosterone production. Significance: Our findings help to provide an ER-stress mediate mechanistic explanation to the impairment of spermatogenesis upon elevation of the testicular temperature. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The testes of most mammals are more susceptible to damage by high temperature than the other organs [29]. In concert, numerous studies across species have reported the adverse effects of hyperthermia on spermatogenesis in the normal adult testis [6,11,22]. The testosterone is produced by the Leydig cells in the testis, which plays an important role in spermatogenesis [7,37,39,42]. Unfortunately, the cellular and molecular mechanism underlying such effects of elevated testicular temperature on the testosterone production and expression of the steroidogenic enzymes in the Leydig cells are poorly described. The testosterone production in turn depends on the secretion of the luteinizing hormone (LH) by the pituitary gland [23]following the binding of LH to receptors on the Leydig cells, promoting the transfer of cholesterol

⁎ Corresponding author at: College of Nature Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea. E-mail address: [email protected] (D.-S. Lee). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.lfs.2015.12.042 0024-3205/© 2015 Elsevier Inc. All rights reserved.

to the inner mitochondrial membrane through the steroidogenic acute regulatory (StAR) protein [32,20]. Next, the conversion of cholesterol to pregnenolone is catalyzed by the P450 side chain cleavage enzyme (CYP11A1). Pregnenolone then moves out of the mitochondria to the endoplasmic reticulum (ER), where it is converted to progesterone by the 3β-hydroxysteroid dehydrogenase enzyme (3β-HSD). Finally, the progesterone is metabolized to testosterone by 17α-hydroxylase (CYP17) and 17β-hydroxysteroid dehydrogenase (17β-HSD) [25]. A testis damaged by heat-stress produces chaperone proteins such as the heat shock protein 70 (HSP70). The increase in the levels of such proteins is a cellular response to adapt and survive under elevated testicular temperatures [15]. Pertinently, the ER is an important organelle required for cell survival and maintenance of the cellular homeostasis under stress conditions. To relieve stress, the ER activates the intracellular signal transduction pathways, collectively termed the unfolded protein response (UPR) [26]. The GRP78/BiP protein is involved in sensing misfolded protein accumulation in the ER, and in conjunction with three other ER transmembrane proteins (ATF6, IRE1, and PERK), is responsible for the UPR [34]. Under normal conditions, these transmembrane proteins exist in a complex with the ER chaperone protein,

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GRP78/Bip [3]. However, under ER stress, the unfolded proteins promote the dissociation of GRP78/BiP by inducing the phosphorylation and relocalization of the transmembrane proteins. This further leads to transcription of chaperone protein, activation of ER stress-mediated degradation, and translational inhibition [28]. However, chronic or unmitigated ER stress induces apoptosis by activating the pro-apoptotic C/EBP homologous protein (CHOP) and caspase through three UPR pathways [38,33]. In the present study, therefore, we have investigated whether hyperthermia induces ER stress, and whether this stress modulates steroidogenic enzyme expression in the mLTC-1 tumor cell line. In addition, we have examined whether repetitive testicular hyperthermia in mice promotes ER stress, thereby leading to apoptosis of the Leydig cells and decreased testosterone production.

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Seoul, Korea) anti-GRP78/BiP, anti-CHOP (Cell Signaling, Beverly, MA, USA), and anti-3β-HSD (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following incubation, the membranes were washed and incubated with anti-goat IgG (Abfrontier, Seoul, Korea), anti-rabbit, and anti-mouse IgGs (Thermo Fisher Scientific, Waltham, MA 02454, USA) conjugated with horseradish peroxidase for 2 h at room temperature. After the removal of the excess antibodies by washing, specific binding was detected using an ECL kit (Bio-Rad, Hercules, USA). The anti-β-actin antibody (Abfrontier, Seoul, Korea) was used as a loading control. The band intensities were analyzed using the Image J software (National Institutes of Health).

2.6. RNA extraction and reverse transcript (RT)-PCR 2. Materials and methods 2.1. Reagents The hCG was commercially obtained from Intervet (Chorulon, Milton Keynes, Buckinghamshire, UK). Brefeldin A (BFA), and thapsigargin (Tg) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Tunicamycin (Tm) and tauroursodeoxycholic acid (TUDCA) were purchased from Calbiochem (La Jolla, CA, USA).

Total RNA was isolated from both the mLTC-1 cells and the testes tissues using TRI solution (Bio Science Technology, Daejeon, Korea) according to the manufacturer's instructions. The cDNAs were synthesized using 1μg of each RNA sample and Accupower® RT-PCR premix (Bioneer, Daejeon, Korea). The PCR was carried out using the Hot Start PCR premix (Bioneer, Daejeon, Korea) containing primers specific to the ER-stress markers and steroidogenic enzymes (Table 1).

2.2. Cell culture

2.7. Progesterone and testosterone assays by EIA

The mLTC-1 mouse Leydig tumor cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured at 37 °C and 5% CO2 in a 95% air incubator. The RPMI 1640 medium was supplemented with 10% fetal bovine serum (FBS) (Hyclone, GE Healthcare Life Sciences, Logan, Utah, USA) and 1% penicillin/streptomycin (Welgene, Daegu, Korea). The cells were subcultured at a density of 2.5 × 105cells/well in 6-well plates.

To measure the progesterone and testosterone levels, the mLTC-1 cell culture media were collected in serum-free culture medium following the respective treatments. Meanwhile, the blood was collected from the abdominal arteries of the mice after the respective heat treatments. Both the media and the sera were separated by centrifugation at 5000 rpm for 10 min, at 4 °C and then stored at −70 °C until used for the assays. The progesterone and testosterone productions were assessed using the respective immunoassay (EIA) kits (ALPCO, Salem, NH, and Enzo Life Sciences Inc., Plymouth Meeting, PA, USA, respectively) according to the manufacturers' instructions. The progesterone and testosterone concentrations for each sample were calculated using the standard graph, and expressed in pg/mL.

2.3. Induction of heat or chemical stress on the ER of mLTC-1 cells When the cells grew to 80% confluency in each of the 6 wells, they were pre-treated with 1% FBS in RPMI 1640 medium for 12 h, treated with 5 IU/ml of hCG for 3h, followed by incubation at 42 °C in 5% CO2 in a 95% air incubator for 1h. For chemical stress induction, the cells grown to 80% confluency were pre-treated with 1% FBS in RPMI 1640 medium and Tm (2 μg/ml), Tg (2.5 μM), and BFA (2 μM) for 12 h. This was followed by treatment with 5 IU/ml of hCG for 3h. 2.4. Induction of testicular heat-stress We maintained in accordance with the institutional guidelines of the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Korea) and male C57/ BL6 mice (9–10 weeks of age) were purchased from Central Animal Laboratory (Korea). The mice were administered TUDCA (250 mg/kg/day) as an ER stress inhibitor by intraperitoneal injection. Control was also administered by i.p injection with PBS. One hour after the administration of TUDCA or saline, the mice were subjected to three, or five cycles of heat-treatment at 42 °C for 15 min per day. Subsequently, the animals were dried, returned to their cages, and allowed to recover from the effect of anesthesia. They were sacrificed 12 h after the heat treatment. 2.5. Western blot analysis Lysates of the mLTC-1 cells and the total testis tissue were prepared in ice-cold PRO-PREP buffer (iNtRON Biotechnology Inc., Daejeon, Korea). The proteins were resolved on 8–12% SDS-polyacrylamide gels, and then transferred to a nitrocellulose membrane (Pall life sciences, NY, USA). The membrane was blocked with blocking buffer, and incubated with the following antibodies: anti-HSP70 (Abfrontier,

2.8. Hematoxylin and eosin (H&E) staining & immunohistochemistry The testes isolated from the mice were fixed with 10% neutral buffered formalin (Sigma-Aldrich) overnight, embedded in paraffin, and processed into 5 μm-thick sections. The sections were then stained with H&E using procedures as described previously [24]. For immunohistochemistry, deparaffinized sections were briefly heated for 4 min in a pressure cooker containing 10 mM citrate buffer (pH 6.0) for antigen retrieval. Subsequent procedures were conducted at room temperature. Sections were pretreated with 3% H2O2 in 0.1 M TBS (pH 7.4) for 30 min to quench endogenous peroxidases. The sections were treated with a protein block solution (Dako, Carpinteria, CA, USA) for 20 min and incubated with antibodies against GRP78/BiP and cleaved caspase-3 (Cell Signaling, Beverly, MA, USA) for 30 min in a humidified chamber. After washing with 0.1 M Tris base saline (TBS) containing 0.01% Tween-20 (TBST), the sections were incubated with an anti-rabbit polymer (Dako) for 30 min. The peroxidases bound to the antibody complex were visualized by treatment with a 3, 30diaminobenzidine (DAB) chromogenic substrate solution (Dako). The DAB reaction was monitored under a microscope to determine the optimal incubation time, and stopped with several washes of 0.1 M TBS. The stained and immunolabeled sections were then dehydrated in a graded ethanol series, defatted in xylene, and mounted. The sections were observed under a BX51 microscope (Olympus, Tokyo, Japan) in a bright field, and the images were acquired with a DP 70 camera (Olympus).

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Table 1 Primer sequences for RT-PCR. Gene

Accession number

Forward (5′- N 3′)

Reverse (5′- N 3′)

GRP78/BiP CHOP StAR CYP11A1 HSD3B1 GAPDH

NM_022310.2 NM_007837.3 NM_011485.4 NM_019779.3 NM_008293.3 NM_008084.2

TGCAGCAGGACATCAAGTTC CGGAACCTGAGGAGAGAGTG CAGGGAGAGGTGGCTATGCA GTCGGAAGGTGTAGGTCAGG GGCAAATTCTCCATAGCCAA ACCACAGTCCATGCCATCAC

CAGCTGCTGTAGGCTCATTG CTGTCAGCCAAGCTAGGGAC CCGTGTCTTTTCCAATCCTCTG CACTGGTGTGGAACATCTGG GCTTCCTCCCAGTTGACAAG TCCACCACCCTGTTGCTGTA

2.9. TUNEL assay The apoptotic cells in the paraffin-embedded sections of the testes were subjected to a TUNEL assay using a commercially available kit (Apop-Tag Peroxidase In Situ Apoptosis Detection Kit; Millipore, Inc., Billerica, MA, USA) according to the manufacturer's protocol. 2.10. Statistical analysis All the measurements were made in triplicate, and all the values are presented as the mean ± standard error of the mean (SEM). The results were subjected to a one way analysis of variance (ANOVA). P values b 0.05 were considered significant. All the calculations were carried out using the Graph Pad Prism 5.0 software package (Graph Pad Software, CA, USA). 3. Results 3.1. Heat treatment induces ER stress in the mLTC-1 cells The mLTC-1 Leydig tumor cell line is known to respond to hCG treatment by producing progesterone [27]. In this study, we investigated whether heat-treatment induces the expressions of the key ER stress genes GRP78/BiP and CHOP, and alters the key steroidogenic enzyme 3β-HSD in mLTC-1 cells. As shown in Fig. 1, the expressions of GRP78/ BiP, and 3β-HSD were significantly increased by hCG treatment compared with the untreated group. With both hCG and heat treatment, the heat stress marker HSP70, GRP78/BiP, and CHOP were more strongly expressed than with only hCG (Fig. 1A, B, C, and D). However, the

hCG-induced 3β-HSD was significantly reduced upon increasing the temperature (Fig. 1A and E). These results indicate that heat stress strongly induce ER-stress in the mLTC-1 cells in contrast to treatment with hCG only. Concordantly, this ER stress affects and decreases the hCG-induced 3β-HSD expression.

3.2. ER stress-inducing chemicals decrease the hCG-induced steroidogenic enzyme expressions and progesterone levels To investigate the changes in the steroidogenic enzymes in the mLTC-1 cells under ER stress, we evaluated their gene expressions by RT-PCR and western blot, following treatment with Tm (inhibitor of N-linked protein glycosylation), Tg (inhibits Ca2 + pumping into the ER), and BFA (inhibitor of ER–Golgi transport). First, as shown in Fig. 2A–C, the expressions of GRP78/BiP and CHOP were significantly increased by Tm and Tg at the RNA level, compared with hCG treatment only. In contrast, the hCG-induced steroidogenic enzyme expressions, particularly those of StAR, CYP11A1, and HSD3B1, significantly decreased at the RNA level by Tm, Tg, and BFA treatments, compared with the untreated controls (Fig. 2A, D–F). Meanwhile, GRP78/BiP was significantly increased at the protein level by Tm, Tg, and BFA treatments, compared with the untreated controls (Fig. 3A and B). Furthermore, as we had expected, the hCG-induced 3β-HSD expression was significantly decreased in the ER stress inducer-treated group (Fig. 3A and C). As shown in Fig. 3D, the ER stress inducers also significantly decreased the hCG-induced progesterone levels. Taken together, these results indicate that ER stress decreases the hCG-induced steroidogenic enzyme expressions and the progesterone levels in the mLTC-1 cells.

Fig. 1. Heat-induced ER stress in mLTC-1 cells. The expressions of HSP70, GRP78/Bip, CHOP, and 3β-HSD were measured by Western blot analysis. The cells were treated with 5 IU/ml of hCG for 3 h and incubated in 42 °C for 1 h (A). The relative band intensities of HSP70, BiP, CHOP, and 3β-HSD are shown (B-E). β-actin was used as a loading control. * indicates P value b 0.05 and ** indicates P value b 0.01 compared with the untreated controls.

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Fig. 2. Changes in the levels of the ER stress markers and steroidogenic enzymes in the mLTC-1 cells treated with ER stress-inducing chemicals. The expressions of the ER stress marker and steroidogenic enzymes in the mLTC-1 cells treated with Tm (2 μg/ml), Tg (2.5 μM), and BFA (2 μM) were measured by RT-PCR (A). The relative band intensities of the stress markers (B and C) and steroidogenic enzymes (D-F) are shown. GAPDH was used as a loading control. * indicates P value b 0.05 and ** indicates P value b 0.01 compared with the hCG-treated controls.

3.3. Testosterone is decreased by repetitive cycles of heat-treatment to the mouse testis Given the above results, we investigated whether heat-stress to mice testes in vivo could also increase the expression of key genes in ER stress and decrease the expressions of steroidogenic enzymes and testosterone. Our results demonstrated that GRP78/Bip, CHOP were significantly increased, and the steroidogenic enzymes such as StAR, P450scc, and 3β-HSD were significantly decreased upon repeated cycles of heat-treatment, particularly by three or five cycles (Fig. 4A-F). The testosterone levels in the mice sera also decreased considerably upon repetitive heat cycles (Fig. 4G). However, the expression of GRP78/Bip, CHOP proteins in testes of mice treated with repetitive cycles of heattreatment were decreased upon the addition of TUDCA (Figs. 4A–C). On the other hand, TUDCA treatment recovered the reduced expression of StAR, P450scc, and 3β-HSD protein (Fig. 4A, D–F). Interestingly, the decreased testosterone levels upon repetitive cycles of heat-treatment were restored by TUDCA treatment (Fig. 4G). Collectively, the reduced expression of steroidogenic enzymes in the testis and subsequent decrease in the serum testosterone levels following repetitive cycles of heat-treatment were restored by ER stress inhibition.

was present in the intertubular space, and in close contact with the lymphatic sinusoid. However, the clusters shrank away from the lymphatic sinusoid in response to three cycles of heat treatment (Fig. 5A). Moreover, the apoptosis signal marker, cleaved caspase-3, was increased in the Leydig cells after three cycles of heat treatment (Fig. 5B). However, disorders of the interstitial spaces and the increased expression of cleaved caspase-3 induced by three cycles of heat treatment were protected against TUDCA treatment (Fig. 5A, B). In fact, by the five cycle, the clusters were randomly scattered and suffered severe damage. Further, the immunohistochemical analyses showed that cleaved caspase-3 was greatly increased in testes, compared with the controls (Supplementary Fig. 3B middle panel). In addition, these results were confirmed by the increased TUNEL-positive staining of testes in the group treated with heat for five cycles (Supplementary Fig. 3B right panel). Meanwhile, the expressions of GRP78/Bip protein as a major ER stress sensor were also generally increased in heat-treated compared to control testes (Supplementary Fig. 3A). Overall, these results indicate that repetitive testicular heat treatment triggers ER stress, which subsequently leads to apoptosis of the Leydig cells, thereby decreasing the testosterone production in the mouse serum. 4. Discussion

3.4. Repetitive testicular heat-treatment shrinks the clusters of Leydig cells and induces cleaved Caspase-3 expression leading to cell death. We next attempted to dig deeper to investigate whether the repeated heat-treatment also induces histological changes, and ER stressmediated apoptosis. In addition, we also studied whether decreasing the testosterone production in mice sera can cause the apoptotic death of the Leydig cells. In control testes, the cluster of Leydig cells

The Leydig cells, which are located between the seminiferous tubules of the testis, synthesize and secrete testosterone [2,21]. It is known that testosterone is essential for normal spermatogenesis and fertility in mammals. When the Leydig cells are unable to produce enough testosterone, spermatogenesis fails altogether [30]. Although many studies have reported the adverse effects of heat on spermatogenesis across diverse mammalian species, it is unknown whether testicular heat-stress affects the cellular condition and steroidogenic ability

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Fig. 3. The ER stress-inducing chemicals decrease 3β-HSD gene expression. The expressions of the ER stress markers and 3β-HSD in the mLTC-1 cells treated with Tm (2 μg/ml), Tg (2.5 μM), and BFA (2 μM) were measured by Western blot analysis (A). The relative band intensities of GRP78/BiP and 3β-HSD (B and C) and progesterone concentrations (D) are shown. β-actin was used as a loading control. ** indicates P value b 0.01 compared with the hCG-treated control.

of the Leydig cells in the mouse testis. In the present study, we demonstrated that heat-induced ER stress in the mLTC-1 Leydig tumor cell line significantly alters the steroidogenic enzyme expressions. In addition, we described that repetitive testicular heat-stress in mice promotes excessive ER stress, thereby leading to ER stress-mediated apoptosis of the Leydig cells. Consequently, the testosterone production is decreased in the mouse serum. In the eukaryotic cells, the ER plays a key role in the synthesis, folding, and assembly of mature proteins [26]. However, extracellular stimuli and changes in the intracellular homeostasis cause protein misfolding in the ER [12]. Indiscriminate accumulation of the misfolded or damaged proteins within the ER lumen results in ER stress [5]. To restore the homeostasis, the ER activates the UPR, which reduces the influx of newly synthesized proteins into the ER through general transcriptional and translational arrest. This induces the transcriptional up-regulation of the genes that enhance the ER protein-folding capacity [26]. The UPR signaling pathways are essential to the normal differentiation and functions of secretory cells such as osteoblasts, pancreatic beta cells, and liver cells [40]. Therefore, the UPR signaling has been shown to regulate the production of specific proteins under homeostatic regulation [17]. Since the Leydig cells are a type of endocrine secretory cells, steroidogenic enzymes are continuously necessary for biosynthesizing testosterone. Therefore, we hypothesized that repetitive heattreatment induces ER stress, and the resulting activated UPR signaling affects the steroidogenic ability of the Leydig cells in mice testes. Induction of GRP78/BiP expression is a marker for ER stress and a central regulator of the activation of the UPR. GRP78/BiP synthesis can be stimulated by a variety of environmental and physiological stress conditions that perturb the ER function and homeostasis [17]. Our data also show that heat treatment induces ER stress, and activates the UPR signaling through GRP78/BiP induction (Fig. 1). Meanwhile, it has been reported that elevated levels of normal protein synthesis induce GRP78/BiP

expression [10]. Our results detected the increased GRP78/BiP expression and progesterone levels following only hCG treatment for the biosynthesis of progesterone in the mLTC-1 cells. In addition, the steroidogenic 3β-HSD was also expressed (Fig. 1 and Supplementary fig. 1, 2). These results suggest that the ER of the Leydig cells activate the UPR signaling as an adaptive response, required to maintain the expressions of the steroidogenic enzymes. The concept of impaired spermatogenesis resulting from testicular hyperthermia is widely known [8,14,19,36]. The exposure to higher body temperatures (as in moderately hot baths) for 30 or more minutes is likely to cause major damage to the testis [29]. Further, several reports have described that excessive heat-stress strongly represses the general transcription and translation, and only the heat shock genes are transcribed [1,9]. However, our data shows that heat treatment induces HSP70 as heat shock response, and also high levels of GRP78/BiP as ER stress response when compared to the mLTC-1 cells induced with hCG only. Moreover, the hCG-induced 3β-HSD expression and progesterone levels were decreased by heat-stress. Also 3β-HSD in the testes of mice, and testosterone in the sera were gradually decreased by repetitive cycles of testicular heat-treatment. (Figs. 1, 3 and Supplementary fig. 2). These observations led us to speculate that the general transcription and translation are not repressed at least not to the extent seen at 42 °C. Possibly, the increase in the expression of HSP70 and GRP78/ BiP, in response to heat, is a strategy adopted by the cells in an attempt to re-establish homeostasis and survive. As shown in the H&E staining data, repeated heat treatment damaged both the interstitial spaces, as well as the inside of seminiferous tubules. Further, through the immunohistochemical analysis, we detected that upon three cycles of heat treatment, cleaved caspase-3 were expressed in Leydig cells (Fig. 5B). Moreover, five cycles of testicular heat treatment, GRP78/BiP and cleaved caspase-3 were expressed not only in the clusters of the Leydig cells, but also in the spermatocyte

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Fig. 4. Repetitive cycles of heat treatment decrease hormone production. The protein expression of GRP78/Bip, CHOP, StAR, P450scc, and 3β-HSD in mice testes treated with repetitive cycles were measured by Western blot analysis. TUDCA as an ER stress inhibitor (250 mg/kg/day) were injected into mice 1 h before heat treatment. Control was administrated PBS by intraperitoneal injection (A). Graph shows that the GRP78/Bip (B), CHOP (C), StAR (D), P450scc (E), and 3β-HSD (F) protein level was quantified and normalized to β-actin for Western blot analysis. Testosterone analysis in serum of mice were analyzed with a testosterone EIA kit (G). Data in the bar graph represent the means ± SEM of three independent measurements. *, P b 0.05; **, P b 0.01; and ***, P b 0.001, compared with control (saline) and non-treated TUDCA.

and sertoli cells (Supplementary fig. 3). Therefore, increased damage in Leydig cells were preceded before increased damage of spermatocytes and sertoli cells were occurred. Based on our results, it is clear that repeated heat treatments induce ER stress and decrease steroidogenic enzyme expression and testosterone production in the Leydig cells. Our previous report demonstrated that repetitive cycles of testicular hyperthermia activates the UPR signaling, and promotes ER stress-mediated apoptosis of the spermatocytes [16]. Based on the combined results of our earlier and current studies, we suggest that repetitive cycles of testicular hyperthermia impair steroidogenesis as well as spermatogenesis. Similarly, frequent hot baths or trips to the sauna might also affect these biological phenomena. Although the UPR fundamentally mediates physiological regulation or homeostasis as a cytoprotective response, excessive or prolonged UPR can trigger cell death, predominantly by inducing apoptosis [4,41]. CHOP, a transcription factor induced under ER stress, has been reported to be involved in ER stress-induced apoptosis [31]. Procaspase-12 is another player in this pathway that is cleaved and activated specifically upon ER stress [13]. The activation of caspase-12, an ER resident protein, causes the downstream activation of cytoplasmic caspase-3 [13]. In concert, our data have shown that the CHOP protein is significantly increased in the mLTC-1 cells by heat stress. Furthermore, we also observed cleaved caspase-3 and TUNEL-positive staining in the Leydig cells, although spermatocytes and sertoli cell were also increased by five cycles heat treatment compared with the controls. These results suggest that heat-stress and repetitive testicular heat-treatment

induces ER stress-mediated apoptosis, and results in decreased testosterone production. We also measured the effects on steroidogenic enzyme expression and progesterone production in the presence of pharmacological inducers of ER-stress such as Tm (inhibitor of Nlinked protein glycosylation), Tg (inhibits Ca2+ pumping into the ER), and BFA (inhibitor of ER–Golgi transport) [41]. These stress inducers increased the expressions of GRP78/BiP and CHOP, and decreased those of the steroidogenic enzymes as well as the progesterone levels. These results indicate that the ER stress induced by each of the inducers impairs the hCG-induced steroidogenic enzyme expressions, which in turn results in the reduced synthesis and secretion of testosterone. On the contrary, TUDCA (an ER stress inhibitor) protect the Leydig cell death and rescued the testosterone levels even under heat-stress conditions (Figs. 4, 5 and Supplementary Fig. 2). Probably, TUDCA enhances the adaptive capacity of the ER and acts as potent steroidogenic factor. Hence, it can be potentially applied to the treatment of Leydig cells disrupted by heat-stress. Meanwhile, the mitochondrion is also an essential organelle in testosterone biosynthesis, being the site of the first enzymatic step in steroidogenesis, involving StAR and CYP11A1. Previous investigations have demonstrated that an intact mitochondrial membrane potential is necessary for cholesterol transfer. When it is damaged by hyperthermia, the signaling events leading to decreased testosterone levels result from the mitochondria-dependent apoptotic pathway [18,35,36]. However, our observations in this study indicate that heat-induced ER-stress might also be an important inhibitor of testosterone production by the

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Fig. 5. Testicular heat treatment increases cell death and rescued by TUDCA in the Leydig cells. Represented images of H&E staining (A) and cleaved caspase-3 staining (B) of the testis section of control (left panel), 3 cycles heat treated (middle panel), and 3 cycles heat and TUDCA treated (right panel). Arrows: Leydig cells, asterisks: spermatocyte, seminiferous tubule. Scale bar = 100 μm.

Leydig cells. We also observed that the ER of the Leydig cells could activate the UPR, an adaptive signaling pathway, in order to maintain the steroidogenic enzyme expression, even under stress. Thus, the ER plays a key role in the synthesis, folding, and assembly of the steroidogenic enzymes. Future investigations are needed to elucidate the correlation between testicular temperature and ER-stress, and whether testicular cooling can retrieve the testosterone production in heat-stressed Leydig cells.

5. Conclusion In conclusion, we demonstrated that heat-induced ER stress significantly regulates the steroidogenic enzyme expressions and testosterone production in the Leydig cell of mice testes. Although it is not yet possible to explain why spermatogenesis is sensitive to heat, our current findings provide a better understanding of impaired spermatogenesis that follows increased testicular temperature. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.lfs.2015.12.042.

Conflict of interest statement The authors declare that there is no conflict of interest.

Acknowledgments This research was supported by a grant (NRF-2014R1A2A1A1105 4095, NRF-2014R1A6A3A01059914, NRF-2015R1A6A3A01061451) from the National Research Foundation of Korea funded by the Republic of Korea government, a grant from the Korea Institute of Bioscience and Biotechnology (KRIBB) Research initiative program (KGM4611512), Republic of Korea.

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Testosterone production by a Leydig tumor cell line is suppressed by hyperthermia-induced endoplasmic reticulum stress in mice.

Leydig cells are characterized by their ability to produce testosterone. When the Leydig cells are unable to produce enough testosterone, spermatogene...
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