Dedifferentiation of Adult Monkey Sertoli Cells through Activation of Extracellularly Regulated Kinase 1/2 Induced by Heat Treatme
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《内分泌学杂志》
State Key Laboratory of Reproductive Biology (X.-S.Z., Z.-H.Z., X.J., P.W., X.-Q.H., M.C., C.-L.L., Z.-Y.H., Y.-X.L.), Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
Division of Endocrinology (Y.-H.H., A.P.S.H., R.S.S., C.W.), Department of Medicine, Harbor-University of California, Los Angeles, Medical Center and Los Angeles Biomedical Research Institute, Torrance, California 90509
Graduate School of the Chinese Academy of Sciences (X.-S.Z.), Beijing 100039, China
Abstract
Sertoli cells play a key role in triggering and regulating the process of spermatogenesis. Failure of a Sertoli cell to mature functionally will presumably render it incapable of supporting germ cell survival and development that appeared after puberty. Expression of cytokeratin 18 (ck-18) intermediate filaments indicates a state of undifferentiation usually observed in Sertoli cells of prepubertal testis. In this study we demonstrated that local testicular heat treatment of adult monkey with water at 43 C for 30 min once daily for 2 consecutive days was capable of activating reexpression of ck-18 in Sertoli cells, which was coincident with activation of ERK1/2 and Akt kinases. Using primary Sertoli cell culture isolated from adult monkey testis, we further confirmed that the heat treatment of the cells at 43 C could also induce ck-18 reexpression, which was similar to the in vivo treatment. ERK MAPK was also induced by the heat treatment in a time- and protein kinase A (PKA)-dependent manner. After blocking the ERK MAPK signaling pathway, an inhibition of ck-18 expression in the cultured Sertoli cells was observed, and this inhibitory effect was also detected by blocking the PKA activation. However, ck-18 activation in Sertoli cells remained unaltered when the phosphatidylinositol 3-kinase/Akt pathway was blocked. In conclusion, the heat treatment of adult monkey Sertoli cells are capable of inducing a reversible change in the Sertoli cells from an adult differentiated state to an immature-like dedifferentiated state through PKA-ERK MAPK-dependent pathways but not via the phosphatidylinositol 3-kinase/Akt pathway.
Introduction
IT HAS BEEN well defined that local treatment of rat and monkey testes in water at 43 C could induce reversible damage to the seminiferous epithelium due to increased germ cell apoptosis (1, 2, 3, 4, 5). But the molecular mechanism is not clear. Reports have shown that elevation of testicular temperature not only causes germ cell loss but also affects the morphology and function of Sertoli cells (6, 7, 8, 9). Mature Sertoli cells are the primary supportive cells of seminiferous epithelium and provide an essential structural and nutritional support for the developing spermatogenic cells in the seminiferous tubules (10, 11). Sertoli cells can also interact with Leydig cells and peritubular cells to regulate spermatogenic process (12, 13, 14). However, Sertoli cells with immature characteristics have been found in the adult human testes in some pathological conditions usually associated with impaired spermatogenesis (15, 16, 17, 18).
Cytokeratin (ck) intermediate filament has been demonstrated to be a Sertoli cell differentiation marker, which is only expressed in immature Sertoli cells and normally lost at puberty (15, 19, 20, 21, 22, 23). Ck-18 is a subtype of the cytokeratin family that represents an excellent marker of immature or undifferentiated Sertoli cells in the seminiferous epithelium (15, 16, 24, 25, 26, 27). We have demonstrated that cryptorchid testis of rhesus monkey could induce expression of ck-18 and other intermediate filaments in Sertoli cells coincidentally with a cessation of spermatogenic activity (8), implying that body temperature is capable of inducing adult Sertoli cells reverting to a dedifferentiated immature state and thus destroys their supportive role in normal spermatogenesis. It is therefore suggested that Sertoli cells undergoing a process of dedifferentiation, as indicated by the reexpression of cytokeratin, may subsequently result in a loss of Sertoli cell function and lead to a cession of spermatogenic activity.
ERK1/2 represent one subfamily of serine/threonine protein kinases collectively referred to as the MAPK family. They have the unique feature of being phosphorylated by an upstream dual-specificity kinase called MAPK kinase or MAPK/ERK kinase (28, 29, 30, 31, 32). The ERK MAPK kinase pathway is essential for controlling cell proliferation and differentiation (33, 34, 35, 36, 37, 38, 39). It has been demonstrated that sustained signaling through the ERK MAPK kinase pathway is capable of driving the dedifferentiation of Schwann cells (40), whereas blocking the ERK MAPK kinase pathway could induce undifferentiated tumor cells to undergo a process of differentiation, but their proliferation activities were inhibited (41). In the neonatal Sertoli cells isolated 5 d after birth, the ERK MAPK pathway was activated by FSH in a protein kinase A (PKA)-dependent manner; the activation, however, was required for Sertoli cell proliferation. In contrast, 19 d after birth, as the Sertoli cells proceeded through their differentiation program, the ERK pathway was dramatically inhibited by FSH treatment (42). These results suggest that the ERK-dependent signaling is involved in the proliferation and differentiation of the Sertoli cells in a stage-specific manner modulated by FSH. However, little is known about the switch between differentiation and dedifferentiation and how the ERK MAPK kinase pathway is affected in Sertoli cells after the heat treatment.
Akt (also known as protein kinase B), a key effector of phosphatidylinositol 3-kinase (PI3K) in another survival signaling pathway, is also a subfamily of serine/threonine protein kinase (43, 44, 45, 46). Interestingly, Akt has been shown to produce synergistic or counteractive regulation on the MAPK-induced cellular responses by modulating Raf, the MAPK kinase kinase (47). The PI3K/Akt pathway has been associated with cell differentiation and dedifferentiation (48, 49, 50) and reported to be phosphorylated in response to cellular stress, such as heat shock (51, 52). However whether the PI3K/Akt pathway plays a role in the differentiation of Sertoli cells after heat treatment has remained unknown.
In this study we designed both in vivo (using adult monkey testicular hyperthermia model) and in vitro (using primary culture of Sertoli cells isolated from adult monkey testes) experiments to examine the possible effect of the well-confirmed heat treatment at 43 C on Sertoli cell dedifferentiation. We have demonstrated for the first time that the heat treatment at 43 C could induce change in the Sertoli cells from an adult differentiated state to an immature-like dedifferentiated state through activation of a PKA-ERK1/2 MAPK-dependent pathways but not via a PI3K/Akt pathway.
Materials and Methods
Materials and animals
DMEM, ERK1/2 inhibitor (U0126), PKA inhibitor (H89), PI3K inhibitor (LY 294002), trypsin (type I), and collagenase (type V) were purchased from Sigma (St. Louis, MO). Ham’s F-12 nutrient mixture was from Invitrogen Corp. (Grand Island, NY). Polyclonal anti-phospho-ERK1/2 (9101), anti-ERK1/2 (9102), anti-phospho-AKT (9271), and anti-AKT (9272) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Monoclonal anti-ck-18 antibody (sc-6259) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Male adult (7–10 yr old) cynomolgus monkeys (Macaca fascicularis) were obtained and housed at the Guangxi Hongfeng Primate Research Center, Institute of Zoology (IOZ), Chinese Academy of Sciences (CAS). Animal handling and experimentation were approved by the Animal Care and Use Review Committee of IOZ, CAS. These monkeys were housed in a standard animal facility under controlled temperature (22 C) and photoperiod (12 h light, 12 h darkness), with free access to water and monkey chow. They were used for mild testicular hyperthermia and testicular tissue and blood collections.
Testes used for isolation and culture of Sertoli cells were from two male adult (5–7 yr old) rhesus monkeys (Macaca mulatta), which were housed in Institute of Biological Products of Beijing, China. The monkeys were housed in the same condition as above. Animal handling and experimentation were approved by the Animal Care and Use Review Committee of IOZ, CAS, and Institute of Biological Products of Beijing.
Heat treatment of cynomolgus monkey testis and testicular tissue and blood collections
Sixteen adult cynomolgus monkeys were used for the experiment. Heating of the scrota of eight adult monkeys was performed as described in rat previously by Lue et al. (3). Briefly, under light sedation, testicular hyperthermia was induced by immersing the monkey scrota containing the testes into a thermostatically controlled water bath at 43 C for 30 min once daily for 2 consecutive days. After the heat treatment, the animals were dried, examined for any redness or injury to the scrota, and then returned to their cages and allowed to recover from the effect of the anesthesia. Inspection of the scrota after heat exposure showed no evidence of thermal injury to the scrotal skin after this short duration of modest increase in temperature. The other eight monkeys were used as the controls. Testicular biopsies or castrations were performed in one testis of the animals on the day before and on d 3, 8, 30, 84, and 144 after heat exposure.
Open testicular biopsies or castrations were performed under general anesthesia with ketamine (10 mg/kg) and atropine (0.05 mg/kg) as premedication. Postoperatively the animals were given oxymorphone (0.1 mg/kg) for analgesia. The operation was performed under aseptic conditions and only one testis from each monkey was used for tissue collection. One portion of the tissue was immersion-fixed in Bouin’s solution and embedded in paraffin, subsequently sectioned for immunohistochemistry, and the other portion snap frozen in liquid nitrogen for protein isolation.
Blood samples were collected from the arm vein of animals before and at wk 2, 4, 6, 8, 10, and 12 after treatment as described above (2), and serum was separated and stored at –20 C for subsequent hormone assays. Serum samples from at least three intact monkeys (without any testicular surgery) in each group were used for hormone assays. Serum FSH was measured by the RIA using reagents supplied by the National Hormone and Pituitary Program through A. F. Parlow, Ph.D. Recombinant cynomolgus FSH (National Institute of Child Health and Human Development, Rec-MoFSH-RP-1, AFP 6940A) was used as a reference preparation. The lower limit of quantitation was 1.0 ng/ml. The intra- and interassay coefficients of variation were relatively high at lower concentrations: 9 and 15% for FSH at 4.3 ng/ml, respectively.
Isolation and primary culture of mature Sertoli cells
Mature Sertoli cells were isolated from adult rhesus monkey testes using a modification of the method as described previously by Majumdar et al. (53). Briefly, the decapsulated testes were minced and washed in cold (4 C) Hanks’ balanced salt solution lacking CaCl2 and MgSO4 (pH 7.4). The suspension was vigorously shaken by hand (eight to 10 times). Seminiferous tubules from suspensions of testes were recovered after sedimentation at unit gravity for 5 min at 4 C. Sedimentation was repeated twice to remove red blood cells and Leydig cells. Then the tubular pieces were incubated with collagenase (0.5 mg/ml) for 10 min at 32 C and monitored closely to limit clumping of tissue that resulted from overdigestion. After washing three times at 150 g for 5 min, the second digestion of adult tissue was performed with 0.05% trypsin for 5 min at 32 C. Upon completion of digestion, fetal bovine serum (FBS) was added to the suspension to terminate the enzyme digestion. After filtration through a 100-mesh stainless steel filter, the filtrate was suspended in DMEM + Ham’s F-12 medium (culture medium) containing 100 U/ml penicillin and 100 μg/ml streptomycin sulfate and washed with Hanks’ balanced salt solution five times (150 g for 5 min) and culture medium three times (150 g for 5 min). The cells were finally suspended in culture medium supplemented with 5% FBS and cultured at 32 C in a humidified atmosphere of 5% CO2 and 95% air. After 12 h, the cells were gently washed to remove unattached germ cells. After an additional 36 h of culture, the cells were digested with pancreatin again and culture medium changed in time to remove unattached germ cells. Then cells were seeded onto 24 x 24-mm coverslips placed in six-well plates (2 x 105 cells/cover-slip) for confocal immunohistochemistry or cultured in six-well plates (2 x 106 cells/well) for protein isolation. The culture medium was changed after every 48 h when necessary and serum starved for 12 h before various treatments.
Immunohistochemical analysis
After deparaffinization and rehydration, 5-μm sections were subjected to antigen retrieval using EDTA buffer [10 mM (pH8.0)] at 98 C for 15 min and then cooled naturally to room temperature. After two washes in PBS, the sections were sequentially incubated with 10% normal blocking serum for 30 min to suppress nonspecific bindings, primary antibodies including anti-ck-18 (1:200), anti-phospho-AKT(1:200), and anti-AKT(1:200) at 4 C overnight, biotinylated secondary antibodies (1:200) at room temperature for 30 min, avidin-alkaline phosphatase complex and Vector Red according to the manufacturer’s protocol (Vectastain ABC-AP kit, Vector Laboratories, Burlingame, CA). Endogenous alkaline phosphatase activity was inhibited with levamisole (Sigma). Intervening PBS washes were performed after incubation when necessary. Sections were counterstained with hematoxylin. For the negative controls, tissue sections were processed without the primary antibodies, which were replaced with the normal rabbit or mouse IgG.
Confocal immunohistochemistry
Sertoli cells cultured on coverslips were treated with control medium, the ERK1/2 inhibitor U0126 (20 μM), PKA inhibitor H89 (10 μM), or PI3K inhibitor LY 294002 (20 μM) followed by heat treatment (43 C, 30 min). The cells were fixed in freshly prepared 4% paraformaldehyde in PBS, followed by washing in PBS (containing 0.1% Tween 20) and incubation with 3% BSA. Then the cells were immunolabeled with the primary anti-ck-18 (1:200) antibody at 4 C overnight, fluorescein isothiocyanate-conjugated antimouse IgG (1:200) at room temperature for 1 h. After three washes in PBS, the cells were incubated with propidium iodide (PI) for 30 min. Slides were finally analyzed by confocal laser scanning microscopy (Carl Zeiss Inc., Thornwood, NY). For the negative controls, the cells were processed without the primary antibody, which was replaced with the normal mouse IgG.
Western blot analysis
For isolation of testicular tissue protein, the snap-frozen testis in liquid nitrogen was homogenized in lysis buffer [5 mM phosphate buffer (pH 7.2), containing 0.1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, and 1 mg/liter chymostatin], whereas for isolation of Sertoli cell protein, the cells were lysed in lysis buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 2 μg/ml aprotinin). The protein content of the supernatant from centrifugation was determined by spectrophotometer, using BSA as a standard. About 40 μg total protein per lane were separated by 10% SDS-PAGE and was electrophoretic transferred to the nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). After blocked in 5% nonfat milk in 0.09% NaCl, 0.1% Tween 20, 100 mM Tris-HCl (pH 7.5), the membranes were incubated with the primary antibodies at the dilutions of 1:1000 each for anti-ck-18, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-AKT, and anti-AKT, respectively, in 5% milk in 0.09% NaCl, 0.1% Tween 20, 100 mM Tris-HCl (pH7.5) at 4 C overnight. The membranes were washed three times and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2500) at room temperature for 1 h. Reactive bands were visualized by SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL), and the membranes were then subjected to x-ray autoradiography. Band intensities were determined by Quantity One software (Bio-Rad).
Results
Animals and serum FSH levels
After scrotal heating, examination of the scrota of the monkeys did not show any significant change. Serum FSH was not significantly different at baseline between the control and heat-treated group. The mean serum FSH levels were not significantly different across time in either group or between the two groups at any time point. In the heat-treated group, baseline mean serum FSH levels were 1.87 ± 1.7 ng/ml and remained not significantly different from baseline at d 14 (1.51 ± 0.17), 28 (1.46 ± 0.17), 42 (1.70 ± 0.17), 56 (1.72 ± 0.17), 70 (1.81 ± 0.17) to d 84 (1.70 ± 0.21 ng/ml) after heat exposure.
Heat-induced ck-18 expression and ERK1/2 activation in mature monkey Sertoli cells
Using immunohistochemical staining in the present study, we examined the effect of the exposure of adult monkey testis to water at 43 C on the expression of ck-18 in the testis. As shown in Fig. 1, no obvious immunoreactivity for ck-18 was observed in the monkey testis before the heat treatment, but it was dramatically induced in the cytoplasm of Sertoli cells on d 3, 8, and 30 after the heat shock. Other testicular cells were all negative for staining of this molecule. On d 84, the expression level of ck-18 seemed to be reduced and it was fully back to the untreated level on d 144. Quantitative analysis of ck-18 protein is shown in Fig. 2. The ck-18 level was significantly increased on d 3, 8, 30, and 84 (P < 0.01) and decreased to the pretreated level on d 144.
To further identify the possible activated signaling molecules involved in the expression of ck-18 by the heat treatment, the total protein levels and phosphorylation status of the two ERK proteins, ERK 1 and 2, were examined in the heat-treated monkey testis by Western blot analysis. Phosphorylation of ERK1/2 was not observed in the untreated samples but obviously up-regulated on d 3 (P < 0.05) after the heat shock. Phosphorylation status reached the maximum on d 8 (P < 0.01) and then decreased to undetectable level at the remaining time points (Fig. 3). The total protein levels remained unchanged all along, suggesting that the heat treatment did not change the total ERK1/2 protein level in the monkey testis.
Heat induces phosphorylation of Akt in mature monkey Sertoli cells
Coincident with the spatiotemporal expression of ck-18, phosphorylated Akt was weakly expressed in the Sertoli cells of the monkey testis before the heat treatment and increased markedly on d 3, 8, and 30 after the heat shock. On d 84 the phosphorylated Akt was also detected transiently in the nuclei of spermatocytes and returned to the basal level on d 144 (Fig. 4). However, the total Akt expression in the Sertoli cells did not change before and after the heat treatment (Fig. 5). The phosphorylation of Akt was also confirmed by Western blot analysis. As shown in Fig. 6, the total Akt protein level remained unchanged, whereas the phosphorylated Akt was up-regulated significantly on d 3, 8, 30, and 84 (P < 0.01) in testis after the heat shock and then decreased to undetectable level on d 144.
Time course of expression of ck-18 induced by heat in the cultured primary mature Sertoli cells
To confirm the in vivo experiment results obtained as shown above, we also isolated Sertoli cells from testes of adult rhesus monkeys. The cells were cultured and then exposed to the similar heat treatment. After heat shock at 43 C for 30 min, the cells were collected at 0, 5, 15, 30, and 60 min after ending the heat treatment and detected with the anti-ck-18 antibody using confocal immunohistochemistry. As shown in Fig. 7A, ck-18 staining was nearly negative in the pretreated Sertoli cells (Pre). Ck-18 staining was detected by just ending the heat shock (0 min); dramatically increased by 5, 15, and 30 min (HS/0, HS/5, HS/15, HS/30); and slightly dropped by 60 min (HS/60) after ending the heat treatment. The result was also confirmed by Western blot analysis (Fig. 7B). Collectively, the in vivo and in vitro results demonstrated that heat could activate expression of ck-18 in mature monkey Sertoli cells.
Heat-induced ERK1/2 activation in cultured primary mature Sertoli cells
Activation of ERK1/2 was assessed by Western blot with an anti-phospho-ERK1/2 antibody that was capable of detecting dually phosphorylated ERK1 and ERK2. As shown in Fig. 8, heat treatment of the Sertoli cells resulted in a transient activation of ERK1/2 kinase. Phosphorylated ERK1/2 was peaked by 0 and 5 min after ending the heat treatment (P < 0.01) and then slowly decreased to undetectable level by 120 min. Reprobing the membranes using an antibody that recognizes total ERK1/2 protein confirmed that the heat treatment specifically altered MAPK activity but did not alter its expression level. Thus, a 5-min period after ending the heat treatment was chosen and used for the subsequent experiments.
To confirm specificity of the heat-induced activation of ERK MAPK, the Sertoli cells were pretreated with 10 μM H89 (PKA inhibitor) or 20 μM U0126 (ERK1/2 inhibitor) for 30 min and then followed by the heat treatment (43 C, 30 min). By 5 min after ending the heat shock, the cells were harvested and the cell lysates were analyzed by Western blot analysis using specific antibodies against either phospho-ERK1/2 or total ERK1/2. The results showed that pretreatment of the Sertoli cells with H89 or U0126 resulted in strong inhibition of the phosphorylation of ERK1/2 induced by the heat (P < 0.01, Fig. 9). These results suggest that heat is capable of inducing ERK1/2 phosphorylation in a time-dependent manner, and the heat-dependent activation of ERK1/2 is predominately mediated by PKA signaling pathway.
Heat-induced Akt activation in cultured primary mature Sertoli cells
Activation of Akt was assessed by Western blot with an anti-phospho-Akt antibody. As shown in Fig. 10A, phosphorylated Akt was significantly up-regulated from 0 to 30 min after ending the heat treatment (P < 0.01) and then slightly decreased by 120 min (P < 0.05). Reprobing the membranes using an anti-Akt antibody confirmed that the heat treatment specifically altered Akt activity but did not alter its expression level. Then the cells were pretreated with 20 μM LY294002 (PI3K inhibitor) for 30 min, followed by heat treatment (43 C, 30 min). By 5 min after ending the heat shock, the cells were harvested, and the cell lysates were analyzed by Western blot analysis using both anti-phospho-Akt and anti-Akt antibodies. The results showed that pretreatment of the Sertoli cells with LY294002 resulted in strong inhibition of the phosphorylation of Akt induced by the heat (P < 0.01, Fig. 10B). These results suggest that heat is also capable of inducing Akt phosphorylation in a time-dependent manner, and the PI3K inhibitor can inhibit the activation of Akt induced by heat treatment.
Effects of inhibitor H89, U0126, or LY294002 on heat-induced expression of ck-18 in the cultured primary mature Sertoli cells
The activation of ERK1/2 and Akt in the Sertoli cells by heat led us to further investigate the causality of these activations in heat-induced expression of ck-18 in the cultured primary mature Sertoli cells. The cells were pretreated with 20 μM U0126, 10 μM H89, or 20 μM LY294002 for 30 min, followed by heat treatment (43 C, 30 min). By 5 min after ending the heat shock, the cells were harvested and examined with anti-ck-18 antibody by confocal immunohistochemistry. As shown in Fig. 11A, the heat treatment strongly activated ck-18 expression (HS/5) in the Sertoli cells as compared with that of the pretreated control (Pre). Addition of H89, U0126, or LY294002 had no obvious influence on the basal ck-18 expression (data not shown), but H89 and U0126 (inactivation of ERK1/2) significantly inhibited the heat-induced ck-18 expression (HS + U0, HS + H89). To our surprise, LY294002 had no inhibitory effect on the activation of ck-18 expression in Sertoli cells (HS + LY). Quantitative analysis of ck-18 protein expression is shown in Fig. 11B. The expression levels in both HS/5 and HS + LY groups were significantly increased (P < 0.01).
Discussion
It is well known that the elevated temperature may be responsible for cellular, histological, and hormonal changes in the testis, which impair spermatogenesis and fertility (3, 5, 54, 55, 56). At the period around onset of puberty, Sertoli cells undergo a radical change in their morphology and function, heralding the switch from an immature, proliferative state to a mature, nonproliferative state (57). As an only supportive cell within the seminiferous epithelium, Sertoli cells perform several physiological functions essential to normal spermatogenesis. But our question is what kind of influence would heat treatment bring to mature Sertoli cells Our previous study in the cryptorchid testis of adult rhesus monkeys has shown that the body temperature could induce reexpression of ck-18, a marker of immature or undifferentiated Sertoli cells (16, 26) and other intermediate filaments in the differentiated Sertoli cells (8), indicating that the adult Sertoli cells have reverted to a dedifferentiated state in the cryptorchid testis and thus lost their supportive role in normal spermatogenesis, leading to a cessation of spermatogenic activity.
Because adult cryptorchid model reflects temperature-induced effects, in the present study, we further demonstrated that local heating the adult monkey testis at 43 C or exposure to the cultured mature monkey Sertoli cells could also induce ck-18 reexpression in the differentiated Sertoli cells. The in vivo data showed that the reexpression of ck-18 in the Sertoli cells of the heat-treated testis was coincident with impairment of seminiferous epithelium. On d 144, the expression of ck-18 nearly completely disappeared in the Sertoli cells when spermatogenesis returned to the normal level (5). It has been reported that secretion of androgen-binding protein, another functional marker of Sertoli cells, was impaired by heat stress (58, 59). These findings suggest that not only germ cells but also Sertoli cells may be affected by heat treatment, inducing Sertoli cells to regain undifferentiated features. The affected Sertoli cells may thus lose their supportive role in spermatogenesis. Clearly future studies on the Sertoli cell functional markers such as measuring the production of testicular androgen-binding protein are needed to test this hypothesis.
MAPKs are serine/threonine kinases that transmit signals from extracellular stimuli to multiple substrates involved in cell growth, differentiation, and apoptosis. Three major subfamilies of MAPKs, ERK, c-Jun N-terminal kinase (JNK), and p38, have been identified. The outcome of MAPK activation depends on the mode of activation, cell type, and threshold of activity. Although the ERK MAPKs generally regulate cell growth and differentiation and the JNK and p38 family MAPKs preferentially mediate stress, there is now increasing evidence that activation of the ERK MAPKs can also be stimulated by a variety of stress stimuli such as heat (60, 61). Heat shock can activate ERK MAPKs via pathways independent (61) or dependent (62) of Ras and Raf activation. We have specifically demonstrated the activation of ERK1/2 in the cryptorchid (our unpublished data) and the locally heated monkey testis.
Our results showed that heat could activate only the phospho-ERK1/2 at the early days after heat treatment but did not alter the total ERK1/2 expression level in the testes. Such change was also found in the activation of Akt. Interestingly, phospho-Akt activation in the Sertoli cells was also induced by the local water treatment at 43 C of the monkey testis in the present study. The active phospho-Akt activity showed a high degree of accumulation in the Sertoli cells on d 3, 8, and 30 after the heat shock; however, the total level of Akt was not altered before and after the heat treatment. We have also detected the expression changes of p38 and JNK in the testes of experimental cryptorchidism of adult rhesus monkeys. Results showed that heat induced by experimental cryptorchidism could activate neither the phospho-p38 MAPK nor the total expression of p38 MAPK in Sertoli cells. The phospho-JNK was detected in neither the scrotal testes nor the cryptorchid testes, whereas the total JNK expression level increased after experimental cryptorchidism (our unpublished data). Therefore, the different expression patterns of p38 and JNK as compared with that of ERK in the testes of experimental cryptorchidism of adult rhesus monkeys gave us hint to further investigate the possible role of ERK in this study. Coincidence of the spatial and temporal activation of both ERK1/2 and Akt with the reexpression of ck-18 in the adult monkey Sertoli cells raises the question whether dedifferentiation of Sertoli cells represented by the ck-18 reexpression is regulated through ERK MAPK kinase pathway and/or PI3K/Akt pathway or they are merely a correlation indicating that ERK 1/2 phosphorylation, Akt phosphorylation and ck-18 re-expression occur at the same time.
To answer these questions, we designed in vitro experiments using differentiated primary Sertoli cells from adult monkey testis to examine the possible signal pathway responsible for the mature Sertoli cell dedifferentiation. The time course of ck-18 expression in the heat-treated Sertoli cells revealed that treatment at 43 C for 30 min could induce expression of ck-18 immediately and peaked by 5 min after ending the treatment, confirming the results obtained in vivo. Our in vivo study showed that the heat could induce the Sertoli cells to regain undifferentiated features. As we expected, the heat treatment also activated ERK1/2 phosphorylation in Sertoli cells immediately after ending the heat shock. Blocking the ERK MAPK signal pathway with prior administration of its inhibitor U0126 could inhibit the activation of ERK1/2 by the heat treatment, and the inhibitor also could remarkably decrease the expression of ck-18 in Sertoli cells induced by the heat shock. Interestingly, although both ck-18 and ERK1/2 activation occurred shortly after heat shock, the activation of ck-18 lasted longer than that of ERK1/2. The first reason is probably that ck-18 needs longer time to adjust to the basal level after heat shock than ERK1/2 does, and the second is that ck-18 reexpression after heat shock in Sertoli cells might be mediated by other signal pathways in addition to ERK MAPK. For example, in our experimental cryptorchidism rhesus monkey models, we found that JNK was up-regulated until d 30 when expression level of ck-18 was still high (our unpublished data). These suggest that other signaling pathways or molecules could modulate the dedifferentiation processes of Sertoli cells after heat treatment.
The cAMP-dependent kinase (PKA)-mediated activation of ERK1/2 in Sertoli cells is modulated by FSH (42). Transient testicular heating did not induce significant changes in serum FSH levels, indicating that heat-induced PKA activation of ERK 1/2 in Sertoli cells may be a FSH-independent process. To our knowledge, however, there is no report available about the PKA activation in heat-treated Sertoli cells. To investigate this point, Sertoli cells obtained from adult monkeys were preincubated with H89, a well-known pharmacological inhibitor of PKA activity, before the heat treatment. As expected, the heat-induced activation of ERK1/2 was completely abrogated by the addition of H89 to the culture, suggesting that heat-induced ERK1/2 activation may be dependent on PKA activation. Furthermore, pretreatment of the cells with H89 also significantly inhibited the heat-induced ck-18 expression in the Sertoli cells. These observations raise the possibility that dedifferentiation of Sertoli cells induced by heat treatment may be mediated by the ERK MAPK pathway, and ERK1/2 activation is partially PKA dependent. To our surprise, blockade of Akt phosphorylation with the PI3K inhibitor LY294002 could inhibit the activation of Akt induced by heat treatment but had no inhibitory effect on activation of ck-18 expression in the cultured Sertoli cells, implying that dedifferentiation of Sertoli cells induced by the heat appears to be independent of the PI3K/Akt pathway.
In conclusion, this study illustrates for the first time in the monkey Sertoli cells that heat treatment could induce a reversible change of the Sertoli cells from an adult-differentiated state to an immature-like dedifferentiated state through activation of PKA-ERK MAPK-mediated pathways but not via the PI3K/Akt pathway-mediated pathway.
Acknowledgments
The authors thank the Guangxi Hongfeng Primate Research Center and the Institute of Biological Products of Beijing for animal health care.
Footnotes
This work was supported by the "973" project (G1999055901), National Nature Science Foundation of China (30230190), and Chinese Academy of Sciences Chuangxi program (KSCX-2-SW-201), and grants from Mellon Reproductive Biology Center (to R.S.S., C.W.), CONRAD-Mellon Twinning grant (MFG-03-67) (to R.S.S., C.W., A.P.S.H., and Y.-H.L.), and RO1-HD 39293 (to R.S.S., C.W., and A.P.S.H.).
All authors have nothing to declare.
First Published Online December 8, 2005
Abbreviations: ck, Cytokeratin; FBS, fetal bovine serum; JNK, c-Jun N-terminal kinase; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A.
Accepted for publication November 30, 2005.
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Division of Endocrinology (Y.-H.H., A.P.S.H., R.S.S., C.W.), Department of Medicine, Harbor-University of California, Los Angeles, Medical Center and Los Angeles Biomedical Research Institute, Torrance, California 90509
Graduate School of the Chinese Academy of Sciences (X.-S.Z.), Beijing 100039, China
Abstract
Sertoli cells play a key role in triggering and regulating the process of spermatogenesis. Failure of a Sertoli cell to mature functionally will presumably render it incapable of supporting germ cell survival and development that appeared after puberty. Expression of cytokeratin 18 (ck-18) intermediate filaments indicates a state of undifferentiation usually observed in Sertoli cells of prepubertal testis. In this study we demonstrated that local testicular heat treatment of adult monkey with water at 43 C for 30 min once daily for 2 consecutive days was capable of activating reexpression of ck-18 in Sertoli cells, which was coincident with activation of ERK1/2 and Akt kinases. Using primary Sertoli cell culture isolated from adult monkey testis, we further confirmed that the heat treatment of the cells at 43 C could also induce ck-18 reexpression, which was similar to the in vivo treatment. ERK MAPK was also induced by the heat treatment in a time- and protein kinase A (PKA)-dependent manner. After blocking the ERK MAPK signaling pathway, an inhibition of ck-18 expression in the cultured Sertoli cells was observed, and this inhibitory effect was also detected by blocking the PKA activation. However, ck-18 activation in Sertoli cells remained unaltered when the phosphatidylinositol 3-kinase/Akt pathway was blocked. In conclusion, the heat treatment of adult monkey Sertoli cells are capable of inducing a reversible change in the Sertoli cells from an adult differentiated state to an immature-like dedifferentiated state through PKA-ERK MAPK-dependent pathways but not via the phosphatidylinositol 3-kinase/Akt pathway.
Introduction
IT HAS BEEN well defined that local treatment of rat and monkey testes in water at 43 C could induce reversible damage to the seminiferous epithelium due to increased germ cell apoptosis (1, 2, 3, 4, 5). But the molecular mechanism is not clear. Reports have shown that elevation of testicular temperature not only causes germ cell loss but also affects the morphology and function of Sertoli cells (6, 7, 8, 9). Mature Sertoli cells are the primary supportive cells of seminiferous epithelium and provide an essential structural and nutritional support for the developing spermatogenic cells in the seminiferous tubules (10, 11). Sertoli cells can also interact with Leydig cells and peritubular cells to regulate spermatogenic process (12, 13, 14). However, Sertoli cells with immature characteristics have been found in the adult human testes in some pathological conditions usually associated with impaired spermatogenesis (15, 16, 17, 18).
Cytokeratin (ck) intermediate filament has been demonstrated to be a Sertoli cell differentiation marker, which is only expressed in immature Sertoli cells and normally lost at puberty (15, 19, 20, 21, 22, 23). Ck-18 is a subtype of the cytokeratin family that represents an excellent marker of immature or undifferentiated Sertoli cells in the seminiferous epithelium (15, 16, 24, 25, 26, 27). We have demonstrated that cryptorchid testis of rhesus monkey could induce expression of ck-18 and other intermediate filaments in Sertoli cells coincidentally with a cessation of spermatogenic activity (8), implying that body temperature is capable of inducing adult Sertoli cells reverting to a dedifferentiated immature state and thus destroys their supportive role in normal spermatogenesis. It is therefore suggested that Sertoli cells undergoing a process of dedifferentiation, as indicated by the reexpression of cytokeratin, may subsequently result in a loss of Sertoli cell function and lead to a cession of spermatogenic activity.
ERK1/2 represent one subfamily of serine/threonine protein kinases collectively referred to as the MAPK family. They have the unique feature of being phosphorylated by an upstream dual-specificity kinase called MAPK kinase or MAPK/ERK kinase (28, 29, 30, 31, 32). The ERK MAPK kinase pathway is essential for controlling cell proliferation and differentiation (33, 34, 35, 36, 37, 38, 39). It has been demonstrated that sustained signaling through the ERK MAPK kinase pathway is capable of driving the dedifferentiation of Schwann cells (40), whereas blocking the ERK MAPK kinase pathway could induce undifferentiated tumor cells to undergo a process of differentiation, but their proliferation activities were inhibited (41). In the neonatal Sertoli cells isolated 5 d after birth, the ERK MAPK pathway was activated by FSH in a protein kinase A (PKA)-dependent manner; the activation, however, was required for Sertoli cell proliferation. In contrast, 19 d after birth, as the Sertoli cells proceeded through their differentiation program, the ERK pathway was dramatically inhibited by FSH treatment (42). These results suggest that the ERK-dependent signaling is involved in the proliferation and differentiation of the Sertoli cells in a stage-specific manner modulated by FSH. However, little is known about the switch between differentiation and dedifferentiation and how the ERK MAPK kinase pathway is affected in Sertoli cells after the heat treatment.
Akt (also known as protein kinase B), a key effector of phosphatidylinositol 3-kinase (PI3K) in another survival signaling pathway, is also a subfamily of serine/threonine protein kinase (43, 44, 45, 46). Interestingly, Akt has been shown to produce synergistic or counteractive regulation on the MAPK-induced cellular responses by modulating Raf, the MAPK kinase kinase (47). The PI3K/Akt pathway has been associated with cell differentiation and dedifferentiation (48, 49, 50) and reported to be phosphorylated in response to cellular stress, such as heat shock (51, 52). However whether the PI3K/Akt pathway plays a role in the differentiation of Sertoli cells after heat treatment has remained unknown.
In this study we designed both in vivo (using adult monkey testicular hyperthermia model) and in vitro (using primary culture of Sertoli cells isolated from adult monkey testes) experiments to examine the possible effect of the well-confirmed heat treatment at 43 C on Sertoli cell dedifferentiation. We have demonstrated for the first time that the heat treatment at 43 C could induce change in the Sertoli cells from an adult differentiated state to an immature-like dedifferentiated state through activation of a PKA-ERK1/2 MAPK-dependent pathways but not via a PI3K/Akt pathway.
Materials and Methods
Materials and animals
DMEM, ERK1/2 inhibitor (U0126), PKA inhibitor (H89), PI3K inhibitor (LY 294002), trypsin (type I), and collagenase (type V) were purchased from Sigma (St. Louis, MO). Ham’s F-12 nutrient mixture was from Invitrogen Corp. (Grand Island, NY). Polyclonal anti-phospho-ERK1/2 (9101), anti-ERK1/2 (9102), anti-phospho-AKT (9271), and anti-AKT (9272) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Monoclonal anti-ck-18 antibody (sc-6259) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Male adult (7–10 yr old) cynomolgus monkeys (Macaca fascicularis) were obtained and housed at the Guangxi Hongfeng Primate Research Center, Institute of Zoology (IOZ), Chinese Academy of Sciences (CAS). Animal handling and experimentation were approved by the Animal Care and Use Review Committee of IOZ, CAS. These monkeys were housed in a standard animal facility under controlled temperature (22 C) and photoperiod (12 h light, 12 h darkness), with free access to water and monkey chow. They were used for mild testicular hyperthermia and testicular tissue and blood collections.
Testes used for isolation and culture of Sertoli cells were from two male adult (5–7 yr old) rhesus monkeys (Macaca mulatta), which were housed in Institute of Biological Products of Beijing, China. The monkeys were housed in the same condition as above. Animal handling and experimentation were approved by the Animal Care and Use Review Committee of IOZ, CAS, and Institute of Biological Products of Beijing.
Heat treatment of cynomolgus monkey testis and testicular tissue and blood collections
Sixteen adult cynomolgus monkeys were used for the experiment. Heating of the scrota of eight adult monkeys was performed as described in rat previously by Lue et al. (3). Briefly, under light sedation, testicular hyperthermia was induced by immersing the monkey scrota containing the testes into a thermostatically controlled water bath at 43 C for 30 min once daily for 2 consecutive days. After the heat treatment, the animals were dried, examined for any redness or injury to the scrota, and then returned to their cages and allowed to recover from the effect of the anesthesia. Inspection of the scrota after heat exposure showed no evidence of thermal injury to the scrotal skin after this short duration of modest increase in temperature. The other eight monkeys were used as the controls. Testicular biopsies or castrations were performed in one testis of the animals on the day before and on d 3, 8, 30, 84, and 144 after heat exposure.
Open testicular biopsies or castrations were performed under general anesthesia with ketamine (10 mg/kg) and atropine (0.05 mg/kg) as premedication. Postoperatively the animals were given oxymorphone (0.1 mg/kg) for analgesia. The operation was performed under aseptic conditions and only one testis from each monkey was used for tissue collection. One portion of the tissue was immersion-fixed in Bouin’s solution and embedded in paraffin, subsequently sectioned for immunohistochemistry, and the other portion snap frozen in liquid nitrogen for protein isolation.
Blood samples were collected from the arm vein of animals before and at wk 2, 4, 6, 8, 10, and 12 after treatment as described above (2), and serum was separated and stored at –20 C for subsequent hormone assays. Serum samples from at least three intact monkeys (without any testicular surgery) in each group were used for hormone assays. Serum FSH was measured by the RIA using reagents supplied by the National Hormone and Pituitary Program through A. F. Parlow, Ph.D. Recombinant cynomolgus FSH (National Institute of Child Health and Human Development, Rec-MoFSH-RP-1, AFP 6940A) was used as a reference preparation. The lower limit of quantitation was 1.0 ng/ml. The intra- and interassay coefficients of variation were relatively high at lower concentrations: 9 and 15% for FSH at 4.3 ng/ml, respectively.
Isolation and primary culture of mature Sertoli cells
Mature Sertoli cells were isolated from adult rhesus monkey testes using a modification of the method as described previously by Majumdar et al. (53). Briefly, the decapsulated testes were minced and washed in cold (4 C) Hanks’ balanced salt solution lacking CaCl2 and MgSO4 (pH 7.4). The suspension was vigorously shaken by hand (eight to 10 times). Seminiferous tubules from suspensions of testes were recovered after sedimentation at unit gravity for 5 min at 4 C. Sedimentation was repeated twice to remove red blood cells and Leydig cells. Then the tubular pieces were incubated with collagenase (0.5 mg/ml) for 10 min at 32 C and monitored closely to limit clumping of tissue that resulted from overdigestion. After washing three times at 150 g for 5 min, the second digestion of adult tissue was performed with 0.05% trypsin for 5 min at 32 C. Upon completion of digestion, fetal bovine serum (FBS) was added to the suspension to terminate the enzyme digestion. After filtration through a 100-mesh stainless steel filter, the filtrate was suspended in DMEM + Ham’s F-12 medium (culture medium) containing 100 U/ml penicillin and 100 μg/ml streptomycin sulfate and washed with Hanks’ balanced salt solution five times (150 g for 5 min) and culture medium three times (150 g for 5 min). The cells were finally suspended in culture medium supplemented with 5% FBS and cultured at 32 C in a humidified atmosphere of 5% CO2 and 95% air. After 12 h, the cells were gently washed to remove unattached germ cells. After an additional 36 h of culture, the cells were digested with pancreatin again and culture medium changed in time to remove unattached germ cells. Then cells were seeded onto 24 x 24-mm coverslips placed in six-well plates (2 x 105 cells/cover-slip) for confocal immunohistochemistry or cultured in six-well plates (2 x 106 cells/well) for protein isolation. The culture medium was changed after every 48 h when necessary and serum starved for 12 h before various treatments.
Immunohistochemical analysis
After deparaffinization and rehydration, 5-μm sections were subjected to antigen retrieval using EDTA buffer [10 mM (pH8.0)] at 98 C for 15 min and then cooled naturally to room temperature. After two washes in PBS, the sections were sequentially incubated with 10% normal blocking serum for 30 min to suppress nonspecific bindings, primary antibodies including anti-ck-18 (1:200), anti-phospho-AKT(1:200), and anti-AKT(1:200) at 4 C overnight, biotinylated secondary antibodies (1:200) at room temperature for 30 min, avidin-alkaline phosphatase complex and Vector Red according to the manufacturer’s protocol (Vectastain ABC-AP kit, Vector Laboratories, Burlingame, CA). Endogenous alkaline phosphatase activity was inhibited with levamisole (Sigma). Intervening PBS washes were performed after incubation when necessary. Sections were counterstained with hematoxylin. For the negative controls, tissue sections were processed without the primary antibodies, which were replaced with the normal rabbit or mouse IgG.
Confocal immunohistochemistry
Sertoli cells cultured on coverslips were treated with control medium, the ERK1/2 inhibitor U0126 (20 μM), PKA inhibitor H89 (10 μM), or PI3K inhibitor LY 294002 (20 μM) followed by heat treatment (43 C, 30 min). The cells were fixed in freshly prepared 4% paraformaldehyde in PBS, followed by washing in PBS (containing 0.1% Tween 20) and incubation with 3% BSA. Then the cells were immunolabeled with the primary anti-ck-18 (1:200) antibody at 4 C overnight, fluorescein isothiocyanate-conjugated antimouse IgG (1:200) at room temperature for 1 h. After three washes in PBS, the cells were incubated with propidium iodide (PI) for 30 min. Slides were finally analyzed by confocal laser scanning microscopy (Carl Zeiss Inc., Thornwood, NY). For the negative controls, the cells were processed without the primary antibody, which was replaced with the normal mouse IgG.
Western blot analysis
For isolation of testicular tissue protein, the snap-frozen testis in liquid nitrogen was homogenized in lysis buffer [5 mM phosphate buffer (pH 7.2), containing 0.1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, and 1 mg/liter chymostatin], whereas for isolation of Sertoli cell protein, the cells were lysed in lysis buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 2 μg/ml aprotinin). The protein content of the supernatant from centrifugation was determined by spectrophotometer, using BSA as a standard. About 40 μg total protein per lane were separated by 10% SDS-PAGE and was electrophoretic transferred to the nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). After blocked in 5% nonfat milk in 0.09% NaCl, 0.1% Tween 20, 100 mM Tris-HCl (pH 7.5), the membranes were incubated with the primary antibodies at the dilutions of 1:1000 each for anti-ck-18, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-AKT, and anti-AKT, respectively, in 5% milk in 0.09% NaCl, 0.1% Tween 20, 100 mM Tris-HCl (pH7.5) at 4 C overnight. The membranes were washed three times and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2500) at room temperature for 1 h. Reactive bands were visualized by SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL), and the membranes were then subjected to x-ray autoradiography. Band intensities were determined by Quantity One software (Bio-Rad).
Results
Animals and serum FSH levels
After scrotal heating, examination of the scrota of the monkeys did not show any significant change. Serum FSH was not significantly different at baseline between the control and heat-treated group. The mean serum FSH levels were not significantly different across time in either group or between the two groups at any time point. In the heat-treated group, baseline mean serum FSH levels were 1.87 ± 1.7 ng/ml and remained not significantly different from baseline at d 14 (1.51 ± 0.17), 28 (1.46 ± 0.17), 42 (1.70 ± 0.17), 56 (1.72 ± 0.17), 70 (1.81 ± 0.17) to d 84 (1.70 ± 0.21 ng/ml) after heat exposure.
Heat-induced ck-18 expression and ERK1/2 activation in mature monkey Sertoli cells
Using immunohistochemical staining in the present study, we examined the effect of the exposure of adult monkey testis to water at 43 C on the expression of ck-18 in the testis. As shown in Fig. 1, no obvious immunoreactivity for ck-18 was observed in the monkey testis before the heat treatment, but it was dramatically induced in the cytoplasm of Sertoli cells on d 3, 8, and 30 after the heat shock. Other testicular cells were all negative for staining of this molecule. On d 84, the expression level of ck-18 seemed to be reduced and it was fully back to the untreated level on d 144. Quantitative analysis of ck-18 protein is shown in Fig. 2. The ck-18 level was significantly increased on d 3, 8, 30, and 84 (P < 0.01) and decreased to the pretreated level on d 144.
To further identify the possible activated signaling molecules involved in the expression of ck-18 by the heat treatment, the total protein levels and phosphorylation status of the two ERK proteins, ERK 1 and 2, were examined in the heat-treated monkey testis by Western blot analysis. Phosphorylation of ERK1/2 was not observed in the untreated samples but obviously up-regulated on d 3 (P < 0.05) after the heat shock. Phosphorylation status reached the maximum on d 8 (P < 0.01) and then decreased to undetectable level at the remaining time points (Fig. 3). The total protein levels remained unchanged all along, suggesting that the heat treatment did not change the total ERK1/2 protein level in the monkey testis.
Heat induces phosphorylation of Akt in mature monkey Sertoli cells
Coincident with the spatiotemporal expression of ck-18, phosphorylated Akt was weakly expressed in the Sertoli cells of the monkey testis before the heat treatment and increased markedly on d 3, 8, and 30 after the heat shock. On d 84 the phosphorylated Akt was also detected transiently in the nuclei of spermatocytes and returned to the basal level on d 144 (Fig. 4). However, the total Akt expression in the Sertoli cells did not change before and after the heat treatment (Fig. 5). The phosphorylation of Akt was also confirmed by Western blot analysis. As shown in Fig. 6, the total Akt protein level remained unchanged, whereas the phosphorylated Akt was up-regulated significantly on d 3, 8, 30, and 84 (P < 0.01) in testis after the heat shock and then decreased to undetectable level on d 144.
Time course of expression of ck-18 induced by heat in the cultured primary mature Sertoli cells
To confirm the in vivo experiment results obtained as shown above, we also isolated Sertoli cells from testes of adult rhesus monkeys. The cells were cultured and then exposed to the similar heat treatment. After heat shock at 43 C for 30 min, the cells were collected at 0, 5, 15, 30, and 60 min after ending the heat treatment and detected with the anti-ck-18 antibody using confocal immunohistochemistry. As shown in Fig. 7A, ck-18 staining was nearly negative in the pretreated Sertoli cells (Pre). Ck-18 staining was detected by just ending the heat shock (0 min); dramatically increased by 5, 15, and 30 min (HS/0, HS/5, HS/15, HS/30); and slightly dropped by 60 min (HS/60) after ending the heat treatment. The result was also confirmed by Western blot analysis (Fig. 7B). Collectively, the in vivo and in vitro results demonstrated that heat could activate expression of ck-18 in mature monkey Sertoli cells.
Heat-induced ERK1/2 activation in cultured primary mature Sertoli cells
Activation of ERK1/2 was assessed by Western blot with an anti-phospho-ERK1/2 antibody that was capable of detecting dually phosphorylated ERK1 and ERK2. As shown in Fig. 8, heat treatment of the Sertoli cells resulted in a transient activation of ERK1/2 kinase. Phosphorylated ERK1/2 was peaked by 0 and 5 min after ending the heat treatment (P < 0.01) and then slowly decreased to undetectable level by 120 min. Reprobing the membranes using an antibody that recognizes total ERK1/2 protein confirmed that the heat treatment specifically altered MAPK activity but did not alter its expression level. Thus, a 5-min period after ending the heat treatment was chosen and used for the subsequent experiments.
To confirm specificity of the heat-induced activation of ERK MAPK, the Sertoli cells were pretreated with 10 μM H89 (PKA inhibitor) or 20 μM U0126 (ERK1/2 inhibitor) for 30 min and then followed by the heat treatment (43 C, 30 min). By 5 min after ending the heat shock, the cells were harvested and the cell lysates were analyzed by Western blot analysis using specific antibodies against either phospho-ERK1/2 or total ERK1/2. The results showed that pretreatment of the Sertoli cells with H89 or U0126 resulted in strong inhibition of the phosphorylation of ERK1/2 induced by the heat (P < 0.01, Fig. 9). These results suggest that heat is capable of inducing ERK1/2 phosphorylation in a time-dependent manner, and the heat-dependent activation of ERK1/2 is predominately mediated by PKA signaling pathway.
Heat-induced Akt activation in cultured primary mature Sertoli cells
Activation of Akt was assessed by Western blot with an anti-phospho-Akt antibody. As shown in Fig. 10A, phosphorylated Akt was significantly up-regulated from 0 to 30 min after ending the heat treatment (P < 0.01) and then slightly decreased by 120 min (P < 0.05). Reprobing the membranes using an anti-Akt antibody confirmed that the heat treatment specifically altered Akt activity but did not alter its expression level. Then the cells were pretreated with 20 μM LY294002 (PI3K inhibitor) for 30 min, followed by heat treatment (43 C, 30 min). By 5 min after ending the heat shock, the cells were harvested, and the cell lysates were analyzed by Western blot analysis using both anti-phospho-Akt and anti-Akt antibodies. The results showed that pretreatment of the Sertoli cells with LY294002 resulted in strong inhibition of the phosphorylation of Akt induced by the heat (P < 0.01, Fig. 10B). These results suggest that heat is also capable of inducing Akt phosphorylation in a time-dependent manner, and the PI3K inhibitor can inhibit the activation of Akt induced by heat treatment.
Effects of inhibitor H89, U0126, or LY294002 on heat-induced expression of ck-18 in the cultured primary mature Sertoli cells
The activation of ERK1/2 and Akt in the Sertoli cells by heat led us to further investigate the causality of these activations in heat-induced expression of ck-18 in the cultured primary mature Sertoli cells. The cells were pretreated with 20 μM U0126, 10 μM H89, or 20 μM LY294002 for 30 min, followed by heat treatment (43 C, 30 min). By 5 min after ending the heat shock, the cells were harvested and examined with anti-ck-18 antibody by confocal immunohistochemistry. As shown in Fig. 11A, the heat treatment strongly activated ck-18 expression (HS/5) in the Sertoli cells as compared with that of the pretreated control (Pre). Addition of H89, U0126, or LY294002 had no obvious influence on the basal ck-18 expression (data not shown), but H89 and U0126 (inactivation of ERK1/2) significantly inhibited the heat-induced ck-18 expression (HS + U0, HS + H89). To our surprise, LY294002 had no inhibitory effect on the activation of ck-18 expression in Sertoli cells (HS + LY). Quantitative analysis of ck-18 protein expression is shown in Fig. 11B. The expression levels in both HS/5 and HS + LY groups were significantly increased (P < 0.01).
Discussion
It is well known that the elevated temperature may be responsible for cellular, histological, and hormonal changes in the testis, which impair spermatogenesis and fertility (3, 5, 54, 55, 56). At the period around onset of puberty, Sertoli cells undergo a radical change in their morphology and function, heralding the switch from an immature, proliferative state to a mature, nonproliferative state (57). As an only supportive cell within the seminiferous epithelium, Sertoli cells perform several physiological functions essential to normal spermatogenesis. But our question is what kind of influence would heat treatment bring to mature Sertoli cells Our previous study in the cryptorchid testis of adult rhesus monkeys has shown that the body temperature could induce reexpression of ck-18, a marker of immature or undifferentiated Sertoli cells (16, 26) and other intermediate filaments in the differentiated Sertoli cells (8), indicating that the adult Sertoli cells have reverted to a dedifferentiated state in the cryptorchid testis and thus lost their supportive role in normal spermatogenesis, leading to a cessation of spermatogenic activity.
Because adult cryptorchid model reflects temperature-induced effects, in the present study, we further demonstrated that local heating the adult monkey testis at 43 C or exposure to the cultured mature monkey Sertoli cells could also induce ck-18 reexpression in the differentiated Sertoli cells. The in vivo data showed that the reexpression of ck-18 in the Sertoli cells of the heat-treated testis was coincident with impairment of seminiferous epithelium. On d 144, the expression of ck-18 nearly completely disappeared in the Sertoli cells when spermatogenesis returned to the normal level (5). It has been reported that secretion of androgen-binding protein, another functional marker of Sertoli cells, was impaired by heat stress (58, 59). These findings suggest that not only germ cells but also Sertoli cells may be affected by heat treatment, inducing Sertoli cells to regain undifferentiated features. The affected Sertoli cells may thus lose their supportive role in spermatogenesis. Clearly future studies on the Sertoli cell functional markers such as measuring the production of testicular androgen-binding protein are needed to test this hypothesis.
MAPKs are serine/threonine kinases that transmit signals from extracellular stimuli to multiple substrates involved in cell growth, differentiation, and apoptosis. Three major subfamilies of MAPKs, ERK, c-Jun N-terminal kinase (JNK), and p38, have been identified. The outcome of MAPK activation depends on the mode of activation, cell type, and threshold of activity. Although the ERK MAPKs generally regulate cell growth and differentiation and the JNK and p38 family MAPKs preferentially mediate stress, there is now increasing evidence that activation of the ERK MAPKs can also be stimulated by a variety of stress stimuli such as heat (60, 61). Heat shock can activate ERK MAPKs via pathways independent (61) or dependent (62) of Ras and Raf activation. We have specifically demonstrated the activation of ERK1/2 in the cryptorchid (our unpublished data) and the locally heated monkey testis.
Our results showed that heat could activate only the phospho-ERK1/2 at the early days after heat treatment but did not alter the total ERK1/2 expression level in the testes. Such change was also found in the activation of Akt. Interestingly, phospho-Akt activation in the Sertoli cells was also induced by the local water treatment at 43 C of the monkey testis in the present study. The active phospho-Akt activity showed a high degree of accumulation in the Sertoli cells on d 3, 8, and 30 after the heat shock; however, the total level of Akt was not altered before and after the heat treatment. We have also detected the expression changes of p38 and JNK in the testes of experimental cryptorchidism of adult rhesus monkeys. Results showed that heat induced by experimental cryptorchidism could activate neither the phospho-p38 MAPK nor the total expression of p38 MAPK in Sertoli cells. The phospho-JNK was detected in neither the scrotal testes nor the cryptorchid testes, whereas the total JNK expression level increased after experimental cryptorchidism (our unpublished data). Therefore, the different expression patterns of p38 and JNK as compared with that of ERK in the testes of experimental cryptorchidism of adult rhesus monkeys gave us hint to further investigate the possible role of ERK in this study. Coincidence of the spatial and temporal activation of both ERK1/2 and Akt with the reexpression of ck-18 in the adult monkey Sertoli cells raises the question whether dedifferentiation of Sertoli cells represented by the ck-18 reexpression is regulated through ERK MAPK kinase pathway and/or PI3K/Akt pathway or they are merely a correlation indicating that ERK 1/2 phosphorylation, Akt phosphorylation and ck-18 re-expression occur at the same time.
To answer these questions, we designed in vitro experiments using differentiated primary Sertoli cells from adult monkey testis to examine the possible signal pathway responsible for the mature Sertoli cell dedifferentiation. The time course of ck-18 expression in the heat-treated Sertoli cells revealed that treatment at 43 C for 30 min could induce expression of ck-18 immediately and peaked by 5 min after ending the treatment, confirming the results obtained in vivo. Our in vivo study showed that the heat could induce the Sertoli cells to regain undifferentiated features. As we expected, the heat treatment also activated ERK1/2 phosphorylation in Sertoli cells immediately after ending the heat shock. Blocking the ERK MAPK signal pathway with prior administration of its inhibitor U0126 could inhibit the activation of ERK1/2 by the heat treatment, and the inhibitor also could remarkably decrease the expression of ck-18 in Sertoli cells induced by the heat shock. Interestingly, although both ck-18 and ERK1/2 activation occurred shortly after heat shock, the activation of ck-18 lasted longer than that of ERK1/2. The first reason is probably that ck-18 needs longer time to adjust to the basal level after heat shock than ERK1/2 does, and the second is that ck-18 reexpression after heat shock in Sertoli cells might be mediated by other signal pathways in addition to ERK MAPK. For example, in our experimental cryptorchidism rhesus monkey models, we found that JNK was up-regulated until d 30 when expression level of ck-18 was still high (our unpublished data). These suggest that other signaling pathways or molecules could modulate the dedifferentiation processes of Sertoli cells after heat treatment.
The cAMP-dependent kinase (PKA)-mediated activation of ERK1/2 in Sertoli cells is modulated by FSH (42). Transient testicular heating did not induce significant changes in serum FSH levels, indicating that heat-induced PKA activation of ERK 1/2 in Sertoli cells may be a FSH-independent process. To our knowledge, however, there is no report available about the PKA activation in heat-treated Sertoli cells. To investigate this point, Sertoli cells obtained from adult monkeys were preincubated with H89, a well-known pharmacological inhibitor of PKA activity, before the heat treatment. As expected, the heat-induced activation of ERK1/2 was completely abrogated by the addition of H89 to the culture, suggesting that heat-induced ERK1/2 activation may be dependent on PKA activation. Furthermore, pretreatment of the cells with H89 also significantly inhibited the heat-induced ck-18 expression in the Sertoli cells. These observations raise the possibility that dedifferentiation of Sertoli cells induced by heat treatment may be mediated by the ERK MAPK pathway, and ERK1/2 activation is partially PKA dependent. To our surprise, blockade of Akt phosphorylation with the PI3K inhibitor LY294002 could inhibit the activation of Akt induced by heat treatment but had no inhibitory effect on activation of ck-18 expression in the cultured Sertoli cells, implying that dedifferentiation of Sertoli cells induced by the heat appears to be independent of the PI3K/Akt pathway.
In conclusion, this study illustrates for the first time in the monkey Sertoli cells that heat treatment could induce a reversible change of the Sertoli cells from an adult-differentiated state to an immature-like dedifferentiated state through activation of PKA-ERK MAPK-mediated pathways but not via the PI3K/Akt pathway-mediated pathway.
Acknowledgments
The authors thank the Guangxi Hongfeng Primate Research Center and the Institute of Biological Products of Beijing for animal health care.
Footnotes
This work was supported by the "973" project (G1999055901), National Nature Science Foundation of China (30230190), and Chinese Academy of Sciences Chuangxi program (KSCX-2-SW-201), and grants from Mellon Reproductive Biology Center (to R.S.S., C.W.), CONRAD-Mellon Twinning grant (MFG-03-67) (to R.S.S., C.W., A.P.S.H., and Y.-H.L.), and RO1-HD 39293 (to R.S.S., C.W., and A.P.S.H.).
All authors have nothing to declare.
First Published Online December 8, 2005
Abbreviations: ck, Cytokeratin; FBS, fetal bovine serum; JNK, c-Jun N-terminal kinase; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A.
Accepted for publication November 30, 2005.
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