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Endocrinology Vol. 148, No. 3 1255-1265
Copyright © 2007 by The Endocrine Society

Heat Treatment Induces Liver Receptor Homolog-1 Expression in Monkey and Rat Sertoli Cells

Jian Guo, Shi-Xin Tao, Min Chen, Yu-Qiang Shi, Zhu-Qiang Zhang, Yin-Chuan Li, Xue-Sen Zhang, Zhao-Yuan Hu and Yi-Xun Liu

State Key Laboratory of Reproductive Biology (J.G., S.-X.T., M.C., Y.-Q.S., Z.-Q.Z., Y.-C.L., X.-S.Z., Z.-Y.H., Y.-X.L.), Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China; and School of Preclinical Medicine (J.G.), Beijing University of Chinese Medicine, Beijing 100029, China

Address all correspondence and requests for reprints to: Yi-Xun Liu, Professor, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 25, Bei Si Huan Xi Lu, Beijing 100080, China. E-mail: liuyx{at}ioz.ac.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We demonstrated in this study that liver receptor homolog-1 (LRH-1) was expressed in the round spermatids in normal monkey testis, and no LRH-1 signal was observed in the Sertoli cells. After local warming (43 C) the monkey testis, however, LRH-1 expression was induced in the Sertoli cells in coincidence with activation of cytokeratin 18 (CK-18), a Sertoli cell dedifferentiated marker. Furthermore, we isolated rat primary Sertoli cells from testes at various stages of development and treated with 43 C water in vitro. The changes in LRH-1 as well as CK-18 expression were analyzed by confocal immunohistochemistry and Western blot. The results showed that LRH-1 was stage-dependently expressed in the Sertoli cells; no LRH-1-positive signal was detected in the cells obtained from the testes of adult rat on d 60 after birth when mature spermatozoa in the testis was completed. However, the mature Sertoli cells were warmed at the 43 C water bath for 15 min, and the LRH-1 signal was remarkably induced in a time-dependent manner, just like the changes of CK-18 expression in the Sertoli cells, suggesting that the heat-induced dedifferentiation of the mature Sertoli cells might be related to LRH-1 regulation. LRH-1 expression induced by the heat treatment was completely inhibited by the addition of ERK inhibitor U0126 in the culture, indicating that the heat-induced LRH-1 expression in the Sertoli cells may be regulated via ERK1/2 activation pathway. Testosterone was found to have no such effect on LRH-1 expression in the monkey and rat Sertoli cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIVER RECEPTOR HOMOLOG-1 (LRH-1; NR5A2) is a member of the Ftz-F1 subfamily of nuclear orphan receptors and expressed in endoderm of intestine, liver, breast, exocrine pancreas, and ovary of various animals (1, 2, 3, 4, 5). LRH-1 has been suggested to play a role in cancer development, reverse cholesterol transport, bile-acid homeostasis, and steroidogenesis (6, 7). Evidence showed that LRH-1 may have potential role in activation of proliferation by induction of cyclin E and cyclin D1 expression (8). Knockout of LRH-1 in mouse results in early embryonic lethality (9, 10). LRH-1 has been also reported to support Oct4 expression at embryonic development (11).

Evidence has shown that LRH-1 is capable of stimulating estrogen (12, 13, 14, 15), progesterone biosynthesis (16), and steroidogenesis-related enzyme activities (17, 18). One report also showed that LRH-1 is associated with transcriptional activation of inhibin {alpha}-subunit gene expression (19). LRH-1 expression has been observed in rat testis mainly in the germ and Leydig cells, but not Sertoli cells (20). Sertoli cells are the most important cells to provide structure and nutrition support for spermatogenesis (21, 22) and communicate with Leydig and peritubular cells to regulate process of spermatogenesis (23, 24, 25).

DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1) is an orphan receptor member of the nuclear hormone receptor superfamily (26, 27, 28). A report has shown that DAX-1 has three LXXLL-related motifs for interaction with LRH-1 and repression of its expression (29). However, there is no further evidence to show the possible functional relationship between these two molecules.

During testicular development, testis at immature stage is in the abdomen, in which the temperature (37 C) is higher than that in the scrotal (33 C). Testis descends in the cooler scrotal position only at the developing mature stage. Sertoli cell impairment would directly lead to germ cell apoptosis.

CK-18 is a subtype of the cytokeratin family and has been used as a marker of Sertoli cell differentiation (30, 31). It is well known that cytokeratin 18 (CK-18), anti-Mullerian hormone, and M2A antigen are the three markers (32) of immature Sertoli cells. They are coordinately expressed in normal prepubertal testes but absent in normal adult testes. During the process of Sertoli cell maturation, these three molecules have a sequential expression (33). Loss of CK18 expression occurs at very early stage during Sertoli cell differentiation. The second step is the loss of M2A antigen. The anti-Mullerian hormone expression appears to be a marker of immature Sertoli cells at late stage during the maturation process (33). In our previous experiments, we demonstrated that experimental cryptorchidism (34) or 43 C water locally warming the testis (35) in adult monkeys could induce CK-18 reexpression in the Sertoli cells that may be regarded as a dedifferentiated immature feature of the adult Sertoli cells (36). We also demonstrated that the heat stress could induce changes in the intermediate filaments in the Sertoli cells (37). The changes in Sertoli cell features may be important for controlling germ cell apoptosis (22).

Considerable amounts of evidence have shown that heat stress induces germ cell apoptosis through various signal pathways (38, 39, 40, 41, 42). Studies have shown that LRH-1 is capable of regulating steroidogenesis in both the ovary and testis (14, 15, 16, 17, 18, 20), and DAX-1 could interact with LRH-1 (29); therefore, we designed in vivo and in vitro experiments in the present study to examine whether heat stress and testosterone or their combination could affect on LRH-1 and DAX-1 expression in the mature Sertoli cells in relation to the cell dedifferentiation using CK-18 as a molecular marker.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and reagent
DMEM, ERK1/2 inhibitor (U0126), trypsin (type I), collagenase (type V), hyaluronidase, DNaseI, and androstenedione were purchased from Sigma (St. Louis, MO). Ham’s F-12 nutrient mixture was from Invitrogen Corp. (Grand Island, NY). Polyclonal antiphospho-ERK1/2(9101) and anti-ERK1/2(9102) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-DAX-1 antibody, anti-CK-18 antibody, and anti-P450aromatase antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against LRH-1 was generously provided by Dr. Luc Belanger (Quebec University, Canada). Testosterone undecanoate was purchased from Xianju Pharmaceutical (Xianju, China).

Animals
The male adult (7 to 10 yr old) cynomolgus monkeys (Macaca fascicularis) were obtained and housed at the Guangxi Hongfeng Primate Research Center, Institute of Zoology, Chinese Academy of Sciences. Animal handling and experimentation were approved by the Animal Care and Use Review Committee of Institute of Zoology, Chinese Academy of Sciences. These monkeys were housed in a standard animal facility under controlled temperature (22 C) and photoperiod (12 h of light and 12 h of darkness), with free access to water and monkey chow.

The experiment details have been reported previously (35, 41). Briefly, 32 adult male cynomolgus monkeys were divided into four groups: 1) intact normal monkeys without any treatment as the control; 2) exposure of the monkey testes locally at 43 C water bath 30 min each day for successive 2 d (H); 3) testosterone (T) implant (5.5 cm length, inner diameter 0.33 cm, and out diameter 0.46 cm); 4) the T implant plus the 43 C water treatment (T+H). The T-filled capsules subdermally along the dorsal surface near the neck under a heavy sedation with ketamine (10 mg/kg) and atropine (0.05 mg/kg). By the end of 12 wk treatment, the implanted T capsules were removed under light anesthesia and the monkeys were then allowed to complete an 8-wk recovery phase. Testicular biopsies were performed in one testis of the animals on the day before and on d 3, 8, 28, and 84 after the treatments.

The experiment details for experimental cryptorchidism have been reported previously (42). The rats (n = 21) for the experimental cryptorchidism were randomly divided into seven groups. One group (three animals) remained intact as the normal control. To induce unilateral cryptorchidism, the animals were anesthetized and a midline incision made in the abdomen. The gubernaculums was open on the left side to push the testis into the abdomen. The inguinal canal was sutured to prevent the testis from descending into the scrotum. Testis on the other side was remained intact. Both the abdominal testis and the intact scrotum testis of all the animals were collected on d 1, 3, 5, 7, 10, and 15 after operation, respectively. The testes were decapsulated for Sertoli cell isolation.

Isolation and culture of normal rat primary Sertoli cells at various stages
Sertoli cells were isolated from the rat testes on d 0, 20, 40, and 60 after birth. The decapsulated testes were washed twice in PBS. After centrifugation at 300 x g for 3 min, the sedimentation was incubated with collagenase (0.5 mg/ml) about 5 min to disperse the seminiferous tubules. Centrifugation was at 300 x g for 3 min and the sedimentation was washed twice in PBS to remove blood and Leydig cells. The tubular pieces were performed for the second digestion by addition of 0.05% trypsin for about 5 min. Fetal bovine serum (FBS) was used to stop the enzyme digestion. After filtration through a 100-mesh filter and centrifugation, the cells were collected and resuspended in the culture medium (DMEM plus F12 with 10% FBS, 100IU/ml penicillin, and 100 µg/ml strepromycin). The cells were washed and seeded onto 10 x 10 mm coverslips placed in 6-well plates (2 x 105 cells/coverslip) or 6-well plates (2 x 106 cells/well) and cultured at 33 C in a humidified atmosphere of 5% CO2-95% air for the confocal immunohistochemistry or protein extraction, respectively. For aromatase activity analysis, 100 nM androstenedione were added to the culture medium before the heat treatment. The cells were gently washed to remove unattached germ cells after 24 h incubation. After additional 24 h culture, the medium was changed with serum-free medium for 12 h. Then the cells were collected and processed. For the testes of adult rats, hyaluronidase or DNase I were added in the collagenase and trypsin digest mixture.

For the cell heat treatment, the dishes were covered with paraffin membrane and put on 43 C water bath for 15 min, and then the dishes were immediately put back into the 33 C incubator. At various intervals, the cell cultures were terminated and assayed.

Immunohistochemistry
Sections were deparaffinized, immersed in PBS, and treated with 0.3% hydrogen peroxide in PBS for 10 min to block endogenous peroxidase. The sodium citrate buffer was used for retrieval antigen. After three washes in PBS, the sections were exposed in 10% normal horse serum to suppress the nonspecific antigen and incubated in the primary antibodies of LRH-1 (1:300), DAX-1 (1:100) overnight at 4 C. After three washes in PBS, the appropriate biotinylated secondary antibodies were added for 1 h at room temperature and then the avidin-biotin-peroxidase complexes. Immunostaining was developed with the diaminobenzidine kit and counterstained with hematoxyline, dehydrated, and mounted. Negative control was added with the normal IgG without the primary antibodies.

Confocal Immunohistochemistry
The Sertoli cells cultured on the coverslips were treated with the control medium and ERK1/2 inhibitor U0126 (20 µM) and followed by the treatment of the 43 C water bath for 15 min. After washes in PBS three times, the cells were fixed in freshly prepared mixture of methanol and acetone (1:1) for 30 min, followed by washes in PBS, and incubated in 10% normal horse serum. Then the primary antibody LRH-1 (1:300) was added at 4 C and incubated overnight. Fluorescein isothiocyanate-conjugated antirabbit IgG (1:200) was added at room temperature and incubated for 1 h. After three washes in PBS, the coverslips were incubated in propidium iodide (PI) for 10 min to dye the nuclei. Finally, the slides were analyzed by confocal laser scanning microscope (Carl Zeiss Inc., Thornwood, NY). The negative control was performed without the primary antibody.

Western blotting
The snap-frozen testis in liquid nitrogen was homogenized in radioimmunoprecipitation assay lysis buffer containing 50 nM Tris-HCL (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, and 0.1% sodium dodecyl sulfate, supplemented with protease inhibitors (phenylmethylsulfonyl fluoride) and phosphatase inhibitors (1 mM NaF). The supernatants after centrifugation (12000 x g, 15 min) were collected, and the total protein concentrations were determined by spectrophotometer.

For the protein extraction in the culture, the cells were digested with the trypsin buffer and centrifuged at 1000 x g for 5 min. The cell pellets were washed in PBS and incubated in radioimmunoprecipitation assay lysis following the extraction protocol. After boiling at 98 C for 5 min, 50 µg total protein per lane were added and the proteins were separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The nitrocellulose sheet was incubated in 5% nonfat milk for 1 h at room temperature and then exposed in the primary antibodies: LRH-1 (1:1000), DAX-1 (1:1000), ERK1/2 (1:1000), p-ERK1/2 (1:500), and CK-18 (1:1000) at 4 C overnight. After washes in PBST three times, the membranes were incubated in the corresponding peroxidase-conjugated second antibodies for 1 h at room temperature, washing again with PBST. The bands were visualized by Supersignal West Pico chemiluminescent substrate (Pierce Co., Rockford, IL). ß-Actin (1:5000; Sigma) was used as an internal control. Band intensities were determined by Quantity One software (Bio-Rad, Hercules, CA).

Estradiol RIA
The Sertoli cells were cultured in a 24-well plate in 1 ml medium in the presence of 100 nM androstenedione and treated with 43 C water bath for 15 min. The media were then collected at 30 min, 3, 6, 12, 24, and 48 h, respectively, and stored at –20 C for estradiol RIA. The contents of estradiol in the media were measured by the standard RIA procedures.

Data analysis and statistics
Experiments were repeated at least three times. Three different monkey testes for biopsy were used for tissue immunohistochemistry. The primary rat Sertoli cells in vitro experiment from three different preparations were processed. For the immunocytochemistry data, one representative picture of three similar results from at least three separate experiments was presented. The quantitative results were represented as the means ± SEM. Statistical analysis was performed with SPSS (version 13.0; SPSS Inc., Chicago, IL), and one-way ANOVA was used for analyzing the data in different groups. Probability values less than 0.05 were considered as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of LRH-1 in monkey testis after heat or testosterone treatment
As shown in Fig. 1 AGo, in the normal adult scrotal monkey testis, LRH-1 was observed in the round spermatids, but no obvious expression of this molecule was observed in the Sertoli cells. After testosterone treatment, the expression profile of LRH-1 was not obvious changed as compared with that in the normal scrotal testis. Three days after treatment of the monkey testes with 43 C water, the LRH-1 signal almost disappeared in the spermatids, and a strong expression of this molecule was induced in the Sertoli cells. The profile of LRH-1 expression in the testis treated with testosterone plus the 43 C warming was similar as observed with the 43 C treatment alone, indicating that testosterone has no obvious effect on LRH-1 expression in the testis in combination with the heat stress.


Figure 1
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  		FIG. 1. Immunohistochemical staining of LRH-1 (A and C) and DAX-1 (B and D) in monkey testes. A, LRH-1 staining in monkey testes after 43 C warming (Heat), T implant (Testosterone), or in combination (T+H). The scrotal normal testis showed that LRH-1 localized in the round spermatids, but not Sertoli cells (Normal). After treatment of the monkey testes at 43 C water bath for 30 min each day for successive 2 d (Heat), LRH-1 expression was obviously induced in the Sertoli cells. Testosterone treatment (Testosterone) had no obvious effect on LRH-1 expression. The profile of LRH-1 expression in the testosterone combined with heat treatment (T+H) was not obviously different as compared with that of the heat treated alone (Heat). Con, Control section without primary antibody. The arrows and arrowheads indicate the round spermatids and Sertoli cells, respectively. Brown color is positive staining, and blue color is counterstaining. Bar, 50 µm. B, DAX-1 staining in monkey testes treated with T, 43 C water (H), and in combination (T+H). The scrotal normal testis showed that DAX-1 expressed in the round spermatids, Sertoli cells, and Leydig cells (Normal). T treatment had no obvious effect on DAX-1 expression (Testosterone). After treatment of the monkey testes at 43 C water bath for 30 min each day for successive 2 d, with germ cells lost, DAX-1 expression was decreased in germ cells but still observed in the Sertoli cells (Heat). The profile of DAX-1 expression in the T combined with H treatment was not obviously different as compared with that of heat treated alone (T+H). Con, Control section without primary antibody. The arrows, arrowheads, and thin arrows indicate the round spermatids, Sertoli cells, and Leydig cells, respectively. Brown color is positive staining, and blue color is counterstaining. Bar, 50 µm. C, Time-dependent expression of LRH-1 in monkey testes after 43 C warming. a, Normal intact testis; b, c, d and e, testes on d 3, 8, 28, and 84 after 43 C water treatment; f, control section without primary antibody. The arrows and arrowheads, respectively, indicate the round spermatids and Sertoli cells. Brown color is positive staining, and blue color is counterstaining. LRH-1 localized in the round spermatids in the normal intact testis (a) after treatment of the monkey testes at 43 C water bath for 30 min each day for 2 d; LRH-1 expression was obviously induced in the Sertoli cells in a time-dependent manner with the increase in number of germ cell apoptosis, reaching the highest level on d 8and 28 (c and d). LRH-1 expression in the Sertoli cells decreased when the germinal epithelium was recovered on d 84 (e). D, Immunohistochemical staining of DAX-1 in monkey testis after 43 C water treatment. a, Normal testis; b, c, d, and e, testes obtained on d 3, 8, 28, and 84 after 43 C water treatment; f, control section without primary antibody. The arrows, arrowheads, and thin arrows indicate the round spermatids, Sertoli cells, and Leydig cells, respectively. DAX-1 expression was observed in the round spermatids, Sertoli cells, and Leydig cells in the normal testis (a). On d 3 and 8 after treatment with 43 C water, the expression of DAX-1 in these cells obviously decreased. Brown color is positive staining, and blue color is counterstaining. Bar, 50 µm.

 
The LRH-1 expression in the testis (mainly in Sertoli cells) increased after 43 C heat treatment in a time-dependent manner, as shown in Fig. 1CGo, reaching the maximum on d 8 and 28 and considerably decreased on d 84 when the seminiferous epithelium was recovered from germ cell apoptosis. The Western blot data, as shown in Fig. 2AGo, qualitatively confirmed the immunocytochemistry results as shown in Fig. 1CGo.


Figure 2
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  		FIG. 2. Western blot analysis of LRH-1 (A) and DAX-1 (B) in monkey testes after 43 C water treatment. After the treatment of the monkey testes with 43 C water, the testicular tissues obtained on d 3, 8, 28, and 84 were prepared for the Western blot analysis. ß-Actin was used as an internal control. Data are presented as mean ± SEM (n = 3). Bar with * is significantly different (P < 0.05) or ** (P < 0.01) as compared with the intact normal testis (N).

 
These data indicate that heat stress could induce LRH-1 expression in Sertoli cells of adult monkey testis, whereas testosterone had no such action.

DAX-1 expression in monkey testis after heat treatment
The expression of DAX-1 was examined by immunocytochemistry (Fig. 1Go, B and D) and Western blot (Fig. 2BGo) in monkey testes. DAX-1 was expressed in the round spermatids and Sertoli cells as well as in the Leydig cells in the adult normal scrotal monkey testis. Testosterone treatment had no obvious effect on DAX-1 expression. After treatment of the monkey testes at 43 C water bath for 30 min each day for a successive 2 d, DAX-1 staining was decreased in the testicular cells but still observed in the Sertoli cells. The profile of DAX-1 expression in the T plus H treatment was no obviously different as compared with that of the H treatment alone.

The staining of DAX-1 in these cells was decreased after the heat treatment on d 3 and 8 and returned to the normal level on d 28 and 84. DAX-1 showed an inverse correlation with LRH-1 expression in the testis. After testosterone treatment, the DAX-1, like the LRH-1, had no obvious changes as compared with the normal.

Expression of LRH-1 in rat Sertoli cells in a stage-dependent manner
Because immature testis develops in the abdomen (37 C), it is interesting to look at stage-dependent expression of LRH-1 in the Sertoli cells isolated from the testes at various developmental stages. The rat Sertoli cells were obtained from the testes on d 0, 20, 40, and 60 after birth, as shown in Fig. 3AGo, and the LRH-1 signal in the cells was decreased in an age-dependent manner. The Sertoli cells at the immature stages (d 0 and day 20) expressed stronger LRH-1, whereas the signal in the mature Sertoli cells obtained on d 60 almost disappeared. The result by Western blot analysis was consistent with that by immunocytochemistry as shown in Fig. 3BGo. These data indicate that immature Sertoli cells are capable of expressing LRH-1, whereas the mature Sertoli cells are not.


Figure 3
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  		FIG. 3. Stage-dependent and testosterone-independent LRH-1 expression in cultured rat primary Sertoli cells A, Confocal immunohistochemistry of stage-dependent LRH-1 expression. The Sertoli cells were prepared from the rat testes at the ages on d 0, 20, 40, and 60 after birth, respectively, and cultured in DMEM plus F-12 medium with 10% FBS for 48 h. The cells were washed and further cultured in serum-free medium for 12 h. The cells were collected and processed for analysis. The cells were fixed in the mixture of methanol and acetone and incubated in the primary antibody of LRH-1 at 4 C and then with the flurescein isothiocyanate conjugated the antirabbit IgG. The green was the positive signal. The nuclei were stained in red using PI. Bar, 50 µm. B, Western blot analysis. The methods have been described in Materials and Methods. ß-Actin was used as an internal control. Data were presented as mean ± SEM (n = 3). **, Significantly different (P < 0.01). C, Confocal immunohistochemistry of testosterone-independent LRH-1 expression. The Sertoli cells were isolated from the rat testes at the ages on d 20 and 60, respectively, after birth and cultured in DMEM plus F-12 medium with 10% FBS for 48 h. The cells were washed and cultured in serum-free medium for 12 h and then further incubated for 24 h in the presence or absence of testosterone (100 nM). The cells were collected and processed for analysis. After fixed in mixture of methanol and acetone, the cells were incubated in the primary antibody LRH-1 at 4 C and flurescein isothiocyanate-conjugated antirabbit IgG was added and developed. The green was the positive signal. The nuclei were stained in red using PI. Bar, 50 µm. D, Western blot analysis of testosterone-independent LRH-1 expression. ß-Actin was used as an internal control. Data are presented as mean ± SEM (n = 3).

 
Testosterone in vitro had no effect on inducing LRH-1 expression in Sertoli cells
To investigate the possible effect of testosterone on LRH-1 expression in vitro, we isolated the Sertoli cells from rat testes on d 20 (immature) and d 60 (mature). After culture in serum-free medium for 12 h, 100 nM testosterone were added to the culture. The cells were collected and analyzed 24 h later. As shown in Fig. 3CGo, the immature Sertoli cells expressed LRH-1, which was not regulated by addition of testosterone. The cells isolated from adult testes on d 60, however, hardly expressed LRH-1. Addition of testosterone to the culture no LRH-1 expression was observed. The Western blot data confirmed the immunocytochemistry data (Fig. 3DGo). These results indicate that testosterone has no effect on LRH-1 expression in the Sertoli cells in vitro obtained from the rat testes of immature or adult.

Water stress (43 C) induced expression of LRH-1 and CK-18 in cultured primary Sertoli cells
The Sertoli cells obtained from mature rat testes on d 60 were cultured in DMEM/F-12 medium and collected before and at various time points after ending the 15-min treatment of the testes at 43 C (the time point was designated as 0 min). As shown in Fig. 4Go, A and B, the 43 C water stress activated expression of LRH-1 as well as CK-18 in the cultured mature primary Sertoli cells. LRH-1 staining in the Sertoli cells was observed at 0 min and then time-dependently increased, reaching the highest level at 20 min, and gradually decreasing. The change in CK-18 expression in the cells was similar to LRH-1 expression, but the profile of CK-18 to reach the maximum was later than that of LRH-1 expression. CK-18 reaching the maximum level was at 30 min and then dropped to the pretreatment level. Western blot data further confirmed the immunocytochemistry result, as shown in Fig. 5AGo.


Figure 4
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  		FIG. 4. Confocal immunohistochemistry of LRH-1 and CK-18 expression induced by 43 C warming or experimental cryptorchidism in adult rat primary Sertoli cells. A, Confocal immunohistochemistry of LRH-1 expression induced by 43 C warming. The mature Sertoli cells were isolated from rat testes at the age on d 60 after birth and cultured in DMEM plus F-12 medium with 10% FBS for 48 h. The cells were washed and further cultured in serum-free medium for 12 h, and then the cells underwent a 43 C water bath for 15 min treatment. N, Normal Sertoli cells without heat treatment. 0 min, Time justly ending the 43 C water treatment; 10, 20, 30, and 60 min and 2 and 3 h, time points, respectively, after ending the heat treatment. After fixing in mixture of methanol and acetone, the cells were incubated with the primary antibody LRH-1 at 4 C and then with the flurescein isothiocyanate conjugated antirabbit IgG. The green was the positive signal. The nuclei were stained in red using PI. Bar, 50 µm. B, Confocal immunohistochemistry of CK-18 expression induced by 43 C warming. After fixing in mixture of methanol and acetone, the cells were incubated with the primary antibody CK-18 (1:100) at 4 C and then with the flurescein isothiocyanate-conjugated antirabbit IgG. The nuclei were stained in red using PI, and the green is the positive signal. The nuclei were stained in red using PI. Bar, 50 µm. C, Confocal immunohistochemistry of LRH-1 expression in cultured Sertoli cells of cryptorchid testes at various days. The mature primary Sertoli cells were isolated from adult rat testes on d 1, 3, 5, 7, 10, and 15 after experimental cryptorchid, respectively, and cultured in DMEM plus F-12 medium with 10% FBS for 48 h. The cells were washed and further cultured in serum-free medium for 12 h. The cells were then collected and processed. After fixing in mixture of methanol and acetone, the cells were incubated in the primary antibody LRH-1 at 4 C and flurescein isothiocyanate-conjugated antirabbit IgG. N, Sertoli cells isolated from normal testes; Con, negative control without primary antibody. The green was the positive signal. The nuclei were stained in red using PI. Bar, 50 µm. D, Confocal immunohistochemistry of CK-18 expression in cultured Sertoli cells of cryptorchid testes at various days. After fixing in mixture of methanol and acetone, the cells were incubated in the primary antibody CK-18 (1:100) at 4 C and flurescein isothiocyanate-conjugated antirabbit IgG. The green was the positive signal. The nuclei were stained in red using PI. Bar, 50 µm.

 

Figure 5
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  		FIG. 5. Western blot analysis of LRH-1 and CK-18 expression in cultured primary adult rat Sertoli cells induced by 43 C warming or experimental cryptorchidism. A, Western blot analysis of 43 C warming induced LRH-1 and CK-18 expression. The Sertoli cells were isolated from adult rat testes and collected at 0, 10, 20, 30, and 60 min and 2 and 3 h after ending 43 C warming treatment as described in Materials and Methods. ß-Actin was used as an internal control. Data are presented as mean ± SEM (n = 3). Bar with *, Significantly different (P < 0.05); **, P < 0.01. B, Western blot analysis of experimental cryptorchidism activated LRH-1 and CK-18 expression. The mature primary Sertoli cells were isolated from adult rat testes on d 1, 3, 5, 7, 10, and 15 after experimental cryptorchidism respectively. ß-Actin was used as an internal control. Data are presented as mean ± SEM (n = 3). Bar with **, Significantly different (P < 0.01).

 
Experimental cryptorchidism-induced adult Sertoli cell LRH-1 and CK-18 expression
The experimental cryptorchidism in adult rats was performed as described in Materials and Methods. The rats (n = 21) were randomly divided into seven groups. One group (three animals) remained intact as the control. Both the abdominal testis and intact scrotum testis of all the animals were collected on d 1, 3, 5, 7, 10, and 15 after operation, respectively. The testes were decapsulated for Sertoli cell isolation.

The Sertoli cells were isolated respectively from the testes on d 1, 3, 5, 7, 10, and 15 after the operation and incubated in the DMEM/F-12 medium. As compared with the primary Sertoli cells obtained from the normal adult scrotal testis, the LRH-1 and CK-18 expression was observed in the Sertoli cells from the cryptorchid testis on the first day after the operation (Fig. 4Go, C and D), the expression signals of both molecules, however, were weaker as compared with that treated by 43 C warming the testes, and no significant changes in the two molecule expression were observed on d 3, 5, 7, 10, and 15 after the operation, indicating that the body temperature- (37 C) induced LRH-1 and CK-18 expression was more moderated. The data by Western blot analysis also confirmed the immunocytochemistry data as shown in Fig. 5BGo.

Warming (43 C) induced LRH-1 and CK-18 expression through ERK activation in cultured primary Sertoli cells
The primary Sertoli cells isolated from the adult rat testes were cultured in DMEM/F-12 medium with 10% FBS and following starving with serum-free medium for 12 h, the cells were treated with ERK inhibitor, U0126 (20 µM), for 30 min, then put the culture in 43 C water bath for 15 min, and collected the cells 20 min later. As shown in Fig. 6Go, 43 C warming activated LRH-1 expression in the Sertoli cells. Addition of U0126 to the culture dramatically inhibited the molecule expression. Western blot data were consistent with the immunocytochemistry results. The phosphorylated ERK was significantly up-regulated and inhibited by the ERK inhibitor. The profile of ERK activation was coincident with the LRH-1 expression.


Figure 6
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  		FIG. 6. Effect of ERK inhibitor U0126 on 43 C warming-induced LRH-1 expression in cultured mature primary Sertoli cells. The mature Sertoli cells were isolated from the rat testes at age on d 60 after birth and cultured in DMEM plus F-12 medium with 10% FBS for 48 h. The cells were washed and cultured in serum-free medium for 12 h, and then the ERK inhibitor U0126 (20 µM) was added to the cell culture; 30 min later the cells were underwent at 43 C water bath for 15 min treatment. At 20 min after ending the heat treatment, the cells were collected and processed for analysis. N, Normal Sertoli cells without any treatments; U0, Sertoli cells with U0126; H, Sertoli cells with heat treatment; H+U0, Sertoli cells with heat plus U0126 treatment. A, Confocal immunohistochemistry of LRH-1 expression. After fixing in mixture of methanol and acetone, the cells were incubated with the primary antibody LRH-1 at 4 C and then with the flurescein isothiocyanate-conjugated antirabbit IgG. The green was the positive signal. The nuclei were stained in red using PI. Bar, 50 µm. B, Western blot analysis of LRH-1. ß-Actin was used as an internal control. Data are presented as mean ± SEM (n = 3). Bar with **, Significantly different (P < 0.01). C, Western blot analysis of ERK. ß-Actin was used as an internal control. Data are presented as mean ± SEM (n = 3). Bar with **, Significantly different (P < 0.01).

 
Activation of LRH-1 expression by 43 C water in cultured primary rat Sertoli cells is followed by increase in aromatase activity and estradiol production
The Sertoli cells were isolated from the testes of adult rat on d 60 after birth and incubated in the DMEM/F-12 medium in the presence of 100 nM androstenedione, a substrate for aromatase. The cells and media were collected after ending the heat treatment at 30 min, 3 h, 6 h, 12 h, 24 h, and 48 h, respectively. As shown in Fig. 7AGo, in the normal of the adult primary Sertoli cells, the staining of aromatase was weakly observed; after the heat treatment, its expression was gradually increased and reached the maximum 12 h after ending the heat treatment. The LRH-1 expression in the culture was dramatically increased and reached the maximum at 30 min (Fig. 7BGo) and followed by increase in medium estradiol production (Fig. 7CGo), reaching the maximum at 12 h and correlated well with the aromatase activity.


Figure 7
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  		FIG. 7. Effect of 43 C warming on aromatase activity and estradiol production in cultured primary Sertoli cells. The mature Sertoli cells were isolated from the rat testes at age on d 60 after birth and cultured in DMEM plus F-12 medium with 10% FBS for 48 h. The cells were washed and further cultured in serum-free medium for 12 h. The cells were collected before and after heat treatment at 30 min and 3, 6, 12, 24, and 48 h after 43 C water treatment for the LRH-1 and aromatase analysis. For the estradiol RIA, the Sertoli cells were cultured in a 24-well plate in 1 ml medium in the presence of 100 nM androstenedione and treated with 43 C water bath for 15 min. The media were then collected and measured before and after treatment at 30 min and 3, 6, 12, 24, and 48 h, respectively. A, Aromatase expression by confocal immunohistochemistry. After fixed in mixture of methanol and acetone, the cells were incubated with the aromatase antibody (1:80) at 4 C and then with the flurescein isothiocyanate-conjugated antirabbit IgG. N, Normal Sertoli cells without heat treatment; Con, negative control without primary antibody. The green was the positive signal. The nuclei were stained in red using PI. Bar, 50 µm. B, Western blot analysis of LRH-1. ß-Actin was used as an internal control. Data were presented as mean ± SEM (n = 3). Bar with **, Significantly different (P < 0.01). C, Estradiol level. Data were presented as mean ± SEM (n = 3). Bar with **, Significantly different (P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies for the first time report that heat-induced adult Sertoli cell expression of orphan receptor LRH-1 may be involved in the signal pathway of the reversible change of the Sertoli cells from a mature differentiated state to a dedifferentiated state like immature state via activation of ERK MAPK pathway.

LRH-1 is mainly expressed in round spermatids but not in Sertoli cells of normal adult testis. However, experimental cryptorchidism or local warming testis at 43 C water bath induced LRH-1 expression in the Sertoli cells in correlation with CK-18 expression, a marker molecule of undifferentiated Sertoli cells detected in prepubertal testis. Because of the profile of LRH-1, expression in the Sertoli cells is earlier than CK-18 production, implying that the mature Sertoli cells dedifferentiation induced by the heat stress might be partially regulated by LRH-1.

To more closely look at the possible molecular mechanism of heat effect on LRH-1 expression in correlation with CK-18 induction in Sertoli cells, we established various rat primary Sertoli cell cultures and studied their expression in various physiological conditions and examined possible involvement of signal pathways. Our experiments showed that with the addition of U0126, an ERK inhibitor, to the mature Sertoli cell culture, the LRH-1 and CK-18 signals induced by the heat treatment was completely inhibited, indicating that the heat-induced LRH-1 expression in the Sertoli cells may be regulated through ERK activation signal pathway.

Considerable evidences have demonstrated that elevation of temperature could induce germ cell apoptosis and accompany histological and hormonal changes in the seminiferous epithelium, leading to impairment of spermatogenesis and infertility (43, 44). However, only a few publications specifically addressed the changes in Sertoli cells related to the event of heat-induced impairment of spermatogenesis. Sertoli cells perform several physiological functions essential to normal spermatogenesis. It has been reported that Sertoli cells undergo a radical change in their morphology and function, heralding the switch from an immature, proliferative state to a mature, nonproliferative state at the period around onset of puberty (45, 46, 47, 48). Our previous studies have demonstrated that the experimental cryptorchid testis of adult rhesus monkeys or 43 C local warming the adult monkey testis could induce reexpression of CK-18 (34, 35), indicating that the mature Sertoli cells have reverted to a dedifferentiated state in the cryptorchid testis and thus may lose their supportive role in normal spermatogenesis, leading to a cessation of spermatogenic activity. Exposure of the cultured mature monkey Sertoli cells to 43 C water could also induce CK-18 reexpression in the differentiated Sertoli cells that is coincident with impairment of seminiferous epithelium (35). These findings suggest Sertoli cells in vitro are also affected by the heat treatment.

We have specifically demonstrated the activation of ERK1/2 in the Sertoli cells of the cryptorchid or locally heat monkey testis (34, 35). Our in vivo study confirmed that heat could induce the Sertoli cells to regain undifferentiated features. The heat treatment also activated ERK phosphorylation in Sertoli cells immediately after the end of the heat stress. Blocking the ERK MAPK pathway with its inhibitor U0126 beforehand could inhibit the activation of ERK induced by the heat treatment, and this blocking also inhibits the expression of CK18 in Sertoli cells after heat shock.

LRH-1 signal was induced in the in vitro cultured Sertoli cells obtained from the testes of the experimental cryptorchid rats from d 1 after operation; however, the molecular expression of the Sertoli cells did not increased in the following d 3, 5, 7, 10, and 15 after cryptorchidism. This finding is consistent with the result of cryptorchidism-induced germ cell apoptosis in vivo (42, 49). The peak level of monkey germ cell apoptosis induced by the body temperature occurred 30 d after operation, as compared with that of only 8 d induced by the 43 C warming the testis (35). Anyhow, the Sertoli cells from heat-treated adult testes or the mature Sertoli cells in vitro-treated with 43 C warming express LRH-1 in correlation with CK-18 reproduction, indicating the involvement of LRH-1 in the changes of Sertoli cells from a differentiated state back to an immature undifferentiated state.

It is well known that exogenous testosterone leads to deprivation of the intratesticular androgen through suppressing release of FSH and LH, resulting in blockage of spermatogenesis, and germ cell apoptosis mainly occurred at stages VII-VIII (38). Heat-induced germ cell apoptosis occurred at the early (I-IV) and late (XII-XIV) stages (38). We have demonstrated that injection of T (49) or experimental cryptorchidism (42, 50, 51, 52) or 43 C water local warming testis in monkeys (53) could induce germ cell apoptosis, and an additive action of T in combination with heat on germ cell apoptosis could be observed (41). Our data also showed that administration of rat with human chorionic gonadotropin or GnRH antagonist, antide (our unpublished data), or exogenous testosterone in vivo has no obvious effect on LRH-1 and CK-18 localization and expression level in the testes. The serum FSH and testosterone concentrations have no significant changes after heat treatment (35, 41), indicating that testosterone-induced germ cell apoptosis may be through different mechanisms from that induced by heat stress.

Our previous studies have shown that tissue type plasminogen activator (tPA) is expressed in monkey (54, 55) and rat Sertoli cells (56) and may be involved in extracellular matrix degradation during spermatogenesis in the testes. Just recently we further demonstrated that tPA in the Sertoli cells was regulated by testosterone and reached the highest level at stages VII-VIII, which is well correlated with testosterone-induced germ cell apoptosis, suggesting that Sertoli tPA activity regulated by testosterone might be related to the cell supportive structure changes, affecting spermatogenesis (our unpublished manuscript).

Evidence has shown that LRH-1 could enhance conversion of androgen into estrogen biosynthesis by up-regulation of aromatase activity (12, 14, 20). There is growing evidence to show that the action of estrogen on testicular development and function via its specific receptors may be important in regulation of spermatogenesis (57, 58, 59). After the heat treatment of Sertoli cells, the LRH-1 expression reached the maximum between 10 and 30 min and followed by a dramatic increase in aromatase activity and estradiol production at 12 h (Fig. 7Go), when the dedifferentiated Sertoli cells beginning to recover to a differentiated state (Figs. 4Go and 5Go). These data suggest that the increased expression of LRH-1 as a transcription factor might up-regulate the aromatase activity, leading to the increasing estradiol production. The LRH-1-induced aromatase activity may be important for the recovering of the Sertoli cells from a dedifferentiated immature state to return a functional differentiated state. However, the resultant regulation of aromatase activity by LRH-1 is still not clear.

It has been reported that FSH receptor (FSHR) (60, 61), androgen receptor (AR) (62) and inhibin-{alpha} (63, 64) are important functional molecules in Sertoli cells. Using an in vitro-cultured rat Sertoli cells, we examined their changes in correlation with LRH-1 expression after 43 C water treatment. Our results showed that the heat treatment obviously inhibited the FSH-induced FSHR and inhibin-{alpha} expression (our unpublished data). More interestingly, warming the cells at 43 C dramatically decreased AR production in a time-dependent manner (our unpublished data); the heat-induced LRH-1 expression is correlated with the decreasing AR production, but the profile of LRH-1 expression is earlier than AR production, implying that LRH-1 might regulate AR production in the Sertoli cells. These data strongly suggest that the heat-induced decreases in AR, FSHR, and inhibin-{alpha} expression in the Sertoli cells may alter normal spermatogenesis in testis. However, how and via what kind of signal pathways LRH-1 controls such molecules production remain unclear.

One may raise a question whether the heat-induced undifferentiated Sertoli cells from the mature differentiated cells have a proliferation characteristic? Ki67 has been reported to be a marker of cell proliferation (65, 66). Our previous study showed that the changes in Ki67 expression in the monkey testis of experimental cryptorchidism occurred only in the nucleus of spermatogonia, not Ki67, but with a positive CK-18 signal was detected in the Sertoli cells (37). Consistent with our in vivo results, no Ki67-positive signal in the cultured Sertoli cells after 43 C water warming or experimental cryptorchid treatment was detected (data not shown). Our evidence suggests that heat-induced expression of LRH-1 as well as CK-18 represents the mature Sertoli cells returning to the undifferentiated state; however, they have no proliferation feature and are still in the pathological state.

DAX-1 is an orphan receptor member of the nuclear hormone receptor superfamily (26, 27, 28) and has recently been shown to inhibit LRH-1 transcriptional activity (29). In the present study, we also detected its expression in the adult monkey testes treated with 43 C water. DAX-1 signal was observed in round spermatids and Sertoli and Leydig cells in normal scrotal monkey testis. After heat treatment the signal in these cells was decreased in a time-dependent manner, and recovered on d 28 and 84 and correlated well with the profile of germ cell apoptosis (35), but no expected expression profile of a decreasing DAX-1 correlated with an increasing LRH-1 production in the cultured Sertoli cells was observed (data not shown), suggesting that DAX-1, as LRH-1 inhibitor in the testis (but not in the Sertoli cells), may play a role in regulation of the heat-induced germ cell apoptosis. The mechanism of its possible action is not known.

In conclusion, this study has demonstrated for the first time that heat stress is capable of inducing LRH-1 expression in mature Sertoli cells correlated with reproduction of CK-18. However, the profile of heat-induced LRH-1 expression is much earlier than that of the CK-18 re-expression. Furthermore, LRH-1 also sustains its expression in the germ cells after the heat stress. It is therefore suggested that LRH-1 might play a key role in regulation of conversion of mature differentiated Sertoli cells into its dedifferentiated-like immature state, which may be related to the event of germ cell apoptosis induced by the heat treatment. The isolated Sertoli cell in vitro experiment further demonstrated that this process may be regulated through ERK activation pathway. However, the evidence provided in this study is limited; the question still remains for further investigation.


   Acknowledgments
 
We acknowledge that part of the monkey materials used in this work was obtained from the collaborate project with C. Wang, A. P. S. Hikim, R. S. Swerdloff, and Y. H. Lue (Division of Endocrinology, Department of Medicine, Harbor-University of California-Los Angeles Medical Center and Los Angeles Biomedical Research Institute, CA) and partially supported by the grants from Mellon Reproductive Biology Center Grant (to R. S. Swerdloff, C. Wang), CONRAD-Mellon Twinning Grants MFG-03-67 (to R. S. Swerdloff, C. Wang, A. P. S. Hikim, and Y. H. Lue) and RO1-HD 39293 (to R. S. Swerdloff, C. Wang, and A. P. S. Hikim). We also acknowledge Dr. Luc Belanger for his generous supply antibody against LRH-1.


   Footnotes
 
This work was supported by the National "973" Program (2006CB504001), the Chinese Academy of Sciences Knowledge Innovation Project (KSCX2-YW-R-55), the Natural Science Foundation of China-Research Grants Council of Hong Kong joint research project (30618005), and the Natural Science Foundation of China Grant 30230190.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 14, 2006

Abbreviations: AR, Androgen receptor; CK-18, cytokeratin 18; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1; FBS, fetal bovine serum; FSHR, FSH receptor; H, water bath; LRH-1, liver receptor homolog-1; PI, propidium iodide; T, testosterone; tPA, tissue type plasminogen activator.

Received July 26, 2006.

Accepted for publication December 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sirianni R, Seely JB, Attia G, Stocco DM, Carr BR, Pezzi V, Rainey WE 2002 Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes. J Endocrinol 174:13–17
  2. Boerboom D, Pilon N, Behdjani R, Silversides DW, Sirois J 2000 Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process. Endocrinology 141:4647–4656[Abstract/Free Full Text]
  3. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME 2000 A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6:517–526[CrossRef][Medline]
  4. Bouchard MF, Taniguchi H, Viger RS 2005 Protein kinase A-dependent synergism between GATA factors and the nuclear receptor, liver receptor homolog-1, regulates human aromatase (CYP19) PII promoter activity in breast cancer cells. Endocrinology 146:4905–4916[Abstract/Free Full Text]
  5. Falender AE, Lanz R, Malenfant D, Belanger L, Richards JS 2003 Differential expression of steroidogenic factor-1 and FTF/LRH-1 in the rodent ovary. Endocrinology 144:3598–3610[Abstract/Free Full Text]
  6. Fayard E, Auwerx J, Schoonjans K 2004 LRH-1: an orphan nuclear receptor involved in development, metabolism and steroidogenesis. Trends Cell Biol 14:250–260[CrossRef][Medline]
  7. Kim JW, Peng N, Rainey WE, Carr BR, Attia GR 2004 Liver receptor homolog-1 regulates the expression of steroidogenic acute regulatory protein in human granulosa cells. J Clin Endocrinol Metab 89:3042–3047[Abstract/Free Full Text]
  8. Botrugno OA, Fayard E, Annicotte JS, Haby C, Brennan T, Wendling O, Tanaka T, Kodama T, Thomas W, Auwerx J, Schoonjans K 2004 Synergy between LRH-1 and ß-catenin induces G1 cyclin-mediated cell proliferation. Mol Cell 15:499–509[CrossRef][Medline]
  9. Pare JF, Malenfant D, Courtemanche C, Jacob-Wagner M, Roy S, Allard D, Belanger L 2004 The fetoprotein transcription factor (FTF) gene is essential to embryogenesis and cholesterol homeostasis and is regulated by a DR4 Element. J Biol Chem 279:21206–21216[Abstract/Free Full Text]
  10. del Castillo-Olivares A, Campos JA, Pandak WM, Gil G 2004 The role of {alpha}1-fetoprotein transcription factor/LRH-1 in bile acid biosynthesis: a known nuclear receptor activator that can act as a suppressor of bile acid biosynthesis. J Biol Chem 279:16813–16821[Abstract/Free Full Text]
  11. Gu P, Goodwin B, Chung AC, Xu X, Wheeler DA, Price RR, Galardi C, Peng L, Latour AM, Koller BH, Gossen J, Kliewer SA, Cooney AJ 2005 Orphan nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development. Mol Cell Biol 25:3492–3505[Abstract/Free Full Text]
  12. Clyne CD, Speed CJ, Zhou J, Simpson ER 2002 Liver receptor homologue-1 (LRH-1) regulates expression of aromatase in preadipocytes. J Biol Chem 277:20591–20597[Abstract/Free Full Text]
  13. Hinshelwood MM, Repa JJ, Shelton JM, Richardson JA, Mangelsdorf DJ, Mendelson CR 2003 Expression of LRH-1 and SF-1 in the mouse ovary: localization in different cell types correlates with differing function. Mol Cell Endocrinol 207:39–45[CrossRef][Medline]
  14. Annicotte JS, Chavey C, Servant N, Teyssier J, Bardin A, Licznar A, Badia E, Pujol P, Vignon F, Maudelonde T, Lazennec G, Cavailles V, Fajas L 2005 The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene. Oncogene 24:8167–8175[Medline]
  15. Britt KL, Stanton PG, Misso M, Simpson ER, Findlay JK 2004 The effects of estrogen on the expression of genes underlying the differentiation of somatic cells in the murine gonad. Endocrinology 145:3950–3960[Abstract/Free Full Text]
  16. Saxena D, Safi R, Little-lhrig L, Zeleznik AJ 2004 Liver receptor homolog-1 stimulates the progesterone biosynthetic pathway during follicle-stimulating hormone-induced granulosa cell differentiation. Endocrinology 145:3821–3829[Abstract/Free Full Text]
  17. Kim JW, Havelock JC, Carr BR, Attia GR 2005 The orphan nuclear receptor, liver receptor homolog-1, regulates cholesterol side-chain cleavage cytochrome p450 enzyme in human granulosa cells. J Clin Endocrinol Metab 90:1678–1685[Abstract/Free Full Text]
  18. Peng N, Kim JW, Rainey WE, Carr BR, Attia GR 2003 The role of the orphan nuclear receptor, liver receptor homologue-1, in the regulation of human corpus luteum 3ß-hydroxysteroid dehydrogenase type II. J Clin Endocrinol Metab 88:6020–6028[Abstract/Free Full Text]
  19. Weck J, Mayo KE 2006 LRH-1 is associated with transcriptional activation of the inhibin {alpha}-subunit gene. Mol Endocrinol 20:1090–1103[Abstract/Free Full Text]
  20. Pezzi V, Sirianni R, Chimento A, Maggiolini M, Bourguiba S, Delalande C, Carreau S, Ando S, Simpson ER, Clyne CD 2004 Differential expression of steroidogenic factor-1/adrenal 4 binding protein and liver receptor homolog-1 (LRH-1)/fetoprotein transcription factor in the rat testis: LRH-1 as a potential regulator of testicular aromatase expression. Endocrinology 145:2186–2196[Abstract/Free Full Text]
  21. Skinner MK 1991 Cell-cell interactions in the testis. Endocr Rev 12:45–77[Abstract/Free Full Text]
  22. Griswold MD 1995 Interactions between germ cells and Sertoli cells in the testis. Biol Reprod 52:211–216[Abstract]
  23. Liu YX, Du Q, Zhou HM, Liu K, Hu ZY 1996 Regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured rat Sertoli and Leydig cells. Sci China C Life Sci 39:37–44[Medline]
  24. Gnessi L, Fabbri A, Spera G 1997 Gonadal peptides as mediators of development and functional control of the testis: an integrated system with hormones and local environment. Endocr Rev 18:541–609[Abstract/Free Full Text]
  25. De Kretser DM, Loveland KL, Meinhardt A, Simorangkir D, Wreford N 1998 Spermatogenesis. Hum Reprod 13:1–8[Free Full Text]
  26. Bassett MH, Mayhew B, Rehman K, White PC, Mantero F, Arnaldi G, Stewart PM, Bujalska I, Rainey WE 2005 Expression profiles for steroidogenic enzymes in adrenocortical disease. J Clin Endocrinol Metab 90:5446–5455[Abstract/Free Full Text]
  27. Sato Y, Suzuki T, Hidaka K, Sato H, Ito K, Ito S, Sasano H 2003 Immunolocalization of nuclear transcription factors, DAX-1 and COUP-TF II, in the normal human ovary: correlation with adrenal 4 binding protein/steroidogenic factor-1 immunolocalization during the menstrual cycle. J Clin Endocrinol Metab 88:3415–3420[Abstract/Free Full Text]
  28. Tajima K, Dantes A, Yao Z, Sorokina K, Kotsuji F, Seger R, Amsterdam A 2003 Down-regulation of steroidogenic response to gonadotropins in human and rat preovulatory granulosa cells involves mitogen-activated protein kinase activation and modulation of DAX-1 and steroidogenic factor-1. J Clin Endocrinol Metab 88:2288–2299[Abstract/Free Full Text]
  29. Suzuki T, Kasahara M, Yoshioka H, Morohashi K, Umesono K 2003 LXXLL related motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1. Mol Cell Biol 23:238–249[Abstract/Free Full Text]
  30. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R 1982 The catalog of human cytokeratin patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24[CrossRef][Medline]
  31. Miettinen M, Virtanen I, Talerman A 1985 Intermediate filament proteins in human testis and testicular germ-cell tumors. Am J Pathol 120:402–410[Abstract]
  32. Steger K, Rey R, Kliesch S, Louis F, Schleicher G, Bergmann M 1996 Immunohistochemical detection of immature Sertoli cell markers in testicular tissue of infertile adult men: a preliminary study. Int J Androl 19:122–128[Medline]
  33. Steger K, Rey R, Louis F, Kliesch S, Behre HM, Nieschlag E, Hoepffner W, Bailey D, Marks A, Bergmann M 1999 Reversion of the differentiated phenotype and maturation block in Sertoli cells in pathological human testis. Hum Reprod 14:136–143[Abstract/Free Full Text]
  34. Zhang XS, Zhang ZH, Guo SH, Yang W, Zhang ZQ, Yuan JX, Jin X, Hu ZY, Liu YX 2006 Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys. Asian J Androl 8:265–272[CrossRef][Medline]
  35. Zhang XS, Zhang ZH, Jin X, Wei P, Hu XQ, Chen M, Lu CL, Lue YH, Hu ZY, Sinha AP, Swerdloff RS, Wang C, Liu YX 2006 Dedifferentiation of adult monkey Sertoli cells through activation of extracellularly regulated kinase 1/2 induced by heat treatment. Endocrinology 147:1237–1245[Abstract/Free Full Text]
  36. Maymon BB, Yogev L, Paz G, Kleiman SE, Schreiber L, Botchan A, Hauser R, Yavetz H 2002 Sertoli cell maturation in men with azoospermia of different etiologies. Fertil Steril 77:904–909[CrossRef][Medline]
  37. Zhang ZH, Hu ZY, Song XX, Xiao LJ, Zou RJ, Han CS, Liu YX 2004 Disrupted expression of intermediate filaments in the testis of rhesus monkey after experimental cryptorchidism. Int J Androl 27:234–239[CrossRef][Medline]
  38. Lue YH, Hikim AP, Swerdloff RS, Im P, Taing KS, Bui T, Leung A, Wang C 1999 Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity. Endocrinology 140:1709–1717[Abstract/Free Full Text]
  39. Lue Y, Hikim AP, Wang C, Im M, Leung A, Swerdloff RS 2000 Testicular heat exposure enhances the suppression of spermatogenesis by testosterone in rats: the "two-hit" approach to male contraceptive development. Endocrinology 141:1414–1424[Abstract/Free Full Text]
  40. Hikim AP, Lue Y, Yamamoto CM, Vera Y, Rodriguez S, Yen PH, Soeng K, Wang C, Swerdloff RS 2003 Key apoptotic pathways for heat-induced programmed germ cell death in the testis. Endocrinology 144:3167–3175[Abstract/Free Full Text]
  41. Lue Y, Wang C, Liu YX, Hikim AP, Zhang XS, Ng CM, Hu ZY, Li YC, Leung A, Swerdloff RS 2006 Transient testicular warming enhances the suppressive effect of testosterone on spermatogenesis in adult cynomolgus monkeys (Macaca fascicularis). J Clin Endocrinol Metab 91:539–545[Abstract/Free Full Text]
  42. Tao SX, Guo J, Zhang XS, Li YC, Hu ZY, Han CS, Liu YX 2006 Germ cell apoptosis induced by experimental cryptorchidism is mediated by multiple by molecular pathways in cynomolgus macaque. Front Biosci 11:1077–1089[CrossRef][Medline]
  43. Chowdhury AK, Steinberger E 1970 Early changes in the germinal epithelium of rat testes following exposure to heat. J Reprod Fertil 22:205–212[Abstract/Free Full Text]
  44. Mieusset R, Bujan L, Mondinat C, Mansat A, Pontonnier F, Grandjean H 1987 Association of scrotal hyperthermia with impaired spermatogenesis in infertile men. Fertil Steril 48:1006–1011[Medline]
  45. Boitani C, Politi MG, Menna T 1993 Spermatogonial cell proliferation in organ culture of immature rat testis. Biol Reprod 48:761–767[Abstract]
  46. Boitani C, Stefanini M, Fragale A, Morena AR 1995 Activin stimulates Sertoli cell proliferation in a defined period of rat testis development. Endocrinology 136:5438–5444[Abstract]
  47. Schlatt S, Zhengwei Y, Meehan T, De Kretser DM, Loveland KL 1999 Application of morphometric techniques to postnatal rat testes in organ culture: insights into testis growth. Cell Tissue Res 298:335–343[CrossRef][Medline]
  48. Zhou B, Watts LM, Huston JM 1993 Germ cell development in neonatal mouse testis in vitro requires müllerian inhibiting substance. J Urol 150:613–616[Medline]
  49. Zhou XC, Wei P, Hu ZY, Han XB, Zhou RJ, Liu YX 2002 Expression of P16 in testes of rhesus monkey during heat stress and testosterone undecanoate induced azoospermia or oligozoospermia. Contraception 65:377–382[CrossRef]
  50. Zhang ZH, Jin X, Zhang XS, Hu ZY, Zou RJ, Han CS, Liu YX 2003 Bcl-2 and Bax are involved in experimental cryptorchidism induced testicular germ cell apoptosis in rhesus monkey. Contraception 68:297–301[CrossRef][Medline]
  51. Guo CX, Ma J, Zhou XC, Liu YX 2000 Expression of hSP 70–2 gene during germ cell apoptosis in rat unilateral cryptorchid testes. Arch Androl 46:109–115[Medline]
  52. Zhou XC, Han XB, Hu ZY, Zhou RJ, Liu YX 2001 Expression of Hsp70–2 in unilateral cryptorchid testes of rhesus monkey during germ cell apoptosis. Endocrine 16:89–95[CrossRef][Medline]
  53. Zhang XS, Lue YH, Guo SH, Yuan JX, Hu ZY, Han CS, Hikim AP, Swerdolff RS, Wang C, Liu YX 2005 Expression of HSP105 and HSP60 during germ cell apoptosis in the heat-treated testes of adult cynomolgus monkeys (Macaca fascicularis). Front Biosci 10:3110–3121[Medline]
  54. Zhang T, Zhou HM, Liu YX 1997 Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes. Mol Human Reprod 3:223–231[Abstract/Free Full Text]
  55. Liu YX, Liu K, Zhou HM, Du Q, Hu ZY, Zou RJ 1995 Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured monkey Sertoli cell. Hum Reprod 10 :719–727
  56. Liu YX, Du Q, Liu K, Fu GQ 1995 Hormonal regulation of plasminogen activator in rat and mouse seminiferous epithelium. Biol Signals 4:232–240[Medline]
  57. Shetty G, Krishnamurthy H, Krishnamurthy HN, Bhatnagar AS, Moudgal NR 1998 Effect of long-term treatment with aromatase inhibitor on testicular function of adult male bonnet monkeys (M. radiata). Steroids 63:414–420[CrossRef][Medline]
  58. Shetty G, Krishnamurthy H, Krishnamurthy HN, Bhatnagar S, Moudgal RN 1997 Effect of estrogen deprivation on the reproductive physiology of male and female primates. J Steroid Biochem Mol Biol 61:157–166[CrossRef][Medline]
  59. Pentikainen V, Erkkila K, Suomalainen L, Parvinen M, Dunkel L 2000 Estradiol acts as a germ cell survival factor in the human testis in vitro. J Clin Endocrinol Metab 85:2057–2067[Abstract/Free Full Text]
  60. Krishnamurthy H, Danilovich N, Morales CR, Sairam MR 2000 Qualitative and quantitative decline in spermatogenesis of the follicle-stimulating hormone receptor knockout (FORKO) mouse. Biol Reprod 62:1146–1159[Abstract/Free Full Text]
  61. Sairam MR, Krishnamurthy H 2001 The role of follicle-stimulating hormone in spermatogenesis: lessons from knockout animal models. Arch Med Res 32:601–608[CrossRef][Medline]
  62. Vornberger W, Prins G, Musto NA, Suarez-Quian CA 1994 Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis. Endocrinology 134:2307–2316[Abstract/Free Full Text]
  63. Medhamurthy R, Culler MD, Gay VL, Negro-Vilar A, Plant TM 1991 Evidence that inhibin plays a major role in the regulation of follicle-stimulating hormone secretion in the fully adult male rhesus monkey (Macaca mulatta). Endocrinology 129:389–395[Abstract/Free Full Text]
  64. Ramaswamy S, Pohl CR, McNeilly AS, Winters SJ, Plant TM 1998 The time course of follicle-stimulating hormone suppression by recombinant human inhibin A in the adult male rhesus monkey (Macaca mulatta). Endocrinology 139:3409–3415[Abstract/Free Full Text]
  65. Funayama Y, Sasano H, Suzuki T, Tamura M, Fukaya T, Yajima A 1996 Cell turnover in normal cycling human ovary. J Clin Endocrinol Metab 81:828–834[Abstract]
  66. Tabibzadeh S 1990 Proliferative activity of lymphoid cells in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab 70:437–443[Abstract/Free Full Text]



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