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Gamete Biology |
Department of Biochemistry and Molecular Biology3
Department of Urology,4 Yamaguchi University School of Medicine, Ube 755-8505, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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signal transduction, spermatid, spermatogenesis, stress, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cellular proteins are denatured when cells or organisms encounter adverse environments, such as high temperatures. In general, denaturation and the subsequent aggregation of proteins are detrimental to cells and lead to cell death. To survive against these adverse environments, cells possess some mechanisms that enable them to cope with the accumulation of denatured proteins. Heat shock response, which is characterized by the induction of a set of heat shock proteins, is a fundamental response in all organisms to protect themselves [11]. Heat shock proteins bind to denatured proteins, prevent misaggregation, facilitate renaturation [12], and even disaggregate the aggregates [13].
Heat shock response is regulated mainly at the level of transcription by heat shock transcription factors (HSFs) [14]. Only one HSF gene is in yeast and in Drosophila, whereas three HSF genes (HSF1, HSF2, and HSF4 genes) have been identified in mammals [15, 16]. Cells can survive against an exposure to lethal temperatures when they are preincubated at sublethal high temperatures. This phenomenon is known as induced thermotolerance [11]. The heat shock response regulated by HSF1 is necessary for the induced thermotolerance [17–19]. Furthermore, HSF1 promotes cell survival against various stresses by up-regulating a specific heat shock gene in the absence of stress [20]. Moreover, HSF1 can protect against cell death by unknown mechanisms that are independent of the activation of heat shock genes (see Discussion) [21]. All these observations demonstrate that HSFs act as cell-survival factors.
We previously suggested another unique function of HSF1, different from the roles of HSFs that protect against cell death, in male germ cells. Transgenic mice expressing an active HSF1 in the testes are infertile because of a block in spermatogenesis [22]. This observation suggests that HSF1 activated by elevated temperatures may induce cell death of male germ cells. Because HSF1 is one of the components that sense protein denaturation and mediate cellular response [15], we proposed that HSF1 eliminates germ cells suffering from thermal stress. However, we could not rule out artificial effects of overexpressing the mutant HSF1. To extend these observations, we analyzed the testes of HSF1-null mice exposed to thermal stress.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure of mice testis to hyperthermia and tissue preparation were performed as described previously [22], except for the protocol for anesthetizing mice. Anesthetizing solution (20 ml) was made by mixing 1.6 ml of Veterinary Ketalar 50 (Sankyo, Tokyo, Japan), 4 ml of Seractal 2% (Bayer, Leverkusen, Germany), and 14.4 ml of water. Mice were anesthetized by intraperitoneal injection of 8 µl/g of anesthetizing solution. All experimental protocols were reviewed by the Committee for Ethics on Animal Experiments of Yamaguchi University School of Medicine.
In Situ Hybridization
The testes were dissected immediately after the mice were killed, fixed in Bouin fixative at 4°C for 16–24 h, and embedded in paraffin. Paraffin sections (thickness, 5 µm) were dewaxed and rehydrated. After soaking in PBS treated with diethyl pyrocarbonate (DEPC), sections were fixed in 4% paraformaldehyde/PBS at room temperature for 20 min and then washed with three changes of DEPC-PBS. Sections were treated with 0.2 N HCl/DEPC-PBS for 10 min, washed, soaked in 0.1 M triethanolamine-HCl (pH 8.0)/0.25% acetic anhydride at 4°C for 10 min, washed, and then treated with 2 µg/ml of proteinase K/DEPC-PBS at 37°C for 10 min. After washing in three changes of DEPC-PBS, sections were dehydrated and air-dried. To perform hybridization, sections were soaked in hybridization solution (50% formamide, 10 mM Tris-Cl [pH 8.0], 0.2 mg/ml of yeast tRNA, 1x Denhardt solution, 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, and 1 mM EDTA) containing digoxigenin (DIG)-labeled probe (1 µg/ml) at 55°C for 16 h. After washing in 50% formamide/2x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate) at 55°C for 60 min and then in wash buffer (500 mM NaCl, 10 mM Tris-Cl [pH8.0]) at room temperature for 10 min, the sections were treated with 20 µg/ml of RNase in wash buffer at 37°C for 30 min. These were washed in 2x SSC at 55°C for 30 min and then in 0.2x SSC at 55°C for 30 min. To perform immunoreaction, the sections were first blocked with 2% blocking reagent (Roche, Mannheim, Germany)/buffer 1 (150 mM NaCl, 10 mM Tris-Cl [pH 7.5]) and then soaked in alkaline phosphatase-conjugated anti-DIG antibody (Roche)/2% blocking reagent (1:3000 dilution) at 4°C for 24 h or at 37°C for 60 min. After the sections were washed with buffer 2 (100 mM NaCl, 50 mM MgCl2, 100 mM Tris-Cl [pH 9.5]), the immunocomplex was visualized with NBT/BCIP (Roche) in buffer 2 (1:50 dilution) at 37°C for 2–4 h. The reaction was stopped with buffer 3 (1 mM EDTA, 10 mM Tris-Cl [pH 8.0]) and mounted with Pristine Mount (Research Genetics, Huntsville, AL).
The DIG-labeled probes were generated according to the manufacturer's instructions using each specific cDNA inserted in pGEM7 vector (Promega, Madison, WI). The cDNA fragments of mouse Hsp70-1 (306 base pairs [bp]) and Hsp70-2 (433 bp) isolated from EcoRI fragments of pmHsp70-1 and pmHsp70-2 [22], respectively, were ligated into pGEM7 vector. Mouse Hsp27 cDNA isolated from a 540-bp HindIII fragment of pmHsp27 [23] and Hsp110 cDNA isolated from a 400-bp SacI/HindIII fragment of pmHsp105 were ligated into pGEM7 vector. To isolate mouse Hsp90
cDNA, reverse transcription-polymerase chain reaction (RT-PCR) was performed to generate a 564-bp fragment using primers human Hsp90-1 (5'-AAA GAA GGC CTG GAA CT-3') and human Hsp90-2 (5'-TCT ACT TCT TCC ATG CG-3'). The fragment was ligated into pGEM7 vector.
Northern Blot Hybridization
The RNA isolation and Northern blot analysis were performed as described previously [22]. A 603-bp mouse Hsp90ß cDNA fragment was amplified by PCR using an RT reaction generated from total RNA of mouse brain. The primers used were as follows: 5'-mHsp90ß, 5'-CAG AAT TCA TGA AGA TTC CAC TAA CC-3'; 3'-mHsp90ß, 5'-CAG AAT TCG TGC CGC AGG GTC TCC ACG-3'. After being digested with EcoRI, the fragment was ligated into pGEM7 vector. A 619-bp mouse Hsc70 cDNA fragment and a 591-bp mouse Hsp60 cDNA fragment were similarly generated using each specific primer. The primers used were as follows: mHsc70-1, 5'-CGC GGA TCC AGG GCC ATG ACC AAG GAC-3'; mHsc70-2, 5'-CGC GGA TCC CCT CTT CAA TGG TGG GGC-3'; mHsp60-1, 5'-CGC GGA TCC GAG CAG CTA GAC ATC ACA AC-3'; mHsp60-2, 5'-CGC GGA TCC CAT GCC GCC TCC CAT ACC-3'. After being digested with BamHI, the fragments were ligated into pGEM7 vector.
Histopathology and In Situ Detection of Apoptotic Cells
The testes were dissected immediately after the mice were killed, fixed in Bouin fixative at 4°C for 16–24 h, embedded in paraffin, and cut into sections (thickness, 5 µm) [22]. Sections were stained with hematoxylin and eosin, and for apoptotic cells by the TUNEL method using an apoptosis in situ detection kit (Wako Pure Chemical Co., Osaka, Japan). To identify stages of seminiferous tubules, sections were stained with periodic acid-Schiff and counterstained with methyl green.
Experimental Cryptorchidism
Mice were anesthetized as described above. The bilateral testes and the spermatic cords were exposed, paying attention not to injure the vasculatures, and the gubernaculum was cut. Testis descent was prevented by closure of the inguinal canal on both sides by suturing with 3–0 silk [24].
Gel Shift Assay
Testes were dissected 12, 24, 36, and 48 h after cryptorchid surgery. Whole-cell extracts were prepared and gel shift assay performed as described previously using a probe for an ideal heat shock element (HSE) [22].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Anesthetized mice were simply submerged in a water bath at 43°C for 15 min and then allowed to recover for up to 10 h, and the expression of heat shock genes was then analyzed by in situ hybridization. We found that Hsp70-1, Hsp110, and Hsp27 mRNAs were highly induced after heat shock in the interstitial Leydig cells, whereas the induction of these mRNAs was hardly detected in cells within seminiferous tubules containing germ cells and somatic Sertoli cells (Fig. 1, A–G). Northern blot analysis using total RNAs isolated from the whole testes showed that Hsp70-1, Hsp110, and Hsp27 mRNAs were slightly induced compared to the marked induction of these heat shock genes in mouse embryonic fibroblast cells exposed to heat shock (Fig. 1J). To rule out the possibility that the mRNAs in germ cells could not be detected, Hsp90 [25] and testis-specific Hsp70-2 [26] mRNAs were shown to be highly expressed in germ cells (Fig. 1, H and I). We also examined the expression of heat shock genes in transgenic mice expressing an active human HSF1 [22]. Induction of Hsp70-1 (Fig. 1C), Hsp110, and Hsp27 (data not shown) mRNAs was not detected in the testes of the transgenic mice even though the active human HSF1 was expressed in the germ cells. These results clearly demonstrate that mRNAs of major heat shock genes are hardly induced in vivo in response to heat shock in male germ cells.
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Normal Spermatogenesis in HSF1-Null Mice
We previously showed that HSF1 is expressed in most cells in the germinal epithelium [22]. We next analyzed the testes of HSF1-null mice in which the HSF1 gene was mutated by gene targeting [21]. Male mice were fertile, and no abnormality was detected in the weight and histology of the testis of HSF1-null mice (data not shown) (Fig. 3). The level of HSF2 protein in the testis of HSF1-null mice was the same as that in wild-type mice, and HSF4 protein was undetectable in both wild-type and HSF1-null mice (Fig. 2A). The expression of major heat shock genes, Hsp110, Hsp90, Hsp90ß, Hsp70-1, Hsc70, Hsp60, Hsp40, Hsp27, and testis-specific Hsp70-2, was not altered in the testes in HSF1-null mice compared to those in wild-type mice (Fig. 2B).
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To examine whether HSF1 is essential for the heat shock response in the testis, testicles of anesthetized mice were subjected to heat shock, and Northern and Western blot analyses were performed. We observed no induction of the mRNA levels and protein levels of Hsp110, Hsp70, and Hsp27 (Fig. 2, C and D), suggesting that induction of heat shock proteins in response to heat shock is regulated at the level of transcription by HSF1 in the testes.
Cell Death of Spermatocytes Induced by Thermal Stress Is Inhibited in HSF1-Null Mice
We examined the histology of the testes when they were exposed to 43°C for 15 min and then allowed to recover for 12 and 16 h. In wild-type mice, condensation of the nuclei of germ cells was evident 8 h after heat shock (data not shown). Twelve hours after heat shock, some germ cells were detached from the germinal epithelium in most of the seminiferous tubules, and later, the entire tubules were destroyed (Fig. 3) [27]. In marked contrast, the condensation of the nuclei of germ cells and the detachment of germ cells were less evident until 16 h after heat shock in HSF1-null mice. To quantify the cell death of germ cells, TUNEL staining was performed to detect apoptotic cells [22]. Few apoptotic cells were found in the control testes in both wild-type and HSF1-null mice. The numbers of apoptotic cells per tubule in wild-type and HSF1-null mice were 11.0 ± 1.8 and 4.4 ± 1.0, respectively, at 8 h of recovery and 21.4 ± 3.5 and 11.3 ± 2.4, respectively, at 12 h of recovery (Fig. 3M).
We next determined which cells die in response to heat shock. In the tubules of wild-type mice, apoptotic cells were observed mostly in the inner layer of the germinal epithelium, and a few apoptotic cells having relatively large nuclei were observed in the outermost layer (Fig. 3G). The localization and morphology suggested that the apoptotic cells were mostly pachytene spermatocytes [20, 28, 29]. In marked contrast, fewer pachytene spermatocytes were TUNEL-positive in HSF1-null mice (Fig. 3H). We further stained these sections with periodic acid-Schiff to identify stages of seminiferous tubules. Although few pachytene spermatocytes at stage V died after heat shock in wild-type mice (Fig. 3I), many pachytene spermatocytes at stages XI–XII died in the same tubules (Fig. 3K). In contrast, few pachytene spermatocytes at stages XI–XII died in HSF1-null mice (Fig. 3L). The stage-specific susceptibility to heat is mainly caused by the effect of supporting Sertoli cells [29]. These results demonstrate that HSF1 promotes heat shock-induced cell death of pachytene spermatocytes.
Cell Death of Spermatogonia Exposed to Thermal Stress Is Protected by HSF1
It was shown by TUNEL staining that a substantial fraction of the apoptotic cells was observed in the outermost layer of the tubules in HSF1-null mice (Fig. 3H). Furthermore, clusters of cells located in the outermost layer of the tubules at stage V died (Fig. 3J). These results suggest that the apoptotic cells are not pachytene spermatocytes but, instead, are more immature germ cells, probably including spermatogonia. These immature cells do not die in the tubules at stage V in wild-type mice (Fig. 3I), indicating that cell death of undifferentiated germ cells exposed to heat shock is inhibited by HSF1. Interestingly, this function of HSF1 is independent of the induction of heat shock proteins as shown in Figure 1.
Cell Death of Spermatocytes after Surgical Induction of Cryptorchidism Was Inhibited in HSF1-Null Mice
Germ cell death induced in the cryptorchid testes is caused mainly by exposure of the testes to a relatively high abdominal temperature. Pachytene spermatocytes and round spermatids are highly susceptible to the experimental cryptorchidism [30, 31], as they are to exposure to heat [28, 29]. Therefore, we next examined the germ cell death induced by bilateral cryptorchidism for 7 days in wild-type and HSF1-null mice. In wild-type mice, many degenerating pachytene spermatocytes were found (Fig. 4A, a and b). The TUNEL staining showed that 21.5 ± 9.6 cells per seminiferous tubule were TUNEL-positive (Fig. 4B). In marked contrast, only a slight germ cell degeneration (2.7 ± 4.5 cells/tubule) was observed in the cryptorchid testes in HSF1-null mice (Fig. 4, A and B). The DNA-binding activity of HSF1 was clearly activated in the cryptorchid testes examined by gel shift assay (Fig. 4C). These results conclusively showed that HSF1 promotes heat shock-induced cell death of pachytene spermatocytes.
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Magnitude of Cell Death of Spermatocytes Depends on the Activity of HSF1
To address the gene-dosage effect of HSF1, transgenic mice were crossed with HSF1-null mice. The weights of the testes in the transgenic mice were low (33.0 ± 4.8 mg) compared with those in the wild-type mice (71.3 ± 5.1 mg) [22]. As expected, the weight of the testes was partially restored in the transgenic mice lacking an endogenous HSF1 (48.8 ± 0.3 mg) (Fig. 5B). Histological examination also showed partial restoration of spermatogenesis in the transgenic mice lacking an endogenous HSF1 (Fig. 5C). The diameters of the seminiferous tubules were larger in the transgenic mice lacking an endogenous HSF1 compared to those in the transgenic mice expressing an endogenous HSF1. In the transgenic mice, many pachytene spermatocytes and only a few spermatids were observed in the tubules (Fig. 5C, b and h) [22]. Many of the spermatids were TUNEL-positive (Fig. 5C, e). In contrast, in the transgenic mice lacking an endogenous HSF1, many round spermatids were observed in most seminiferous tubules, and many of the spermatids were TUNEL-positive (Fig. 5C, c, f, and i). These results demonstrate the gene-dosage effect of HSF1 on spermatogenesis and that HSF1 promotes cell death of round spermatids in addition to pachytene spermatocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The HSF1 is activated in response to stresses that induce protein denaturation [14, 15]. The roles of HSF1 in male germ cells may be analogous to a transcription factor, p53, that is activated by genotoxic stresses. In response to the genotoxic stresses, p53 induces cell-cycle arrest to allow the opportunity for DNA repair to occur before replication or mitosis. Alternatively, it induces apoptotic cell death to eliminate irreparably damaged cells (for review, see [32, 33]). The choice between growth arrest and apoptosis is determined by many factors, including the cell type. For example, T lymphocytes undergo extensive apoptosis in response to DNA damage, whereas fibroblasts enter cell-cycle arrest (for review, see [34]). The p53 is expressed in male germ cells [35], and cell death of p53-deficient germ cells is delayed in experimental cryptorchidism [9], suggesting that the p53 pathway is involved in germ cell apoptosis in response to thermal stress. Thus, in germ cells, HSF1 and p53 monitor abnormalities of protein and of DNA, respectively. The present study establishes the notion that the HSF1 pathway is one of the quality-control mechanisms in spermatogenesis.
The expression of most heat shock proteins, including testis-specific isoforms, is generally high, but controversy exists regarding whether heat shock genes are activated in germ cells in response to heat shock (for review, see [36]). The Hsp70 protein was first reported to be induced in response to heat shock in isolated germ cells [37]. This result was confirmed by immunohistochemical and DNA microarray analyses using heat-treated testes [38]. In contrast, Hsp70 protein was reported not to be induced by in vitro heat shock in isolated pachytene spermatocytes [39]. Consistent with this result, ß-galactosidase activity was not induced by in vivo heat shock in spermatocytes of transgenic mice in which the Hsp70 genes were replaced by a ß-galactosidase gene [40]. In the present study, we clearly showed that heat shock genes were not activated in male germ cells and that protein level of Hsp70 was not induced. The activation of HSF1 is not accompanied with the increased expression of heat shock proteins in male germ cells. These results demonstrate that HSF1 determines the fate of germ cells independent of the expression of heat shock proteins.
Recently, we demonstrated that HSF1 protects against cell death caused by a single exposure to heat. This function is independent of the activation of heat shock genes [21]. In avians, HSF1 does not have the ability to activate heat shock genes under heat shock conditions, whereas an avian-specific factor, HSF3, activates heat shock genes. The induction of heat shock proteins through HSF3, as well as Drosophila HSF or mammalian HSF1 [17, 18], is necessary for induced thermotolerance in chicken B lymphocyte DT40 cells [19]. In contrast, the induction of heat shock proteins does not inhibit cell death of DT40 cells subjected to a single exposure of heat [21]. Rather, HSF1 strongly suppresses cell death of DT40 cells in the absence of heat shock gene activation. Increased cell death of HSF1-null DT40 cells at high temperatures is restored by ectopically expressing human HSF1 [21]. In the present study, we found a similar function of HSF1 in vivo in immature germ cells, probably including spermatogonia. Spermatogonia are stem cells of sperm, and they undergo mitotic cell division like somatic cells do. Taken together, HSF1 supports cell survival through the activation not only of heat shock genes but also of unknown target genes.
Embryonic weight was reduced when normal female mice were mated with males exposed to thermal stress [41]. This result suggests that embryo quality is linked to sperm quality and that injury caused by thermal stress is passed on to the next generation. Therefore, HSF1 should actively eliminate injured spermatocytes, which are committed to differentiate to sperm. On the other hand, HSF1 supports the survival of undifferentiated germ cells. These two functions of HSF1 might have played major roles in maintaining species during evolution against various stresses causing protein denaturation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Akira Nakai, Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Minami-Kogushi 1-1-1, Ube 755-8505, Japan. FAX: 81 836 22 2315; anakai{at}yamaguchi-u.ac.jp
Received: 5 June 2003.
First decision: 7 July 2003.
Accepted: 18 August 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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