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Genes to Cells (2006) 11, 1161-1171. doi:10.1111/j.1365-2443.2006.01013.x
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The testes-specific bZip type transcription factor Tisp40 plays a role in ER stress responses and chromatin packaging during spermiogenesis

Ippei Nagamori1, Kentaro Yomogida2, Masahito Ikawa3, Masaru Okabe3, Norikazu Yabuta1 and Hiroshi Nojima1,*

1 Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita City, Osaka 565-0871, Japan
2 School of Human Environmental Sciences, Mukogawa Women's University, Ikekai-chou 6-46, Nishinomiya, Hyogo 663-8558, Japan
3 Genome Information Research Center, Osaka University, Yamadaoka 3-1, Suita City, Osaka 565-0871, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
We previously reported that the spermatid-specific transcription factor Tisp40 functions through UPRE and CRE. To investigate Tisp40 function in vivo, we generated TISP40(–/–) mice. TISP40(–/–) mice were born at expected ratios, were healthy, and mutant males bred normally. However, the ER stress-response protein Grp78/BiP accumulated in the TISP40(–/–) testis and RAMP4 (Ribosome-associated membrane protein 4) mRNA level was up-regulated. Disruption of TISP40 caused ER stress and activation of caspase 12 but not caspase 9, leading to apoptosis of meiotic/postmeiotic germ cells. On the other hand, DAPI staining and electron microscopy revealed that epididymal sperm nuclei were abnormally relaxed in the TISP40(–/–) testis, a phenotype that was independent of the expression and maturation of transition proteins and protamines but due to abnormally retained histones. Histones localized to the cytoplasm as well as to the nucleus and were also retained in epididymal sperm. Histones H2A and H4 were dramatically up-regulated and the acetylation of H2A, H2B and H4 was also enhanced in the TISP40(–/–) testis. Taken together, we conclude that Tisp40 plays an important role in the unfolded protein response of the testis and in regulating the maturation of sperm head nuclei.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
In eukaryotic cells, secretory and transmembrane proteins are synthesized in the endoplasmic reticulum (ER). Newly synthesized proteins are folded and/or modified by N-linked glycosylation in the ER with the assistance of ER chaperones and are then translocated into other organelles. In cells exposed to chemical reagents or heat shock, proteins may remain unfolded and accumulate in the ER, which leads to ER stress (Schroder & Kaufman 2005). Eukaryotic cells can reduce the level of unfolded proteins in the ER by the attenuation of de novo translation and the up-regulation of ER chaperones. These processes are part of the unfolded protein response (UPR), which is widely conserved from yeast to humans (Patil & Walter 2001; Mori 2003). In mammalian cells, ATF6 (Activating transcription factor 6), PERK (PKR-like ER kinase), and IRE1 (Inositol-requiring-1) are the most important factors for the UPR to regulate ER stress (Harding et al. 2002; Mori 2003; Schroder & Kaufman 2005).

Upon ER stress, PERK phosphorylates the eukaryotic initiation factor 2{alpha} (eIF2{alpha}), which diminishes the overall efficiency of translation except for that of ATF4 (Harding et al. 2000). The ATF4 protein is efficiently translated by phosphorylated eIF2{alpha}, and ATF4 directly or indirectly up-regulates ER chaperones (Harding et al. 2000). Thus, the PERK pathway inhibits translation but not transcription until the level of unfolded proteins is reduced (Harding et al. 2002).

When unfolded proteins accumulate in the ER, ATF6 up-regulates the transcription of ER chaperone genes by binding to ER stress response element (ERSE) (Haze et al. 1999). Ire1, another type I ER transmembrane protein, contains an RNase-like domain and a kinase domain (Mori et al. 1993; Yoshida et al. 2001). Upon ER stress, IRE1 generates the spliced (activated) variant of XBP-1 (X-box binding protein 1) (Lee et al. 2002; Yoshida et al. 2003). XBP-1up-regulates the transcription of several ER chaperone genes through the ERSE, and some protein degradation-related genes through the unfolded protein response element (UPRE) (Yoshida et al. 2003).

Although the ERSE and UPRE are important for the expression of UPR genes in cultured cells, it is unclear whether these UPR regulatory pathways operate during mammalian organogenesis. Three transcription factors localized in the ER, OASIS, CREB-H, and Tisp40, were reported to function through the UPRE and are expressed predominantly in astrocytes, hepatocytes, and male germ cells, respectively (Chin et al. 2005; Kondo et al. 2005; Nagamori et al. 2005). The expression profiles of these transcription factors imply the existence of tissue or differentiation specific UPR.

Spermatogenesis is one of the complicated differentiation models of organogenesis from spermatogonia to sperm cells in testis. During spermatogenesis, male germ cells proceed to meiosis, and then haploid spermatids undergo dramatic changes that include sperm tail elongation and tightly regulated chromatin condensation (Hecht 1998). Prior to chromatin condensation, the spermatid nucleosomes are modified by hyperacetylation, thus inducing the relaxation of chromatin (Eddy 1999). Finally, these transition proteins are replaced by highly basic proteins, protamines (for a review see Sassone-Corsi 2002). These sequential changes result in spermatozoa maturation that generates sperm-specific mobility and fertility. We previously proposed that testis contains unique UPR system to reduce the ER stress-induced apoptosis (Nagamori et al. 2005). Recently, TISP40/CREB3l4-deficient male mice were reported to be almost normal, except for a slight increase of apoptosis in male germ cells, because they are fertile and grow normally (Adham et al. 2005). However, using the TISP40-deficient mice we constructed, we found some important abnormalities which they did not describe in the TISP40(–/–) testis. Here, based on our analysis of the TISP40-deficient mice, we propose that the testis specific UPR and the maturation of sperm head nuclei are regulated by Tisp40.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Generation of TISP40-deficient mice

To investigate TISP40 functions in vivo, we generated TISP40-deficient mice by homologous recombination. As shown in Fig. 1A, we replaced the genomic region from exon 5–10, which codes for the basic zipper (bZip) and transmembrane regions, with a neomycin cassette. We confirmed homologous replacement by genomic PCR and Southern blot analysis (Fig. 1B,C). We also confirmed the absence of Tisp40 mRNA and protein from TISP40(–/–) testes (Fig. 1D,E). We found that healthy TISP40(–/–) mice were born at expected ratios and that mutant males bred normally, which is consistent with recent observations of TISP40/CREB3l4-deficient mice (Adham et al. 2005). We confirmed that the average number of offspring was not affected: 7.6 ± 2 (n = 7) for TISP40(+/+) mice, 8.6 ± 1.9 (n = 8) for TISP40(+/–) mice, and 8.9 ± 1.3 (n = 7) for TISP40(–/–) mice (WT males crossed with WT females, KO males crossed with WT females, and KO males crossed with KO females, respectively).


Figure 1
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  		Figure 1  Targeted disruption of TISP40. (A) Schematic presentation of the TISP40 genomic region, targeting vector, and recombinant TISP40 genome. The positions of primers used for analytic PCR are indicated as arrowheads; the fragment used as a probe for Southern analysis is indicated by the bar. (B) Targeted recombination was confirmed by genomic PCR. (C) Southern blot analysis of EcoRI-digested genomic DNA hybridized with the 5' probe. (D) Northern blot analysis shows that Tisp40 mRNA is not expressed in the TISP40(–/–) testis. GAPDH was used as a loading control. (E) Western blot analysis shows that Tisp40 protein is absent from the TISP40(–/–) testis. Tubulin was used as a loading control.

 
Regulation of the UPR in the TISP40(–/–) testis

We previously reported that Tisp40 functions through the UPRE but not the ERSE (Nagamori et al. 2005). The UPRE is an important element that mediates the UPR by regulating the expression of several genes relevant to ER chaperone and protein degradation (Mori 2003). To confirm if Tisp40 acts as a UPR regulator in the testis, we examined the expression of the ER stress-induced genes Grp78/BiP, Grp94, and calnexin by Western blot analysis. As expected, the expression of Grp78/BiP, but not of Grp94 or calnexin, was greatly augmented in the TISP40(–/–) testis (Fig. 2A, right lanes).


Figure 2
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  		Figure 2  Activation of the UPR in TISP40(–/–) testis. (A) Proteins were extracted from TISP40(+/+) or TISP40(–/–) testes. Expression of Grp78/BiP, Grp94 and calnexin was detected by Western blot analysis. Tubulin was used as a loading control. (B) RNA was isolated from TISP40(+/+) or TISP40(–/–) testes and used for cDNA synthesis. The transcription of RAMP4, ERdj4, EDEM, p58IPK, and HEDJ was monitored by semiquantative RT-PCR. Tubulin was used as a loading control. (C) Luciferase assay to show tunicamycin induced expression through UPRE was inhibited by Tisp40{alpha}{Delta}TM. CHO cells were co-transfected with pGL3-5XUPRE and pEGFP3B(s) or pEGFP3B(s)-Tisp40{alpha}{Delta}TM. After 48 h of transfection, the cells were harvested and the cell lysates were subjected to luciferase assays. Average values of three independent experiments are shown. The results were normalized against Renilla luciferase activities obtained from pTK-hRL, which served as an internal control. All data were expressed as standard deviations of the mean for independent three experiments.

 
To determine why disruption of TISP40 leads to ER stress, we searched for genes whose transcriptions were changed in TISP40(–/–). As described above, Tisp40 functions through the UPRE but not the ERSE. Thus, genes regulated by XBP-1 but not ATF6 are also candidates for Tisp40 targets in the testis. Lee et al. (2003) reported that EDEM, p58IPK, HEDJ, RAMP4, and ERdj4 are XBP-1 specific but independent of ATF6. Thus, we examined these genes by RT-PCR. As shown in Fig. 2B, the mRNA levels of EDEM, p58IPK, HEDJ, and ERdj4 were unchanged, but that of RAMP4 (ribosome associated membrane protein 4) was up-regulated in the TISP40(–/–) testis. RAMP4 acts to stabilize transmembrane proteins (Yamaguchi et al. 1999). Up-regulation of RAMP4 in male germ cells may result in the unusual stabilization or degradation of many proteins in male germ cells. These target proteins of RAMP4 may then cause the ER stress.

It was reported that RAMP4 was transcribed by XBP1 through the UPRE. Therefore, we examined whether RAMP4 was up-regulated by XBP1. For this purpose, we examined the activity of XBP1 by RT-PCR. XBP1 is normally produced as an unspliced and inactive form. However, upon ER stress, the XBP1 mRNA is spliced and then translated to produce a highly active transcription factor (Yoshida et al. 2001). These two forms with different sizes can be distinguished by electrophoresis (unspliced form, 448 bp; spliced form, 422 bp). Indeed, we could detect both unspliced and spliced forms of XBP1 mRNA. We found that the band intensity for the spliced XBP1 mRNA (just above the 421 bp marker) was stronger than that of unspliced form of XBP1 mRNA, and that expression and splicing of XBP1 mRNA were not increased in TISP40(–/–) (Fig. 2B). These data led us to surmise that Tisp40{alpha} could act as a negative regulator through UPRE, and RAMP4 was a direct target of Tisp40 in testis, because transactiavation domain of Tisp40 was encoded in Tisp40ß specific region but not in Tisp40{alpha} specific region. We tried to confirm this hypothesis. First, we investigated whether Tisp40{alpha}{Delta}TM actually displayed a negative effect on transcription through UPRE. As shown in Fig. 2C, GFP-Tisp40{alpha}{Delta}TM suppressed the tunicamycin induced transcription through UPRE in CHO cells, while exogenous expression of GFP alone did not affect the tunicamycin induced transcription through the UPRE. Next, we carefully searched for the promoter region of RAMP4; however, we could not find any UPREs in the promoter region. From these data, we propose that, during spermatogenesis, RAMP4 is in the transcriptional pathway of Tisp40, which is probably mediated through the UPRE in its enhancer region, and disruption of TISP40 leads to activation of the UPR due to release from down-regulation of RAMP4.

On the other hand, it is widely known that bZip type transcription factors can form a homo and/or hetero dimer, and this dimerization often affects their binding ability. Indeed, in contrast to Tisp40 homo dimer, Tisp40-CREM{tau} hetero dimer prefers CRE to UPRE (Nagamori et al. 2006). Moreover, we previously reported that the Tisp40 binding site, T(G/T)ACGT(G/A)(G/T), does not completely coincide with UPRE (TGACGTGG) (Nagamori et al. 2005). Probably due to these differences in binding ability, RAMP4 mRNA but not EDEM, ERdj4, p58ipk and HEDJ was up-regulated in TISP40(–/–) testis. Notably, previously reported that TISP40 generates not only an inactive transcription factor, Tisp40{alpha}, but also an active transcription factor, Tisp40ß (see Discussion).

Relationships among ER stress, apoptosis, caspase 12, caspase 9, and p53 pathway

Prolonged ER stress leads to apoptosis mediated by caspase 12 (Nakagawa et al. 2000). Thus, we examined whether apoptosis occurs in the TISP40(–/–) testis using the TUNEL (TdT mediated dUTP nick end labeling system) assay. In the wild-type testis, cells undergoing meiotic recombination and chromatin remodeling are known to be TUNEL-positive (Fig. 3A,iii), and cells in which histones are being replaced by transition proteins (Tnps) and protamines (Prms) are also TUNEL-positive (Marcon & Boissonneault 2004). As shown in Fig. 3A,iv, the number of TUNEL-positive male germ cells was increased in the TISP40(–/–) testis compared to the wild-type testis (Fig. 3A,iii), consistent with what was seen for CREB3l4-deficient mice (Adham et al. 2005). High magnification image of TISP40(–/–) is shown in Fig. 3A,v. To further explore the significance of this apoptosis and to exclude the possibility that these TUNEL-positive cells arose due to other defects, we observed them at higher magnification and confirmed chromatin fragmentation by DAPI staining (Fig. 3A,vi–viii). Next, we counted the number of apoptotic cells per tubule, and found that their frequency was indeed increased (Fig. 3B). However, this increase did not result in a complete depletion of germ cells (Fig. 3A).


Figure 3
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  		Figure 3  Increased apoptosis in the TISP40(–/–) testis. (A) TISP40(+/+) and TISP40(–/–) testes were fixed by 4% PFA and sectioned. Detection of DNA strand breaks in germ cells by the TUNEL assay (iii, iv, v, vii, and viii). DNA was counterstained with DAPI (i, ii, vi, and viii). High magnification images of TUNEL-positive germ cells in the TISP40(–/–) testis (v-viii). (B) Bar graph that indicates the frequency of TUNEL-positive cells per tubule. (C) Detection of precursor and cleaved forms of caspase 12 and 9. Arrows indicate specific cleavage fragments or active caspases. Tubulin was used as a loading control.

 
In many murine cells, caspase 12 is an important factor for ER stress-induced apoptosis to occur. As expected, we found by Western blot analysis that caspase 12 was activated in the TISP40(–/–) testis (Fig. 3C). However, absence of caspase 9 cleavage in TISP40(–/–) testis indicates that caspase 9, which functions in the downstream of caspase 12, was not activated. These data imply that the mechanism of apoptosis mediated by caspase 12 is not conserved in testis. On the other hand, it was reported that ER stress leads to dephosphorylation of GSK-3ß at the Ser9 residue and that p53 is phosphorylated by GSK-3ß and then degraded (Qu et al. 2004). Considering the inhibition of caspase 12 activation and apoptosis in TISP40(–/–) testis, we wondered if GSK-3ß mediated p53 inactivation also was not conserved in testis. To test this possibility, we examined the expression of p53 target genes such as cytochrome c (Cyt c) and Cyclin G1. As a result, both of Cyt c and cyclin G1 genes were up-regulated in the TISP40(–/–) testis (Fig. S1A). Moreover, we examined the phosphorylation status of GSK3ß Ser9 and found it unaffected in the TISP40(–/–) testis (Fig. S1B). Thus, the ER stress-induced dephosphorylation of GSK-3ß and degradation of p53 are not conserved in the testis. Disruption of TISP40 appears to cause ER stress and activation of caspase 12. However, absence of caspase 9 cleavage in TISP40(–/–) testis indicates that caspase 9 is not activated, probably because it is protected by testis specific chaperons (see Discussion). On the other hand, when ER stress occurs in TISP40(–/–) testis, p53 pathway is activated to cause apoptosis and thus the abnormal germ cells are eliminated (see Discussion). Taken together, these data imply that not all of the UPR systems are conserved in the mouse testis and that p53 pathway is activated in TISP40(–/–) testis to eliminate abnormal germ cells.

Observation of epididymal sperm from TISP40(+/+) and TISP40(–/–) testes by fluorescence and electron microscopy

We next investigated whether disruption of TISP40 confers defects during spermatogenesis, because TISP40 is one of the TISP (transcript induced in spermiogenesis) genes that appear to have important roles in spermiogenesis (Fujii et al. 2002; Nagamori et al. 2005). Unexpectedly, we did not detect any differences in testis size or spermatogenetic defects by inspection of pathological sections (Fig. S2A,B). On the other hand, we found that most nuclei of epididymal sperm in TISP40(–/–) mice were abnormally enlarged when stained with DAPI (Fig. 4A). These data suggest that Tisp40 has a role in chromatin condensation.


Figure 4
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  		Figure 4  Observation of epididymal sperm from TISP40(–/–) testes. (A) Images observed by fluorescence microscopy. Sperms were collected from the epididymis and counterstained with DAPI. Sperm nuclei were strongly stained by DAPI. Bar, 10 µm. (B) Images observed by transmission electron microscopy. Epididymal sperms from TISP40(+/+) and TISP40(–/–) mice were collected and sectioned. Several defects were observed in TISP40(–/–) sperm heads: head membrane is detached from the nucleus (asterisk); head membrane is ruptured (#); nuclei is engulfed by membranes (black arrow); a hollow is detected in the sperm nucleus (white arrow); inner membrane regions were detached (asterisk and arrowhead). (C) Frequency of each abnormality in TISP40(–/–) sperm.

 
To investigate these uncondensed sperm heads in more detail, we observed epididymal sperm by transmission electron microscopy. As shown in Fig. 4B, the nuclei of epididymal sperm from TISP40(–/–) mice were deformed (Fig. 4B,i–iv). Their head membranes were not attached to the nucleus (asterisks in Fig. 4B,i–iv), and some head membranes were degraded (# in Fig. 4B,ii). Some sperm nuclei were engulfed by membranes, forming a hooked shape (Fig. 4B,ii–iv). Even in seemingly normal sperm, an abnormal hollow was detected in the nucleus (Fig. 4B,iii,iv, arrows), and inner membrane regions, such as of the acrosome or theca, were detached (asterisk and arrowheads in Fig. 4B,v) as compared to TISP40(+/+) sperm (Fig. 4B,vii). Such abnormal sperm were frequently observed for TISP40(–/–) mice but almost never for TISP40(+/+) mice (Fig. 4C). Thus, we conclude that Tisp40 is important for chromatin condensation during spermiogenesis.

Expression and maturation of sperm nuclear basic proteins and histone

During the course of differentiation to produce mature sperm, sperm nuclei are sequentially packaged by highly basic proteins, transition proteins (Tnps) and protamines (Prms), which is essential for chromatin condensation and fertilization (Dadoune 2003; Kimura et al. 2003; Meistrich et al. 2003; Tanaka & Baba 2005). We therefore speculated that the enlarged nuclei of TISP40(–/–) sperm may be due to defects in the expression of nuclear basic proteins. To determine the role of Tisp40 in chromatin condensation, we first examined the mRNA levels of Prm1, Prm2, and Tnp2 by Northern blot analysis and found that they were unaffected in the TISP40(–/–) testis (Fig. 5A). Next, we investigated whether maturation of Prm2 was normal in TISP40(–/–) sperm. For this purpose, we purified sperm nuclear proteins and examined their migration in AUT (Acid-urea-tritonX100) gels followed by staining with Coomassie Brilliant Blue. We could detect matured Prm2 and Prm1 proteins but not precursor form of Prm2, indicating that maturation of Prm2 is normal in TISP40(–/–) sperm (Fig. 5B).


Figure 5
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  		Figure 5  Expression of basic nuclear proteins. (A) The transcription of Prm1, Prm2, and Tnp2 was detected by Northern blot analysis. GAPDH was used as loading control. (B) Sperm nuclear proteins were purified and separated on an acid-urea-triton X100 gel followed by Coomassie Brilliant Blue staining. (C) Testes were embedded in 4% PFA followed by methyl methacrylate resin, cut into 5 µm sections and stained with an anti-histone monoclonal antibody and Alexa 594-conjugated anti-mouse IgG. DNA was counterstained with DAPI. (D) Detection of core histones in epididymal sperm. (E and F) Expression and acetylation of H2A, H2B, H3, and H4 were detected by Western blot analysis. Tubulin was used as a loading control.

 
Next, we speculated that the nuclear abnormality of sperm arose at the stage at which histones are replaced with Tnps but not when Tnps are replaced with Prms. Sperm in the mouse mutant germ cell-less (GCL) have nuclei with an abnormal shape that contain immature Prm2, which is partly caused by a delay in replacing Tnps with Prms (Kimura et al. 2003). Furthermore, a precursor form of Prm2 is present in the late spermatids of Tnp1 and Tnp2 double knock out mice (Zhao et al. 2004). Since the maturation of Prm2 was normal in TISP40(–/–) mice, we surmised that there was a defect in the transition of nuclear proteins from histones to Tnps but not from Tnps to Prms. Thus, we examined the localization of histones in testis by immunofluorescence analysis using an antibody that recognizes all histones. In the TISP40(+/+) testis, histones were expressed from spermatogonia to stage 8 haploid spermatids and were predominantly localized in the nucleus (Fig. 5C,upper panels). In TISP40(–/–) mice, however, histones were detected at later stages and slightly retained in epididymal sperm, and they were found in the cytoplasm as well as in the nucleus (Fig. 5C,lower panels and Fig. 5D). These data suggest that Tisp40 is required for the proper localization and timely removal of histones.

We next examined each histone by Western blot analysis and found that H2A and H4 were dramatically up-regulated, H3 was slightly up-regulated, and H2B was unaffected in TISP40(–/–) mice (Fig. 5E). Since hyperacetylation occurred just before histone removal, we examined the acetylation status of each histone and found a dramatically increased level of acetylation of H2A, H2B, and H4, but not of H3 (Fig. 5F). The increases in both acetylation and expression of H2A and H4 were almost proportional, suggesting that most of the increased histones were acetylated. These data imply that acetylated H2A and H4 are not removed during the last stages of spermiogenesis in TISP40(–/–) mice and that H3 was unaffected.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
ATF6 and its target XBP-1(s) act through the ERSE or UPRE to up-regulate many genes whose products reduce the level of unfolded proteins in the ER. The transcription of ER chaperone genes is regulated mainly through the ERSE and partly through the UPRE. The transcription of genes encoding degradation proteins and some ER cochaperones is mediated through the UPRE. These transcriptional responses occur in a time-dependent manner and are regulated by the ATF6 and IRE1-XBP-1 pathways (Yoshida et al. 2003). However, most studies of ER stress responses were conducted using cultured cells. Recently, we and others reported that ER-localized transcription factors (OASIS, CREB-H, and Tisp40) play a UPR role in a tissue-specific manner (Chin et al. 2005; Kondo et al. 2005; Nagamori et al. 2005). Probably, the UPR related genes, such as Grp78/94 or RAMP4, are differentially regulated in these tissues. In the case of testis, RAMP4 was regulated in Tisp40 pathway. Specifically, UPR may play a special role in the testis because elevated levels of a large variety of proteins are constantly produced in the testis, and more importantly, germ cells in the testis are connected by cytosolic bridges. If a differentiating germ cell happens to undergo apoptosis, activated caspases or apoptotic factors may leak from it to neighboring cells where they may induce apoptosis. We previously proposed that testes are equipped with testis-specific UPR to prevent an apoptotic chain reaction (Nagamori et al. 2005).

Tisp40, a haploid spermatid-specific transcription factor, functions as a regulator of the testis-specific UPR through UPRE (Nagamori et al. 2005). Indeed, disruption of TISP40 caused the accumulation of Grp78/BiP, which is an ER stress marker. In the TISP40(–/–) testis, the transcription of RAMP4 but not of spliced XBP-1 increased (Fig. 2). This down-regulation of RAMP4 in TISP40(–/–) testis was curious because TISP40 encodes not only Tisp40{alpha} as a negative regulator, but also Tisp40ß as a positive regulator. Very recently, it was reported that Tisp40{alpha}/Atce1 but not Tisp40ß is the main product of TISP40/CREB3l4 gene in mouse testis (El-Alfy et al. 2006). Consistent with their results, our analysis on TISP40(–/–) mice showed that Tisp40{alpha} but not Tisp40ß plays an important role in testis. Thus, we surmise that Tisp40{alpha} in TISP40(+/+) mice suppresses the transcription of RAMP4 gene in testis by covering the UPRE in its enhancer region. In contrast, absence of this suppression in TISP40(–/–) testis causes RAMP4 gene up-regulation, which may lead to ER stress in testis because RAMP4 is a transmembrane protein stabilizer (Yamaguchi et al. 1999). Prolonged ER stress activates the UPR, and affected cells are eventually eliminated by apoptosis. On the other hand, the testis-specific transcription of Tisp40 mRNA begins in stage 5 spermatids (Stelzer & Don 2002; Nagamori et al. 2005), and disruption of TISP40 leads to ER stress (Fig. 2A). These results imply that spermatid cells in TISP40-deficient mice are exposed to ER stress for 11 days, which allows sufficient time for ER stress to induce apoptosis. Indeed, in the TISP40(–/–) testis, apoptosis in male germ cells was clearly increased and an ER stress-specific caspase, caspase 12, was also activated (Fig. 3C). Nonetheless, the average number of offspring was unchanged. If TISP40 disruption causes ER stress and male germ cells apoptosis, why is this not linked to infertility?

One observation relevant to this question concerns caspase 9, which acts downstream of caspase 12, because activation (cleavage) of caspase 9 was not increased in the TISP40(–/–) testis (Fig. 3C). In case of caspase 3, Hsp70 inhibits processing of caspase 3 and 9 (Mosser et al. 2000). On the other hand, many testis-specific chaperones, such as Calmegin, Msj1, Hsp70-2, and DjA1, are predominantly expressed in male germ cells (Dix et al. 1997; Ikawa et al. 1997; Berruti & Martegani 2001; Terada et al. 2005). One of these chaperones may inhibit caspase 12 or another component of the apoptotic pathway. Consistent with our previous report (Nagamori et al. 2005), analysis of TISP40(–/–) also led us to propose that the role of the testis-specific UPR is to reduce apoptosis during germ cell differentiation.

DAPI staining and electron microscopy revealed that TISP40(–/–) mice produce sperm with uncondensed nuclei (Fig. 4). We speculate that the nuclear defect of sperm in the TISP40(–/–) testis is mainly due to abnormality in a histone but not in Tnps or Prms. Indeed, in the wild-type testis, histones are expressed in nuclei from spermatogonia to stage 8 spermatids (Fig. 5C). After stage 9, histones are completely absent (data not shown). In contrast to what is seen for the wild-type testis, histones are retained in later stages of germ cells in the TISP40(–/–) testis, and they are localized not only in the nucleus but also in the cytoplasm. Furthermore, unlike wild-type sperm, TISP40(–/–) mice produced sperms that contain histones (Fig. 5D). Thus, we surmise that Tisp40 is required for the proper nuclear localization and removal of histones. On the other hand, many histone variants are reported to show testis specific expressions, and some of these histones are important factors for spermatogenesis (Lin et al. 2004; Martianov et al. 2005; Tanaka et al. 2005). We, recently reported that histone H3.3 specific chaperone, HIRA, was recruited to CRE by Tisp40 with CREM{tau} (Nagamori et al. 2006). In mouse, there has been no report on where H3.3 is deposited in male germ cells. However, H3.3 is properly deposited in chromatin during first meiotic prophase cells and elongating spermatids cells in Drosophila, and HSP70 genes rapidly lose histone H3 and acquire variant H3.3 histones (Akhmanova et al. 1997; Schwartz & Ahmad 2005). As described above, expression and acetylation of H2A and H4 but not H3 increased in TISP40(–/–) testis, and HIRA was recruited to CRE by Tisp40 and CREM. These data led us to propose a possible model for histone removal; during mid spermatogenesis, HIRA was recruited to CRE by Tisp40-CREM for proper deposition and/or removal of H3.3, and this Tisp40-CREM mediated proper deposition and/or removal of H3.3 in chromatin is required for the removal of other histones and sperm head maturation.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Generation of TISP40-deficient mice

To construct the TISP40 targeting vector, a fragment covering the locus was isolated from a C57BL/6 genomic library using the Tisp40 full length cDNA. The two PCR amplified AscI and EagI/NotI fragments covering the locus were inserted into the vector pMulti flanking the neomycin cassette. The pMulti-TISP40 vector was electroporated into D3 mouse embryonic stem cells, and targeted clones were obtained by positive/negative selection with G418 and DT, respectively. Selected embryonic stem cells were screened by PCR and confirmed by Southern blot analysis. The karyotypes of targeted embryonic stem cells were confirmed to be normal (2N chromosomes). Chimeric mice were obtained from blastocytes injected with targeted embryonic stem cells and were mated with C57BL/6 mice to obtain TISP40 progeny. We used 2- to 4-month aged mice in all these experiments.

Pathological analyses of testis sections and sperm

For morphological observations of testis sections and epididymal sperm, testes were fixed in 4% paraformaldehyde (PFA) at 4 °C followed by methyl methacrylate resin. The embedded testes were cut into 5 µm sections and stained with hematoxylin and eosin. Sperm was collected from the epididymis and spotted on to glass slides coated with 3-aminopropyltriethoxysilane followed by a dry up procedure at 65 °C for 5 min. Sperm was stained with 2 µg/mL 4',6-diamidino-2-phenylindole (DAPI) for 5 min. Purification of sperm nuclear proteins was essentially described as (Herrada & Wolgemuth 1997).

RT-PCR, Northern blot, and Western blot analyses

RNA was isolated from testes by the standard AGPC method (Fujii et al. 2002). PCR amplifications consisted of 30–35 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min, and a final extension at 72 °C for 5 min with the following oligonucleotides: 5'-gtggtggtgctacacccttt-3' and 5'-gcttgggaaaagtctgcaag-3' for ERdj4, 5'-gcaatgggagtccttttgaa-3' and 5'-gtgtcgggctgagttttgtt-3' for p58IPK, 5'-cgagcctggattgaacattt-3' and 5'-agcaggcttctctccattca-3' for RAMP4, 5'-cggttccgaatcaaagttgt-3' and 5'-ccagacgttaaatccccaga-3' for HEDJ, 5'-tgggctggattccttctatg-3' and 5'-ggtgggtctccttctccttc-3' for EDEM, and 5'-ccttgtggttgagaaccagg-3' and 5'-gaggcttggtgtatacatgg-3' for spliced XBP1. Tubulin was used as a loading control.

Northern and Western blot analyses were performed as described (Nagamori et al. 2005) with antibodies against the following proteins: core histones (Chemicon, Temecula, CA, USA), tubulin (Sigma, Milwaukee, WI, USA), H2A, H2B, H3, H4, acetylated H2A, acetylated H2B, acetylated H3, acetylated H4 (Cell Signaling, Technology Inc., Danvers, MA, USA) and CREM (Santa Cruz, Biotechnology, Santa Cruz, CA, USA).

Electron microscopy

Electron microscopy was conducted by the Hanaichi Ultra Structure Research Institute (http://www.kenbikyo.com/top/top.html). Briefly, testes were fixed in cacodylate-buffered 2% glutaraldehyde for 2 h and treated with 2% osmium tetroxide for 3 h, dehydrated in serially graded ethanol, and embedded in an epoxy resin. After ultrathin sectioning, the sections were stained with 2% uranyl acetate in distilled water for 5 min and with a lead stain solution for 5 min. Specimens were examined by a JEOL JEM 2000EX electron microscope operating at 100 kV. The electron micrographs were recorded on FG films.

TUNEL assay and luciferase assay

We used DeadEnd Fluorometric TUNEL system (Promega) to detect apoptotic cells. Luciferase assay was performed as previously described (Nagamori et al. 2006). Briefly, we co-transfected pGL3–5XUPRE and pEGFP3B(s) or pEGFP3B(s)-Tisp40{alpha}{Delta}TM plasmids into CHO cells. pGL3–5XUPRE is a gift from Dr Kazutoshi Mori (Kyoto University), and pEGFP3B(s) or pEGFP3B(s)-Tisp40{alpha}{Delta}TM were described as in Nagamori et al. (2005). After 24 h transfection, we replace the medium with or without 2 µg/mL Tunicamycin. After more 24 h incubation, we harvested and measured luciferase activity. Renilla luciferase activity derived from Thimidine kinase promoter was used as internal control.


   Acknowledgements
 
We are grateful to Dr Hiromitsu Tanaka for supporting in preparation of frozen section, Dr Kazutoshi Mori, Dr Kazunori Imaizumi and Dr Laurie H Glimcher for plasmids. We also thank Ms. Hisae Takema, Yumiko Maruyama, and Akiko Kawai for technical assistance in the generation of TISP40(–/–) mice and Dr P. Hughes for critically reading the manuscript. This work was supported by Innovation Plaza Osaka of the Japan Science and Technology Agency (JST), and grants-in-aid for Scientific Research on Priority Areas, Scientific Research (S), Exploratory Research and Science, and Technology Incubation Program in Advanced Regions, from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Hisato Kondoh

* Correspondence: E-mail: snj-0212{at}biken.osaka-u.ac.jp


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Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 14 February 2006
Accepted: 10 July 2006




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