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1 Biomedicum Helsinki, Institute of Biomedicine, University of Helsinki, FI-00014 Helsinki, Finland
2 Department of Clinical Chemistry, University of Helsinki, FI-00014 Helsinki, Finland
3 Departments of Physiology and Pediatrics, University of Turku, FI-20520 Turku, Finland
4 Department of Medical Biochemistry, University of Kuopio, FI-70211 Kuopio, Finland
5 Institute of Developmental Genetics, GSF-National Research Center for Environment and Health, Neuherberg, Germany
6 Laboratory of Gene Transfer, National Institute for Cancer Research and DOBIG, University of Genova, Genova, Italy
(Requests for offprints should be addressed to J J Palvimo; Email: jorma.palvimo{at}helsinki.fi)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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and ß splice variants of PIASx (Nakagawa & Yokosawa 2002, Schmidt & Müller 2003). PIAS proteins show predominantly nuclear localization and are characterized by an N-terminal SAP (scaffold attachment factor, acinus, and PIAS) and a C-terminal SP-RING domain (Schmidt & Müller 2003). PIAS proteins were first identified as inhibitors of activated STAT (Chung et al. 1997), but they have thereafter been shown to interact with and modulate several other proteins, such as steroid receptors and p53 (Schmidt & Müller 2003). Recently, PIAS proteins have been shown to act as E3 ligases in sumoylation (Melchior et al. 2003), but they also appear to possess functions beyond the modification process of sumoylation.
Sumoylation is a posttranslational modification resembling ubiquitination, where a SUMO (small ubiquitin-related modifier) protein is attached to a lysine residue of a target protein. Several consequences for sumoylation have been found. Sumoylation may block ubiquitination at the same lysine residue and thereby prevent degradation of a target protein such as I
B (inhibitor of nuclear factor B alpha) or PCNA (proliferating cell nuclear antigen; Desterro et al. 1998, Hoege et al. 2002). Sumoylation also plays a role in nuclear import and export as well as in the regulation of subnuclear localization of a number of proteins (Melchior et al. 2003, Seeler & Dejean 2003). Many of the sumoylation targets are transcription factors, and sumoylation may regulate their transcriptional activity (Verger et al. 2003). Mammals possess three different SUMO proteins; SUMO-1 and the nearly identical SUMO-2 and SUMO-3 (Seeler & Dejean 2003). E1-activating enzyme Aos1/Uba2 and E2-conjugating enzyme Ubc9 are needed for the attachment of SUMOs to target proteins (Seeler & Dejean 2003), and SUMO E3 ligases appear to facilitate the reaction. In addition to PIAS proteins, nucleoporin RanBP2 and polycomb protein Pc2 have been shown to possess SUMO E3 ligase activity (Pichler et al. 2002, Kagey et al. 2003).
In Saccharomyces cerevisiae, PIAS protein family consists of Siz1 and Siz2. Deletion of either Siz1 or Siz2 leads to reduced levels of SUMO conjugates, and the compound mutant shows retarded growth and a delay or arrest in the cell cycle (Johnson & Gupta 2001). In Drosophila melanogaster, only one PIAS gene, termed dPIAS/Zimp/Su(var)2–10, is currently known. It is an essential gene; cells in which dPIAS is mutated show severe abnormalities in chromosome condensation and segregation (Mohr & Boswell 1999, Betz et al. 2001, Hari et al. 2001).
PIASx is highly expressed in testis, and the protein can be detected in spermatogonia and pachytene spermatocytes up to stage XII of the seminiferous epithelium as well as in Sertoli cells, suggesting that PIASx has a role in testicular function (Moilanen et al. 1999, Yan et al. 2003). We have disrupted the PIASx gene in a large-scale gene-trap mutagenesis of the mouse genome (Hansen et al. 2003). The preliminary analysis of PIASx gene-targeted mice, however, did not reveal an obvious phenotype (Hansen et al. 2003). In this study, we have examined in more detail the consequences of PIASx deficiency on testicular function. We show that homozygote PIASx mutants display a reduction in testis size although their fertility is normal.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The production of the PIASx-knockout mouse has been described before (Wiles et al. 2000, Hansen et al. 2003). Briefly, ES (embryonic stem) cells were electroporated with PT1ßgeo gene-trap vector (Wiles et al. 2000), which contains an En-2 splice acceptor site upstream of a ß-galactosidase/neomycin-resistance fusion gene (ßgeo). Gene-trap vector integration occurred in the second intron of the PIASx gene as determined by sequencing of the fusion transcript, PCR, and Northern- and Southern-blot analyses (Fig. 1
and results not shown). Genotyping of mice was performed using genomic DNA from a tail-clip biopsy by using PCR with sense primer 5'-GTTCTCATCAAGCCCACGAG-3' and antisense primers 5'-CTAGACATTCATACTCTCAGGTC-3' and 5'-CTTGAAGGACTCCAATAGGGTA-3' for wild-type and mutant alleles, respectively. Mice were backcrossed with C57BL/6 mice for several generations. All animal experiments were approved by the Institutional Review Board on animal experiments at the University of Helsinki. Mice were analyzed at the age of 3 months if not specified otherwise. The wild-type control animals were either littermates or other wild-type mice with the same genetic background and housing conditions.
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Cauda epididymis was excised from 3-month-old mice and pierced with a 25-gauge needle in a cell-culture dish containing 0.5 ml Brinster’s BMOC-3 medium (Invitrogen, Carlsbad, CA, USA). Sperm suspensions were incubated at 37 °C in a humidified 5% CO2/95% air environment for 30 min. Sperm suspensions were diluted 1:10 in the medium and sperm counts were determined using a hemocytometer. Sperm count is expressed as the number of sperm cells per epididymis.
Acridine Orange staining
Spermatozoa from cauda epididymides were obtained by placing a piece of cauda epididymis in a drop of Tris-buffered saline (TBS; 100 mM Tris/HCl and 150 mM NaCl, pH 7.5) and squeezing with forceps. Spermatozoa were smeared on glass slides, air-dried, fixed with acetic alcohol (1:3, v/v, glacial acetic acid/methanol) for 2 h, and air-dried again. Slides were stained for 5 min in a solution containing 0.02% Acridine Orange (Sigma-Aldrich, Steinheim, Germany) in a citrate/phosphate buffer (pH 2.5) protected from light. Slides were rinsed with deionized water, covered with a coverslip, and sealed with Permount (Fisher Scientific, Fairlawn, NJ, USA). Slides were kept in the dark and examined using an Olympus AX70 fluorescence microscope (Olympus, Tokyo, Japan) with 490 nm excitation filter and 530 nm barrier. Spermatozoa with normal DNA structure displayed a green fluorescent color, whereas yellow to red marked abnormal DNA structure.
Hormone measurements
Blood was collected by cardiac puncture with a 25-gauge needle, was allowed to clot for at least 30 min, and then centrifuged for 10 min at 1000 g. Serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were determined by immunofluorometric assays as described previously (Haavisto et al. 1993, van Casteren et al. 2000). Intratesticular testosterone was determined by homogenizing approximately half of a testis in 0.5 ml PBS (4.3 mM sodium phosphate, 1.4 mM potassium phosphate, 137 mM NaCl, and 2.7 mM KCl, pH 7.3). 300 µl of the gonadal homogenate was extracted twice with 2 ml diethyl ether and evaporated to dryness over night. After reconstitution into 200 µl PBS, testosterone was measured by standard RIA (Huhtaniemi et al. 1985). Protein concentration in homogenates was measured using the Bradford method (Bradford 1976), and testosterone concentration was related to protein concentration.
Histological examination
Testes were immersed in Bouin’s fixative overnight at 4 °C, embedded in paraffin, and sectioned at 5 µm thickness. Sections were mounted on poly-lysine-coated slides, stained with hematoxylin and eosin, and examined with light microscope. Diameter of seminiferous tubules was measured from tubules at stage VII–VIII of the seminiferous epithelial cycle with the help of a scale bar.
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling) staining
Testes were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 µm thickness. Sections were mounted on poly-lysine-coated slides and TUNEL staining was performed with the DeadEnd colorimetric TUNEL system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The numbers of TUNEL-positive cells and seminiferous tubules were counted from four sections and the number of apoptotic cells was expressed in terms of cells per 100 tubules.
Northern blotting
Probe templates for PIASx were PCR-amplified from testicular cDNA using the following oligonucleotides: upstream of gene-trap insertion, 5'-AAATGGCGGA TTTCGAGGAGTTG-3' (nucleotides 188–210 of NM_008602
[GenBank]
.2) and 5'-TGTCCGTGGGTATCGGC GTCT-3' (nucleotides 393–373 of NM_008602
[GenBank]
.2); downstream of gene-trap insertion, 5'-ATGAAAGG TATTAGAAATCCTGATCATTCC-3' (nucleotides 1117–1146 of NM_008602
[GenBank]
.2) and 5'-TCCTTTTGG CGGGAGGGTCTT-3' (nucleotides 1654–1634 of NM_008602
[GenBank]
.2). The oligonucleotides that were used in construction of the probe templates for PIAS1, PIAS3, PIASy, and Pem are shown in Supplementary data table 1 (see http://jme.endocrinology-journals.org/content/vol34/issue3/). The amplified products were cloned into pGEM-T Easy vector (Promega) and the resulting insert was verified by sequencing. The plasmids were linearized and antisense RNA probes were prepared using either SP6 or T7 RNA polymerase (Promega). [-32P]UTP (Amersham Biosciences) was used for labeling of the antisense probe. 28 S rRNA cDNA probe was labeled with [-32P]dCTP (Amersham Biosciences) by the random-priming method (Ready-To-Go DNA Labeling Beads; Amersham Biosciences). Total RNA was isolated from testes using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. 10 µg of total RNA were size-fractionated in 1% denaturing agarose gel and transferred on to Hybond-N+ nylon membrane (Amersham Biosciences). Prehybridization and hybridization were performed with ULTRAhyb (Ambion, Austin, TX, USA) according to the manufacturer’s instructions.
Immunoblot analysis
Mouse tissues were homogenized in a buffer containing 50 mM Tris/HCl, pH 7.8, 300 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, and a 1:100 dilution of protease-inhibitor cocktail (Sigma-Aldrich). The tissue lysates were centrifuged at 16 000 g for 20 min at 4 °C, the supernatants were transferred to new tubes, and the concentration of protein was measured using the Bradford method (Bradford 1976). Samples from the supernatants (50 µg protein/lane) were resolved on SDS-PAGE and electrophoretically transferred on to nitrocellulose membranes (Hybond ECL; Amersham Biosciences). The membrane was blocked and washed according to the instructions of the manufacturer (Amersham Biosciences). PIASx protein was detected with a polyclonal rabbit anti-ARIP3 antibody (1:5000 dilution; Moilanen et al. 1999). The immunocomplexes were visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:10 000 dilution; Zymed Laboratories, South San Francisco, CA, USA) and the ECL detection system (Amersham Biosciences). Mouse anti--tubulin monoclonal antibody (sc-5286; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to monitor sample loading.
Microarray analysis
Total RNA was isolated from testes of three mutant and three wild-type mice with Trizol reagent and the RNeasy kit (Qiagen, Hilden, Germany) according to manufacturers’ instructions. 20 µg total RNA was labeled with Cy3-dUTP (wild-type) and Cy5-dUTP (mutant) using the Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions. cDNAs were subsequently hybridized on Mouse (Development) Oligo Array slides (Agilent Technologies) representing over 20 000 known mouse genes. The fluorescence intensities on the slides were analyzed using a laser confocal scanner and Feature Extraction Software (Agilent Technologies). Low-quality measurements (i.e. the signal was lower than 2.6 times the background, more than 50% of pixels of the spot were saturated, or either the spot or the background were not uniform) were excluded from the analysis. After background subtraction, the ratio was calculated by dividing the average intensity of each spot in mutant hybridization by the average intensity of the corresponding spot in the wild-type hybridization. For transcripts with two or three high-quality measurements from separate slides, the mean of log-transformed ratios was calculated. Transcripts that corresponded to a Unigene cluster, had a mean of log-transformed ratios below –0.3 (–2-fold-change) or above 0.3 (2-fold change), and differed significantly from zero (P< 0.05 as assessed with t-test) were regarded as differentially expressed.
RT (reverse transcriptase)-PCR
3 µg total RNA (three wild-type and three mutant testes) and 50 ng oligo(dT)12–18 primer (Amersham Biosciences) were heated to 70 °C for 10 min. cDNA synthesis was carried out with 9 U AMV (avian myeloblastosis virus) RT (Promega) at 50 °C for 1 h and subsequently at 42 °C for 1 h. PCRs were performed with the Lightcycler system using SYBR Green I dye (Roche Molecular Systems, Indianapolis, IN, USA; Wittwer et al. 1997). Thermocycling was carried out in a final volume of 20 µl, containing 2 µl cDNA sample (1:50–100 dilution) or standard cDNA, 3.5 mM MgCl2, 0.5 µM primers, and 2 µl FastStart DNA master SYBR Green I mix. After a 10-min initial denaturation at 95 °C, the 45-cycle run consisted of a 10-s denaturation step and a 5-s annealing step followed by an extension step at 72 °C. Annealing temperatures, extension times, melting temperatures, and oligomers that were used in RT-PCR are shown in Supplementary data table 1 (see http://jme.endocrinology-journals.org/content/vol34/issue3/). A standard curve for PCR test was constructed using serial 1:5 dilutions of normal murine testis cDNA. The concentrations of samples, analyzed in triplicate, were determined with the help of standard curve using the fit-points method in data-analysis software (Wittwer et al. 1997, Woo et al. 1998). The mean of two repeated PCR values was used in the statistical analyses. Results were normalized to porphobilinogen deaminase (PBGD) by dividing the individual RT-PCR values by the mean of three repeated PBGD test values of the respective sample to reduce variability between RNA amounts introduced into the RT-PCR reactions (Schrader et al. 2002). To distinguish the specific PCR products from non-specific products and primer dimers, a melting-curve analysis was performed as described earlier (Woo et al. 1998). Additionally, samples were analyzed by agarose gel electrophoresis to verify that the amplified products exhibited correct sizes.
Statistical analysis
Data are presented as means ± S.E.M. and were analyzed by t-test using SPSS version 10.0 statistical software. P <0.05 was considered as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We have used the gene-trap approach to disrupt the PIASx gene (Wiles et al. 2000, Hansen et al. 2003). The gene-trap vector contains an En-2 splice acceptor site to allow the fusion of the ßgeo fusion gene to the target gene. The murine PIASx gene is located on chromosome 18. It is composed of 15 exons and gives rise to and ß forms of PIASx mRNA through alternative splicing of the last three exons. The gene-trap vector insertion occurred in the second intron of the PIASx gene (Fig. 1A
). Northern blotting of testicular RNA with a probe that recognizes the second exon of the PIASx gene showed that PIASx+/– mice indeed produced an additional longer transcript that is likely to correspond to the fusion of the 5' part of PIASx transcript and the ßgeo transcript (Fig. 1B). The amount of authentic PIASx mRNA was reduced in PIASx+/– mice. In PIASx–/– mice, the authentic PIASx mRNA was totally absent, and the intensity of the longer transcript was stronger than in PIASx+/– mice (Fig. 1B). Northern analysis with a probe that recognizes exons downstream of the gene-trap vector insertion site demonstrated that the native PIASx transcript was completely absent in PIASx–/– mice, but a shorter form of the PIASx transcript was present in PIASx+/– and PIASx–/– mice (Fig. 1C). Immunoblotting showed a gene-dose-dependent disappearance of PIASx-derived signals in testis, and no additional shorter forms of PIASx proteins, which might have been encoded by the shorter form of the PIASx transcript, were evident in PIASx+/– or PIASx–/– mice (Fig. 1D).
Testis weight is reduced in PIASx mutant mice
The PIASx mutant mice did not show any obvious phenotype in the preliminary analysis; the animals were viable, grew normally, and were fertile (Hansen et al. 2003). Since PIASx is highly expressed in Sertoli cells and developing spermatogonia and spermatocytes, it was pertinent to examine testicular function of PIASx mutant mice in more detail. Although the body weights were similar in PIASx+/+ and PIASx–/– mice, there was a clear difference in the weight of their testes: the absolute and relative testicular weights were decreased by 23% (P< 0.001) and 25% (P< 0.001), respectively, in homozygote PIASx mutants compared with their wild-type littermates (Table 1). PIASx mutants also exhibited a tendency for diminished production of spermatozoa as their epididymal sperm counts were 19% lower (P=0.12) when compared with wild-type mice (Table 1). However, spermatozoa of PIASx–/– mice were motile, and Acridine Orange staining did not reveal any gross disturbances in the spermatozoa DNA structure (results not shown).
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Histological examination of testes of 3- and 9-month-old mutant mice revealed no apparent abnormalities in their spermatogenesis (Fig. 2B and C). However, in 3-month-old mutant mice, the average diameter of stage VII–VIII seminiferous tubule was decreased by 6% (P=0.151; Fig. 2A), with both seminiferous tubule lumen size and wall thickness being reduced. We next performed TUNEL staining to examine whether PIASx gene disruption influenced testicular cell apoptosis. We measured the number of TUNEL-positive cells and seminiferous tubules in cross-sectioned testes and observed that the number of apoptotic cells was increased by 74% (P< 0.05) in PIASx mutant mice compared with wild-type mice (Fig. 2D). However, the apoptotic cells did not appear to accumulate in any specific step or stage of spermatogenesis (Fig. 2E and F). Most of the TUNEL-positive cells were primary spermatocytes both in wild-type and PIASx-knockout mice.
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Testis is a target tissue of several endocrine hormones. FSH has a stimulatory role in spermatogenesis and LH regulates testosterone production of Leydig cells (Cooke & Saunders 2002). Furthermore, testosterone, produced intratesticularly by Leydig cells, is required for normal spermatogenesis. Therefore, we studied serum levels of FSH and LH as well as the intratesticular level of testosterone. As shown in Table 2, we did not find significant differences in the levels of FSH and LH between PIASx+/+ and PIASx–/– mice. However, the intratesticular testosterone concentration was elevated by 67% in PIASx–/– mice, but the difference was not statistically significant. Even though the trend to intratesticular testosterone is not likely to explain the increased testicular apoptosis, we cannot rule out the possibility that it may have contributed to the reduction of testis weight. A reminiscent situation in humans is testicular atrophy and azoospermia secondary to supraphysiologic androgen levels.
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PIASx is a member of the PIAS family, raising the possibility that the amounts of other members of the family may increase to compensate for the loss of PIASx. We addressed this issue by examining the levels of mRNAs coding for PIAS1, PIAS3, and PIASy by real-time quantitative RT-PCR and Northern blotting. We did not detect changes in the mRNA levels of PIAS1 or PIAS3, whereas the amount of testicular PIASy mRNA was reduced by 30% in PIASx–/– mice (Fig. 3).
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Many interaction partners of PIASx, such as AR (androgen receptor) and p53, are transcription factors. Depending on the studied protein and promoter context, overexpressed PIASx can either increase or decrease transcriptional activity of the target protein (Schmidt & Müller 2003). In order to study the effect of PIASx gene disruption on AR-dependent gene expression, we examined the mRNA level of the Pem gene, whose expression is androgen-regulated and Sertoli-cell-specific (Rao et al. 2003), and found that the amount of Pem mRNA was increased by ~33% in PIASx–/– animals compared with wild-type mice (Fig. 3). In order to study the effect of PIASx gene disruption on the transcriptome, we compared testicular mRNAs in PIASx+/+ and PIASx–/– mice by microarray analysis and found several differentially expressed genes (showing at least 2-fold change; Supplementary data table 2 (see http://jme.endocrinology-journals.org/content/vol34/issue3/). However, these genes did not accumulate to any specific functional category. Among the differentially expressed genes that have a possible testicular function, DNA polymerase (Polk; Unigene Mm.89926) and X-linked zinc-finger protein (Zfx; Mm.919) were upregulated, whereas DEAD box polypeptide 24 (Ddx24; Mm.3935), DNA polymerase 
(Poll; Mm.46509), trafficking protein particle complex 2 (Trappc2; Mm.279752), tissue plasminogen activator (Plat; Mm.154660), and growth factor/erv1 (S. cerevisiae)-like (Gfer; Mm.28124) were downregulated in the mutant mice. As assessed by quantitative RT-PCR, the Gfer mRNA was decreased by 2.4-fold in PIASx–/– mice compared with PIASx+/+ mice.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Studies on D. melanogaster, which has only one PIAS gene, have demonstrated clearly the physiological importance of PIAS proteins for multicellular organisms, in that disruption of dPIAS/Zimp/Su(var)2–10 leads to embryonic lethality due to severe abnormalities in chromosome condensation and segregation (Hari et al. 2001). In S. cerevisiae, mutation of either Siz1 or Siz2 fails to cause a striking phenotype, but a strain that harbors mutations in both genes grows poorly at a lower temperature (Johnson & Gupta 2001). PIASy- and PIAS1-deficient mice were being generated while this paper was being prepared for publication (Liu et al. 2004, Roth et al. 2004, Wong et al. 2004). PIASy-knockout mice appear to be phenotypically normal and show protein-sumoylation patterns comparable with those of wild-type mice (Roth et al. 2004, Wong et al. 2004). Interestingly, PIAS1–/– mice, especially males, are smaller than the wild-type mice and are born at a frequency lower than the expected Mendelian ratio, but they do not have any histological defects and they are also fertile (Liu et al. 2004). These results, together with the subtle phenotype of PIASx-knockout mice, suggests that functional redundancy is a general phenomenon among PIAS proteins in vivo. In line with this, several in vitro experiments have shown that more than one PIAS protein may promote sumoylation of the same target protein, such as PIAS1 and PIASxß towards p53 (Schmidt & Müller 2002) and PIAS1 and PIASx towards AR (Kotaja et al. 2002, Nishida & Yasuda 2002). In keeping with this, we did not detect changes in the overall sumoylation pattern of proteins in PIASx mutant mice (data not shown).
PIASx–/– mice had qualitatively normal spermatogenesis and were fertile, although their testes were smaller than in wild-type littermates. PIAS1 and PIASy are, however, normally highly expressed in the testis and their expression patterns overlap with that of PIASx, although their highest levels are detected in different cells of the seminiferous epithelium (Tan et al. 2000, 2002, Gross et al. 2001, Yan et al. 2003). Members of the PIAS protein family are known to interact with each other and be capable of modulating each others’ functions (Tan et al. 2002). Therefore, the mild phenotype of PIASx–/– mice may also, at least in part, be a result of an imbalance among the PIAS proteins in the mutant mice.
The extent of apoptosis was increased in PIASx mutant mice testis, as judged by their higher number of TUNEL-positive cells. This finding is interesting because several sumoylation targets, such as topoisomerase I, NEMO (nuclear factor B essential modulator), Smads, and p53, are involved in apoptosis (Gostissa et al. 1999, Rodriguez et al. 1999, Horie et al. 2002, Huang et al. 2003, Lin et al. 2003). Notably, both p53 and Smads are also interaction partners for PIAS proteins (Kahyo et al. 2001, Megidish et al. 2002, Schmidt & Müller 2002, Lin et al. 2003, Ohshima & Shimotohno 2003), although the role of PIAS proteins in the regulation of p53 function has remained elusive. Although PIAS1 and PIASxß promote sumoylation of p53, repression of p53 activity by PIAS proteins is not entirely dependent on p53 sumoylation (Schmidt & Müller 2002). Likewise, PIASy is able to block induction of p53 target genes (Nelson et al. 2001). Other studies have, however, pointed out that PIAS1 and PIAS3 L may in fact activate p53-dependent gene expression (Kahyo et al. 2001, Wible et al. 2002). Smads act as downstream mediators of transforming growth factor ß and activin signaling and are thus potential regulators of apoptosis. PIAS proteins are capable of facilitating sumoylation of Smads. PIASy tends to decrease transcriptional activity of Smad3 and Smad4 (Lee et al. 2003, Long et al. 2003), whereas PIASxß and PIAS3 activate the function of Smad4 and Smad3 respectively (Ohshima & Shimotohno 2003, Long et al. 2004). Interestingly, PIAS3L and PIAS1 themselves have been demonstrated to possess proapoptotic activities (Liu & Shuai 2001, Wible et al. 2002). Increased apoptosis in the testis of PIASx-deficient mice suggests that, unlike PIAS1 and PIAS3L, PIASx can act as a negative regulator of apoptosis. The mechanism of increased apoptosis remains to be elucidated, since we could not detect differential expression of apoptosis-related genes in our microarray analysis.
Majority of the reported interaction partners of PIAS proteins are transcription factors (Schmidt & Müller 2003). Even though PIAS proteins can act as SUMO E3 ligases, all of their functions are not necessarily related to sumoylation (Horie et al. 2002, Schmidt & Müller 2002). For example, the effects of PIAS proteins on AR activity depend on cell and promoter context and may be either positive or negative (Kotaja et al. 2000). We studied the expression of Pem that is an AR target gene in Sertoli cells and found that the Pem mRNA level was increased in PIASx–/– mice, which is in line with the notion that PIASx-promoted SUMO modification of AR may repress the receptor function in murine testis. Unfortunately, the currently available antibodies do not allow direct measurement of sumoylation of endogenous AR in mice.
In this study, we found Gfer among the genes that were repressed in the testes of PIASx–/– mice. Gfer is a sulphydryl oxidase capable of generating disulphide bridges in proteins (Klissenbauer et al. 2002). Gfer accumulates in mitochondria of spermatogonia and primary spermatocytes, indicating that Gfer may be an important protein in the early stages of spermatogenesis (Klissenbauer et al. 2002). Interestingly, Gfer has been found to augment liver regeneration and enhance the success rate of pancreatic transplantation, potentially by regulating intracellular redox status (Hagiya et al. 1994, Adams et al. 1998, Wu et al. 2003). However, the function of Gfer in context of spermatogenesis and its possible role in apoptosis need further studies.
In conclusion, we show that PIASx–/– mice produce viable spermatozoa. This finding indicates that PIASx is not absolutely required for the spermatogenesis, and the deficiency of PIASx may be substituted by other PIAS proteins. However, the increased number of apoptotic spermatogenic cells, the reduced epididymal sperm count and diameter of seminiferous tubules indicates mildly disturbed spermatogenesis, implying that spermatogenesis is quantitatively rather than qualitatively dependent on functional PIASx.
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Acknowledgements |
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Received 30 November 2004
Accepted 25 January 2005
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