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J Biol Chem, Vol. 275, Issue 20, 14791-14794, May 19, 2000

ACCELERATED PUBLICATION
Insertional Mutation of the Murine Kisimo Locus Caused a Defect in Spermatogenesis^*

Noriyuki Yanaka, Kinji Kobayashi^§, Koji Wakimoto^¶, Eriko Yamada^¶, Hiroshi Imahie^§, Yuji Imai^¶, and Chisato Mori

From the Discovery Research Laboratory, ^§ Safety Research Laboratory, and ^¶ Department of Advanced Medical Research, Tanabe Seiyaku Co., Ltd., 16-89, Kashima 3-chome, Yodogawa-ku, Osaka 532-8505 and the  Department of Anatomy, Faculty of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Spermatogenesis is a developmental process that occurs in several phases and is regulated by a large number of gene products. An insertional transgenic mouse mutant (termed kisimo mouse) has been isolated that results in abnormal germ-cell development, showing abnormal elongated spermatids in the lumina of seminiferous tubules. We cloned the disrupted locus of kisimo and identified a novel testis-specific gene, THEG, which is specifically expressed in spermatids and was disrupted in the transgenic mouse. The yeast two-hybrid screening method revealed that THEG protein strongly interacts with chaperonin containing t-complex polypeptide-1, suggesting that THEG protein functions as a regulatory factor in protein assembly. Our findings indicate that the kisimo locus is essential for the maintenance of spermiogenesis and that a gene expression disorder may be involved in male infertility.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The integration of foreign DNA into the mouse germ line by retroviral infection or microinjection can result in insertional disruption of endogenous genes with important roles in development (1-3). Foreign DNA insertion provides an approach to the cloning of disrupted host loci. By using the introduced DNA as a probe to screen genomic libraries from mutant animals, it has been possible in a few instances to isolate clones that contain DNA flanking the exogenous integrated material and, thus, include portions of the interrupted gene (4, 5). We have produced a series of 12 transgenic mouse lines with the human phosphodiesterase 5A (PDE5A)¹ gene (6). As the mice were bred, it became evident that many males of one line were sterile and that the sterility arose from a defect in spermatogenesis (we named this mutant kisimo for a Japanese goddess of easy delivery). Because the sterility segregated with the hemizygous transgene and occurred in the absence of the detectable expression of the transgene, we concluded that the abnormal phenotype was due to mutagenesis by insertion of the transgene. In this study, to clone the junctions between the inserted transgene and adjoining cellular DNA, we used thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) (7) and defined a genomic locus important for spermatogenesis.

The process of spermatogenesis in the mouse has been well characterized at the morphological level. The spermatogenic process can be subdivided into three main phases. Spermatogonia, the germinal stem cells, undergo mitosis to produce additional spermatogonia, a portion of which develop into primary spermatocytes. The spermatocytes enter meiosis and proceed through two cell divisions to give rise to haploid round spermatids. These, in turn, undergo a complex morphological transformation involving nuclear condensation and elongation resulting in the production of mature spermatozoa. However, at the molecular level, relatively little is known about the control of cellular differentiation and the architectural changes during spermatogenesis.

In this study, we found that an insertion of foreign DNA results in abnormal male germ-cell development, showing abnormal elongated spermatids in the lumina of seminiferous tubules with severely abnormal or absent flagella, and that a novel testis-specific gene was disrupted in the transgenic mouse. This novel mouse autosomal recessive mutant exhibited a phenotype similar to asthenospermia and provides us an approach to understand the mechanisms underlying the formation of flagella during spermiogenesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Restriction endonucleases and DNA-modifying enzymes were purchased from Takara Shuzo (Kyoto, Japan). 293 cells were from Dainippon Pharmaceutical Co. (Osaka, Japan). Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were obtained from Life Technologies, Inc. [-³²P]ATP was from Amersham Pharmacia Biothech. The MATCHMAKER II two-hybrid system and the mouse testis MATCHMAKER cDNA library were obtained from CLONTECH.

Transgenic Mice-- A DNA fragment carrying the full-length human PDE5A cDNA downstream of cytomegalovirus promoter was generated by digestion with PvuI. The DNA was used to inject the male pronuclei of (C3H × C3H) F₁ embryos by standard techniques. The transgenic mice were selected after screening tail DNA by dot or Southern blot analyses with ³²P-labeled human PDE5A cDNA.

Histological and Electron-microscopic Examinations-- Five male ki/ki mice aged 12 weeks were examined histologically together with age- and sex-matched +/+ mice. Organs were exiced, fixed in Bouin's solution, embedded in paraffin, sectioned in 4-µm slices, and stained with hematoxylin/eosin. The testes were also stained with periodic acid-Schiff, and quantitative evaluation of spermatogenic cells was performed using the simplified morphological method (8). For electron-microscopic examination, testes pieces of ki/ki and +/+ mice were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer at pH 7.4 and post-fixed in buffered 1% osmium. The fixed samples were dehydrated and embedded in an epoxy resin. Ultrathin sections were prepared and stained with lead citrate and uranyl acetate. The specimen was examined by a transmission electron microscope (JEM-100CX, JEOL).

Fluorescence in Situ Hybridization (FISH)-- FISH was done as described previously (9). Probes (the entire transgene carrying the human PDE5A cDNA) were biotin-labeled by nick translation.

TAIL-PCR-- TAIL-PCR was performed according to the method originally developed by Liu and Whittier for chromosomal walking (7). Four relatively short arbitrary degenerate primers were designed according to a previous report (10).

Genomic and cDNA Library Screening-- A mouse genomic library, constructed on lambda EMBL3 (CLONTECH), was used to clone the ki locus. About 1 × 10⁶ plaques were screened with ³²P-labeled 400b mouse genome DNA obtained by TAIL-PCR as a probe according to the standard procedure. Plaques of the mouse genomic library (CLONTECH) plated onto 20 plates at approximately 5 × 10⁴ plaque-forming units/plate were lifted using Hybond-N+ membrane and then screened by hybridization with the ³²P-labeled probe in 6× SSC (1× SSC: 0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 0.5% SDS, 5× Denhardt's solution (1× Denhardt's: 0.02% each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll 400), and 100 µg/ml salmon sperm DNA at 65 °C for 16 h. The filters were washed in 2× SSC, 0.5% SDS at room temperature for 10 min followed by a two 30-min washes in 0.1× SSC, 0.5% SDS at 65 °C. The filters were exposed to x-ray film at 70 °C for 1 day. Mouse cDNAs were cloned from mouse testis cDNA library (CLONTECH) by screening 5 × 10⁵ plaques with ³²P-labeled KpnI/XhoI 5-kb mouse genome DNA as a probe. Human cDNA was isolated from human testis cDNA library (CLONTECH) by screening 1 × 10⁶ plaques with ³²P-labeled full-length mouse cDNA as a probe. Library screening was performed according to the standard procedure. The inserted DNA digested with EcoRI was subcloned into pBluescript II SK (+) (Stratagene) and determined for the nucleotide sequence.

Northern and in Situ Hybridization-- Human multiple tissue Northern blots were purchased from CLONTECH. Hybridization was performed in 50% formamide, 4× SSC, 0.5% SDS, 5× Denhardt's, 100 µg/ml salmon sperm DNA, and the probe at 42 °C for 16 h. Finally, the membrane was washed in 0.2× SSC and 0.1% SDS at 60 °C for 1 h and exposed to x-ray film at 70 °C for 2 days. In situ hybridization using digoxigenin-labeled probes was performed as described previously (11). Digoxigenin-labeled cRNA probes (antisense and sense) were made by in vitro transcription using cDNAs subcloned into pGEM-T vector (Promega) as templates in the presence of digoxigenin-labeled dUTP (Roche Molecular Biochemicals) according to the manufacturer's instructions.

Yeast Two-hybrid Screening-- The yeast two-hybrid screening was performed as described by the manufacturer. A cDNA encoding full-length mouse THEG was subcloned into the yeast expression vector pAS2-1 (CLONTECH) fused in frame with the DNA binding domain of yeast transcriptional activator GAL4, which generated pAS2-1-mTHEG1b. The yeast strain Y190 was co-transformed with pAS2-1-mTHEG1b and the mouse testis cDNA libraries in pGAD10 using the lithium acetate method. Transformants were selected on synthetic dropout agar plates lacking tryptophan, leucine, and histidine but including 25 mM 3-aminotriazole. Yeast colonies were transferred onto nylon membrane and processed by the -galactosidase filter assay. Plasmid DNA was isolated from positive colony and re-transformed into the yeast strain Y190 with either pAS2-1 empty vector or pAS2-1-mTHEG1b. The -galactosidase assay was again conducted to ensure the THEG-dependent activity.

In Vivo Binding Analysis-- 293 cells were cultured in DMEM supplemented with 10% FCS, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 °C in 5% CO₂. The full-length mouse THEG cDNA in the expression vector pFLAG-CMV-2 (Eastman Kodak Co.) and/or the full-length mouse CCT cDNA in pcDNA3.1/HisA (Invitrogen) were transiently expressed in 293 cells using LipofectAMINE PLUS reagent as described by the manufacturer (Life Technologies, Inc.). Cells were washed with ice-cold phosphate-buffered saline 24 h after transfection and scraped in an ice-cold TNE buffer (10 mM Tris-HCl at pH 7.5, 1% Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, 10 mg/ml aprotinin, 10 mM leupeptin, and 1 mM dithiothreitol). Cell extracts were centrifuged at 16,000 × g for 15 min at 4 °C to remove cellular debris and immunoprecipitated with anti-Xpress polyclonal antibody (Invitrogen) with protein G-Sepharose. The beads were washed five times with TNE buffer, and immune complexes were further analyzed by immunoblotting with anti-FLAG M5 monoclonal antibody (Kodak) as described previously (12).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

All twelve independent transgenic lines carrying the human PDE5A gene (6) were bred to homozygosity to screen for recessive insertional mutations. One of these mice transmitted the transgene to its progeny, but when they were intercrossed the resulting homozygotes (ki/ki mice) were infertile. The average testis weight of ki/ki mice (46.9 ± 8.2 mg; n = 5) was 60% that of wild-type littermates (78.0 ± 6.3 mg; n = 5) at 12 weeks of age. In wild and heterozygous mice, none of the spermatogenic cell types at stages II-III, V, VII, and XI showed either pathological or quantitative changes. On the contrary, ki/ki mice had virtually no spermatozoa in the lumina of seminiferous and epididymal tubules. As shown in Fig. 1B, elongated spermatids in the vicinity of the lumina of seminiferous tubules showed vacuolation and were occasionally phagocytosed by Sertoli cells. Several studies on male mice that were sterile because of blocked spermatogenesis have demonstrated the appearance of multinucleated giant cells and numerous spermatocytes undergoing apoptotic cell death (13, 14). However, analysis of cross-sections of these testes by Tdt-mediated dUTP-biotin nick-end labeling assay showed the absence of apoptotic cells in the lumina of seminiferous tubules from ki/ki mice (data not shown). Further characterization of spermatids by electron microscopy revealed the elongated spermatids to have abnormal or completely nonexistent flagella. In the elongated spermatids of wild-type mice, the axoneme was composed of microtubules emanating straight from the centriole at the base of the spermatid nucleus (Fig. 1C). On the contrary, the microtubules and coarse fibers were arranged in a whirl, the nuclei were misshapen, and the cytoplasmic electron density was increased in elongated spermatids of ki/ki mice (Fig. 1D). Intracytoplasmic vacuoles and autophagolysosomes were increased in elongated and some round spermatids. In addition, in the quantitative evaluation of spermatogenic cells in seminiferous tubules, elongated spermatids were significantly decreased at all stages (stage VII is shown in Fig. 1E). Neither the number of spermatogonia nor that of spermatocytes exhibited significant differences. Furthermore, we analyzed testis RNAs derived from wild-type and ki/ki mice for markers of testis development, post-meiotic-specific cAMP-responsive element modulator (CREM), and haploid-specific protamine 2 transcripts. Neither CREM nor protamine 2 RNA levels were significantly affected by the insertional mutation (Fig. 2C), also indicating that spermatogenesis was not suppressed in the proliferative and meiotic phases.

View larger version (70K): [in this window] [in a new window]   Fig. 1.   Spermatogenesis abnormality in ki/ki mice. Cross-sections of testes (stage VII) from wild-type (A) and ki/ki mice (B) are shown. Staining with hematoxylin and eosin revealed the abnormal elongate spermatids. The arrowhead indicates the increased intracytoplasmic vacuoles in elongated spermatids in ki/ki mice. Electron microscopy of spermatids from wild-type (C) and ki/ki mice (D) are shown. In ki/ki mice, dense fibers are shown to form a circular array (arrowhead). Bar, 2 µm. E, quantitative evaluation of spermatogenic cells in seminiferous tubules (stage VII). The ratios of each type of spermatogenic cell to Sertoli cell per seminiferous tubule cross-section were calculated, and the obtained values were analyzed using a Dunnett-type multiple comparison test, with p < 0.01 (**) as minimum significance.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Identification of the mouse ki locus. A, physical map of the ki locus. The direction of THEG transcription is indicated by an arrow. The genomic DNA flanking the transgene insertion site obtained by TAIL-PCR is shown by a bar. The hatched box represents a DNA fragment used as a probe for Northern blot analysis. Restriction enzymes are indicated as follows: K, KpnI; X, XhoI. B, fluorescence in situ hybridization. The transgene insertion locus was determined to be localized chromosome 10. C, Northern blot analysis probed with the KpnI/XhoI 5-kb genomic fragment from ki locus (top panel) or with testis-specific probes corresponding to protamine 2 (prm-2) or CREM cDNA.

Several copies of the transgene were integrated into the ki/ki genome (data not shown). The chromosomal localization of the transgene insertion site was determined by FISH using the whole transgene as a probe, which resulted in a single pair of symmetrical signals mapped to the C1 region of chromosome 10, showing multiple copies of the transgene to be integrated in tandem at a single locus (Fig. 2B). To clone the junctions between the inserted transgene and adjoining cellular DNA, we employed TAIL-PCR by using degenerated primers and specific primers corresponding to the nucleotide sequence of the transgene. One junction fragment containing DNA flanking the transgene insertion site was identified. A wild-type mouse genomic phage library was screened using the flanking DNA fragment as a probe. These isolated phage clones covered about 25 kb of the mouse genome, showing that the integration of the transgene produced a deletion of about 20 kb in the genomic locus (Fig. 2A).

To identify exons in the genomic locus, Northern blot analysis was performed using several DNA fragments (3-6 kb) obtained by endonuclease digestion, covering the deleted region, as a probe. When using 5 kb of the KpnI/XhoI fragment as a probe, a 1.6-kb transcript was detected in the testes from wild-type mice. However, Northern blot analysis showing no signal in testis RNA from ki/ki mice revealed ki/ki mice to be a null strain for this transcript (Fig. 2C). Next, a mouse testis cDNA library was screened with the 5-kb KpnI/XhoI fragment to isolate the corresponding cDNA. We cloned three cDNAs generated by alternative splicing. The proteins predicted from these nucleotide sequences are 313, 351, and 375 amino acids long (Fig. 3A). In vitro transcription/translation analysis demonstrated that the mouse cDNAs encode corresponding proteins (data not shown). To isolate a human counterpart, a human testis cDNA library was screened at a reduced stringency using the mouse full-length cDNA as a probe. The isolated human cDNA encoded a 380-amino acid protein and showed 59.6% identity with the mouse protein (Fig. 3B). A search of the database using the nucleotide and amino acid sequences revealed that the isolated cDNA is identical to a novel THEG (15). Expression of the THEG is highly specific to the testes of mice (data not shown) and humans (Fig. 4A). To identify the cell types that express the THEG, we performed in situ hybridization with a cRNA probe (Fig. 4B). In testis, the transcripts were detectable only in round and elongated spermatids, consistent with the abnormal spermiogenic process of ki/ki mice.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   A, three isoforms of the mouse THEG protein (mTHEG1a, mTHEG1b, and mTHEG1c) are produced by alternative splicing. The hatched line represents the 24-amino acid insertion. mTHEG1c has an independent typical polyadenylation signal in the 3' untranslated region. B, alignment of amino acid sequences of mTHEG1a (m1a) and its human homologue (h). Identical amino acids are indicated by an asterisk.

View larger version (59K): [in this window] [in a new window]   Fig. 4.   Expression of THEG. A, Northern blot analysis probed with full-length human THEG cDNA. Each lane contained 2 µg of poly(A) + RNA (human MTN blot, CLONTECH). B, In situ hybridization analysis of mouse THEG mRNA expression in testis. Antisense probe gave no signal in testis from ki/ki mice (data not shown).

To investigate the potential functions of THEG protein and to determine the mechanism by which these functions are carried out, we employed the yeast two-hybrid system using THEG as bait and a mouse testis MATCHMAKER cDNA library. We found a single positive clone, CCT, that could interact with the THEG protein (Fig. 5A). To confirm this interaction between THEG protein and CCT in intact cells, we co-expressed a FLAG epitope-tagged THEG (FLAG-mTHEG1b) with an Xpress epitope-tagged CCT (Xpress-CCT) in 293 cells. The ability of antibody against the Xpress epitope to precipitate a complex of THEG and CCT suggests that the two proteins interact in the cytoplasm (Fig. 5B). The TCP-1 gene is located in the mouse t-complex on chromosome 17 (16, 17). A mouse t-complex mutation was discovered to produce a phenotype with a tail-less sperm and, to date, TCP-1 identical to the  subunit of CCT has been shown to be highly expressed during haploid stages of spermatogenesis (18). The CCT complex is reported to be required for the proper folding or assembly of cytoskeletal proteins under both in vitro and in vivo conditions (19-21). In the testis, one possible candidate for such a substrate may be  tubulin; -tubulin has constitutively expressed subtypes and testis-specific subtypes (22, 23). Taken together with the previous observations that mutations in individual CCT subunits affect microtubule assembly and produce morphologically abnormal structures detected by anti--tubulin antibodies in yeast (24), abnormal or absent flagella in ki/ki mice appear to be associated with impairment of the assembly of cytoskeletal proteins such as the tubulins that are major structural proteins of flagella.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   A, two-hybrid analysis of THEG and CCT stained by in situ -galactosidase assay. B, interaction of THEG with CCT in vivo. 293 cells were transiently transfected with FLAG-mTHEG1b an/or Xpress-CCT constructs, and cell extracts were immunoprecipitated (IP) with antibody specific to Xpress (IP: Xpress) and immunoblotted with anti-FLAG (Blot: FLAG). Total cell extracts were blotted with anti-Xpress or anti-FLAG antibody.

In the sperm flagellum, as in the epithelial cilia, the cytoskeleton is highly differentiated to permit motility. The basic element of the cytoskeleton is the axoneme, which has an identical structure in both cilia and flagella. Structural anomalies of the axoneme, which are associated with impaired motility, have been described in Chlamydomonas (25). On the other hand, asthenozoospermia is a very frequent cause of male infertility. Previous reports demonstrated that ultrastructural abnormality of spermatozoa is observed in asthenozoospermic or teratozoospermic sterile men (26, 27). A high incidence of flagellar pathology was found to be the underlying cause of motility disorders in severely asthenozoospermic patients (28). In mammalian spermatozoa the flagellum is distinguished by adjoining structures, particularly the dense fibers and fibrous sheath. In particular, poor development of outer dense fibers is considered to be a major cause of tail abnormality; however, research in this area has been limited by the lack of appropriate animal models. The present study indicated that availability of this novel mouse autosomal recessive mutant showing a phenotype similar to asthenospermia enables us to investigate the developmental mechanisms underlying the formation of flagella.

	ACKNOWLEDGEMENTS

We thank Y. Hamasaki, H. Chiba, and K. Muguruma for their technical support. We are grateful to Y. Kondo, H. Sakurai, K. Omori, T. Nishimura, and C. Aruga for their technical advice. We also thank S. Nito and M. Sugiura for their continuous kindness.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB033128 and AB033129.

To whom correspondence should be addressed. Tel.: 81-6-6300-2577; Fax: 81-6-6300-2593; E-mail: n-yanaka@tanabe.co.jp.

Published, JBC Papers in Press, March 20, 2000, DOI 10.1074/jbc.C901047199

	ABBREVIATIONS

The abbreviations used are: PDE, phosphodiesterase; THEG, testicular haploid expressed gene; TCP, t-complex polypeptide; CCT, chaperonin containing TCP-1; TAIL-PCR, thermal asymmetric interlaced polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; ki, kisimo; FISH, fluorescence in situ hybridization; kb, kilobase; CREM, cAMP-responsive element modulator.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/20/14791    most recent C901047199v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yanaka, N. Articles by Mori, C. Search for Related Content PubMed PubMed Citation Articles by Yanaka, N. Articles by Mori, C. Social Bookmarking                      What's this?