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BOR - Papers in Press, published online ahead of print July 30, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032789
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BIOLOGY OF REPRODUCTION 71, 1927–1935 (2004)
DOI: 10.1095/biolreprod.104.032789
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Characterization of a Novel Postacrosomal Perinuclear Theca-Specific Protein, CYPT1

Kouichi Kitamura2, Naoko Iguchi, Yoshioki Kaneko, Hiromitsu Tanaka, and Yoshitake Nishimune1

Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Disease, Osaka University, Osaka 565-0871, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The perinuclear theca (PT) is a unique cytoskeletal structure that surrounds the nucleus of the sperm. The posterior acrosome segment of the PT (postacrosomal PT) is thought to play roles in shaping the nucleus during differentiation of the spermatid and in activating the oocyte during fertilization. We isolated a cDNA clone that encoded a novel haploid germ cell-specific cysteine-rich perinuclear theca protein, CYPT1. The transcripts were expressed exclusively in testicular germ cells after meiotic division. Sequence analysis revealed that CYPT1 comprised 168 amino acids and that the N-terminal was rich in basic amino acids, including cysteine clusters. Immunohistochemical and biochemical analyses localized CYPT1 to the postacrosomal PT of elongated spermatids and mature sperm. The cypt1 had three paralogs that were expressed in adult testis. A comparison of genomic structure suggested that two of the three cypt1 paralogs were generated by gene triplication on the X chromosome, while one paralog was retrotransposed to an autosome. Interestingly, the 5'-flanking regions of these genes were highly homologous with the promoter region of the spermatid-specific gene Zfy-2. CYPT1 and the proteins of the paralogous genes constitute a novel, basic cysteine-rich sperm protein family that may contribute to the function of the postacrosomal PT during nuclear shaping.

gametogenesis, gene regulation, perinuclear theca, postacrosomal region, spermatogenesis, sperm maturation, testis, X-linked gene


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the testis, spermiogenesis (late stage of spermatogenesis) is a complex developmental process that includes the dynamic morphological changes in postmeiotic haploid germ cells that are required for the production of sperm. During spermiogenesis, the nucleus is shaped, mitochondria are rearranged, the flagellum develops, and the acrosome is generated [1]. The perinuclear theca (PT) also develops during spermiogenesis and is believed to contribute to the formation of the sperm head [2].

The PT lies between the nuclear envelope (NE) and plasma membrane (or inner acrosomal membrane). The morphology of the PT is remarkably different among mammalian species [3] and the differences in PT architecture may determine interspecies differences in the shape of the sperm head. The cytoskeletal structure of the PT continuously surrounds the nucleus and comprises three segments, namely the subacrosomal, equatorial, and postacrosomal regions, each of which is associated with a particular function (reviewed in [4]). The postacrosomal PT (also known as the calyx) is also thought to be involved in oocyte activation during fertilization [5–7].

Several postacrosomal PT-specific molecules have been identified, including the highly-basic proteins calicin [8], cylicin I [9], and cylicin II [10], as well as the actin-capping proteins CPbeta3 [11] and CPalpha3 [12, 13]. Actin-related proteins were recently found in the postacrosomal PT [14], which suggested that actin filaments were nucleated during formation of the PT during spermiogenesis. However, the molecular composition, mechanism of formation, and functional role of the postacrosomal PT remain largely unknown.

Previously, we isolated cDNA clones that were expressed specifically in mouse haploid germ cells [15]. Many of these genes are thought to be involved in differentiation during spermiogenesis or in sperm function [16–24]. Here, we report the characterization of one of these novel genes, namely cypt1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Ethics

Animals were purchased from SLC (Shizuoka, Japan). All experiments were carried out in dedicated facilities at the Research Institute for Microbial Diseases at Osaka University. All experimental procedures were approved by the institutional Animal Experimentation Committee.

Construction of Subtracted cDNA Library, Screening, and DNA Sequencing

A haploid germ cell-specific cDNA library in the vector pAP3neo (Takara, Shiga, Japan) was generated by subtracting the mRNA of a testis of a 17-day-old mouse from a cDNA library of an adult (35-day-old) mouse testis, as described previously [12]. Plasmid DNAs of clones that were selected randomly from the subtracted cDNA library were screened in a Northern blot analysis using testicular RNA from 17- and 35-day-old mice.

We designated testis-specific cDNA sequences as those transcripts for which expression increased during spermiogenesis (TISP) [15]. One such clone, namely TISP55, was used as a probe to screen an adult mouse testis cDNA library to isolate the complete sequence of the clone. An adult mouse testicular pAP3neo cDNA library in Escherichia coli MC1061A cells [12] was diluted and seeded onto a nitrocellulose filter on a Luria broth plate at a final concentration of 1 x 105 cfu. After incubation at 37°C, colonies were transferred onto two nylon replica filters, and the filters were soaked sequentially at room temperature in the following solutions: 0.5 M NaOH and 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.4) and 1.5 M NaCl, and 2 x SSC (5 min in each). After incubation at 80°C for 2 h, the filters were washed and bacterial debris was removed. A [{alpha}-32P] dCTP-labeled probe was prepared from a 0.6-kilobase (kb) EcoRI-NotI fragment of the TISP55 cDNA using the BcaBEST random primer kit (Takara). Filters were hybridized with the probe at 65°C for 20 h in a solution containing 4 x SSC, 10 x Denhardt solution, 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA. Dideoxy chain-termination sequencing reactions were performed with fluorescent dye-labeled primers and thermal cycle sequencing kits purchased from Applied Biosystems (Foster City, CA). The reaction products were analyzed using an ABI-PRISM 310 Genetic Analyzer (Applied Biosystems). Several independent, positive clones were isolated and sequenced.

Northern Blot Analysis

Freshly removed organs of adult mice (C57BL/6 strain) were homogenized in RNAzol B (Tel-Test, Tokyo, Japan). Germ cells and other somatic cells from testes were prepared as described previously [25]. Briefly, seminiferous tubules were placed in 20 mM HEPES buffer containing 0.1% collagenase. The tubule suspension was left standing to sediment tubules. The supernatant containing separated cells was used as the Leydig cell fraction. The remaining tubules were cut into small fragments in phosphate-buffered saline (PBS) and the suspension was again left standing. The supernatant was used as the germ cell fraction, and sedimented tubules, which contained mainly Sertoli cells, were used as the Sertoli cell fraction.

Total RNA was extracted according to the manufacturer's recommendations (Tel-Test) and was quantified by measuring the optical density. RNA samples (10 µg per lane) containing 2.2 M formaldehyde were electrophoresed on a 1.1% agarose gel containing 0.66 M formaldehyde. RNA was transferred to a nitrocellulose filter in 20 x SSC, and the filter was baked at 80°C for 1 h. Hybridization was performed with 32P-labeled cDNA prepared with the BcaBEST random primer kit (Takara) at 42°C for 16 h in a solution containing 4 x SSC, 5 x Denhardt solution, 0.2% SDS, 12 µg/ml denatured salmon sperm DNA, and 50% formamide. Filters were washed twice in 0.1 x SSC and 0.1% SDS at 60°C. Band signals were detected with a Fuji image analyzer (Fuji Film, Tokyo, Japan).

Antibodies

Synthetic peptides (SKLEKTTKRFKLIK, residues 24–37) were designed from the deduced amino acid sequence of CYPT1 and were purified using high-performance liquid chromatography for immunization of Japanese white rabbits (MBL, Nagoya, Japan). Polyclonal antiserum was obtained by injecting peptide conjugated to keyhole limpet hemocyanin, followed by booster injections at 3-wk intervals. Anti-CYPT1 antibodies were affinity purified from the whole antisera. Anti-CYPT1 antibody recognized a band corresponding to a molecular weight of 19 kDa in a Western blot analysis (Fig. 3).



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  		FIG. 3. Western blot analysis of TISP55-encoded protein cysteine-rich postacrosomal perinuclear theca (CYPT1) protein expression. Protein was extracted from the organs of adult mice. The blots were probed with a specific anti-CYPT1 antibody. Bars at the left are molecular weight markers. The arrowhead indicates the position of the CYPT1 protein

Western Blot Analysis

Samples of protein from various organs of adult C57BL/6 mice were lysed in RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 1 mM PMSF), centrifuged, and quantified using the Bradford method (Nacalai Tesque, Kyoto, Japan). Protein samples (~50 µg per lane) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel before being transferred to polyvinylidene difluoride filters (Millipore, Bedford, MA). The filters were blocked with 5% skim milk in Tris-buffered saline (TBS: 2 mM Tris HCl, pH 7.5, 150 mM NaCl, and 50 mM KCl) and were incubated with antibody diluted in TBS (1:500). The filters were washed with TBS containing 0.2% Tween20 and were then treated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Amersham Biosciences, Tokyo, Japan). After a final wash, the filters were developed using a POD staining kit (Wako, Osaka, Japan).

Immunohistochemical Analysis

Testes were immersed in OTC embedding compound (Tissue-Tek, Sakura, Tokyo, Japan) and were frozen at –20°C. Sections (10 µm thick) were prepared using a cryomicrotome (HM 500 OM; Microm, Walldorf, Germany). Mature sperm were removed from the cauda epididymis and spotted onto glass slides (Matsunami Glass, Osaka, Japan). Samples were fixed with 4% paraformaldehyde-PBS at 4°C for 10 min before being permeabilized in PBS containing 0.1% Triton X 100 for 5 min. Each sample was incubated with 10% normal goat serum in PBS for 1 h at room temperature. After washing, the samples were incubated with anti-CYPT1 rabbit antiserum (1:300) and were then incubated with anti-rabbit Igs goat serum conjugated to fluorescein isothiocyanate (FITC) (Amersham Biosciences, Buckinghamshire, UK) diluted at 1:500 in PBS. The sections were counterstained with 4'-6-diamidino-2-phenylindole (DAPI; Nacalai Tesque), washed, and examined under a fluorescence microscope. As the monoclonal MN13 antibody labels the postacrosomal PT [26], we analyzed the colocalization of CYPT1 and MN13 in mildly sonication sperm. MN13 labeling was detected with FITC-conjugated anti-mouse Igs raised in sheep.

Subcellular Fractionation of Sperm

Subcellular fractionation of sperm was performed according to a method described previously [27], with minor modifications. All buffers contained a protease inhibitor mixture (Sigma, Tokyo, Japan). Sperm from the cauda epididymis were suspended in ice-cold PBS and centrifuged at 5000 x g for 20 min. Pellets were then washed twice in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA to avoid contamination with blood and epithelial cells. Sperm were resuspended in mild lysis buffer (50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 1 mM EGTA, 1 mM Na3VO4, 50 mM NaF, and 150 mM NaCl) and centrifuged at 15 000 x g for 10 min. The supernatant was used as the membrane/cytoplasm fraction. After washing with PBS, the pellets were resuspended in 50 mM Tris-HCl (pH 7.4) and 1% SDS to solubilize the acrosome and were centrifuged. The pellets were washed three times in PBS, resuspended in 50 mM Tris-HCl (pH 8.0) and 25 mM DTT, and were incubated at room temperature for 20 min with agitation. Cetyltrimethylammonium bromide was added at final concentration of 1% to extract the PT. After an additional 20-min incubation, the remaining nuclear and perinuclear matrix proteins were pelleted and extracted in 50 mM Tris-HCl (pH 8.0), 132 mM CaCl2, 88 mM MgCl2, and 50 mg/ml DNase I at 37°C for 1 h. The proteins in the supernatant of each fraction were precipitated overnight with three volumes of ethanol at –20°C, washed with acetone, and air dried. The samples were solubilized in SDS buffer and subjected to Western blot analysis. A control antibody (CD46) was used for the membrane fraction [28].

Database Search and Sequences

We performed BLAST searches of the DNA Data Bank of Japan, GenBank, European Molecular Biology Laboratory, Swiss-Prot, and Protein Identification Resource databases using the cypt1 cDNA sequence as the query. The searches identified three cDNA sequences that were similar to cypt1, namely ckt1, ckt1r1, and ckt1r2 (National Center for Biotechnical Information [NCBI] accession numbers AF463499, AF463500, and AF463501, respectively).

Reverse Transcriptase-PCR

First-strand cDNAs of ckt1, ckt1r1, and ckt1r2 were generated from 5 µg of testis total RNA using the Thermoscript reverse transcriptase-polymerase chain reaction (RT-PCR) system (Invitrogen, Carlsbad, CA) with a specific primer for the sequence that was common to all of the cypt1 paralogs (5'-TTCGATCAGCTCCAGTTGG-3'). The mRNA-cDNA hybrids were then digested with RNase H and were used as template for PCR amplification of 116- to 176-base pair (bp) internal fragments of cypt1 and the paralog cDNAs using one pair of primers (forward: 5'-TGGCCAAGAAAGTCCACTGGT-3'; reverse: 5'-AGATCTCTTGGGAAGCTTAGA-3'). PCR conditions were as follows: 30 cycles at 96°C for 45 sec, 55°C for 45 sec, and 72°C for 1 min. The PCR products were separated in an 8% polyacrylamide gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Sequencing of cypt1

We isolated a new haploid germ cell-specific cDNA clone of 624 bp from the subtracted mouse testis cDNA library, which we designated TISP55 (NCBI accession number AB046308) [15]. The clone did not have a full-length open reading frame; therefore, we rescreened an adult mouse testis cDNA library in the vector pAP3neo using TISP55 as a probe. We identified a 696-bp cDNA clone (NCBI accession number AF463502) that encoded 168 amino acids (Fig. 1). A stop codon was present 30 bp upstream of the first ATG. The sequence of the full-length TISP55 cDNA was deposited in GenBank and named ckt1r3.



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  		FIG. 1. The cypt1 cDNA and deduced amino acid sequences. Asterisks indicate stop codons. Shaded boxes indicate basic amino acids. Alanine and cysteine clusters are indicated in boldface. Arginine-serine dipeptides are underlined. Protein kinase casein kinase II (CK2) phosphorylation sites ([S/T]XX[D/E]) are indicated by wavy lines

The deduced amino acid sequence of the cDNA included a basic region at the N-terminus (residues 1–114) with a predicted isoelectric point (pI) of pH 11.11 (Fig. 1). In contrast, the remainder of the amino acid sequence showed an acidic pI of 3.49 (Fig. 1). The predicted isoelectric point of the whole protein was basic (pI 9.65). The protein had two amino acid clusters in the N-terminal region that consisted of 8 alanine and 14 cysteine residues. The N-terminal region also contained the following potential target sites for protein kinases: arginine/serine (RS) dipeptides, phosphorylation sites for serine- and arginine-rich (SR) protein-specific kinases (SRPKs) [29], and target sites for protein kinase casein kinase II (CK2) (consensus [S/T]XX[D/E]) [30]. As we had isolated the protein from the postacrosomal PT, we renamed the TISP55-encoded protein cysteine-rich postacrosomal PT protein (CYPT1).

Expression Pattern of cypt1

Northern blot analysis revealed that cypt1 was expressed specifically within the testis as a single 0.7-kb transcript; cypt1 was not detected in any other organ, including the brain, heart, intestine, kidney, liver, lung, muscle, ovary, and spleen (Fig. 2A). In testis, the cypt1 transcript was detected in the germ cell fraction, but not in the somatic cell fractions (i.e., Sertoli or Leydig cells) (Fig. 2B).



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  		FIG. 2. Distribution and developmental expression of cypt1 mRNA. Northern blot analysis of cypt1 mRNA in mouse organs (A), fractionated cells from mouse testis (B), and mouse testis from mice of different ages (days) (C). Adults were older than 2 mo. Total RNA was extracted from several different organs. Filters were rehybridized with GAPDH cDNA as a control

To identify the developmental expression pattern of cypt1, prepubertal mouse testes were examined. The transcript was not expressed in the testis until 4 wk (29 days) postpartum, at which time elongated spermatids appeared; thereafter, the expression increased with age (Fig. 2C). Western blot analysis of testis extracts with anti-CYPT1 rabbit antiserum revealed a single band with a molecular mass of 19 kDa (Fig. 3). A band of the same size was observed in sperm from the epididymis (Fig. 6). A band at ~45 kDa detected in heart extracts was an unidentified cross-reactive antigen because no cypt1 transcript was detected in this organ.



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  		FIG. 6. Western blot analysis of CYPT1 from sperm. Protein samples were obtained from the following fractions of sperm (see Methods): NP-40, membrane/soluble fraction; SDS, acrosome fraction; DTT & CTAB, perinuclear theca fraction; DNase I, nuclear fraction. A control antibody (CD46) was used for the membrane fraction

Localization of CYPT1 in Testis and Sperm

Immunofluorescence analysis of CYPT1 expression in adult mouse testis revealed that CYPT1 was expressed predominantly within elongated spermatids during the late stages (steps 9–16) of haploid germ cell development (Fig. 4C), which was consistent with the results of the Northern blot analysis. The subcellular localization of CYPT1 was restricted to the postacrosomal region of spermatids (Fig. 4E). There was no immunopositive signal in immature germ cells (spermatogonia and spermatocytes) or in somatic cells (Sertoli and Leydig cells). In sperm, CYPT1 expression was limited to the postacrosomal region of the sperm head after detergent permeabilization (Fig. 5). CYPT1 was also detected in mildly sonicated sperm and was colocalized with the MN13 epitope, which labeled postacrosomal PT [26]. After subcellular fractionation, CYPT1 was detected in the PT fraction but not in the membrane/soluble or nuclear fraction (Fig. 6). These results suggested that CYPT1 was expressed specifically within the postacrosomal PT.



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  		FIG. 4. Immunofluorescent localization of CYPT1 protein in testis. Sections of adult mouse testis were incubated with anti-CYPT1 antibody and Texas Red-labeled secondary antibody (red). Sections were counterstained with 4'-6-diamidino-2-phenylindole (DAPI) to visualize nuclei (blue). A, B) Control (preimmune rabbit serum). C, D) Anti-CYPT1 antibody. E) Higher magnification image of CYPT1 immunofluorescence (x1000). Scale bar = 100 µm



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  		FIG. 5. Immunofluorescent localization of CYPT1 protein in sperm. A) Detergent-permeabilized sperm were incubated with anti-CYPT1 antibody and Texas Red-labeled secondary antibody (red). Samples were counterstained with DAPI to visualize nuclei (blue). Red and blue images were merged. B–D) Mildly sonicated sperm were incubated with anti-CYPT1 antibody (red) and antiMN13 monoclonal antibody (green). Original magnification x400

Comparison of cypt1 and Paralogous Gene Sequences

We found three cDNA sequences in the mouse testis cDNA library and expressed sequence tag (EST) databases that were similar to cypt1 (see Materials and Methods). These cDNAs, which had been named ckt1, ckt1r1, and ckt1r2, are likely functional genes because the reading frames are conserved within the gene sequences. To confirm gene expression, RT-PCR analysis was performed, and each of the cDNA fragments was found to be expressed in mouse testis (Fig. 7A). The putative proteins encoded by the cDNA sequences were 58%, 68%, and 59% (CKT1, CKT1R1, and CKT1R2, respectively) identical to CYPT1 and included various specific domains, such as an alanine cluster, a cysteine cluster, RS dipeptides, CK2 phosphorylation sites, and an N-terminal that was rich in basic amino acids (Fig. 7B).



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  		FIG. 7. Comparison of cypt1 and paralogous genes. A) Alignment of the predicted amino acid sequence of CYPT1 and the paralogous gene proteins. Asterisks indicate residues that are identical to CYPT1. Shaded boxes indicate basic amino acids. Alanine and cysteine clusters are indicated in boldface. Arginine-serine dipeptides are underlined. CK2 phosphorylation sites ([S/T]XX[D/E]) are enclosed in boxes. B) Expression of the cypt1 paralogs in mouse testis was analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR). Left margin, molecular size markers (bp). Right margin (arrowheads), amplified genes

Chromosome Localization and Genomic Structure of cypt1 and Paralogous Genes

The genes ckt1, ckt1r1, and cypt1 were mapped to the X chromosome of the mouse genome (X D, X F3, and X A1.2, respectively) and each gene contained two exons (Fig. 8). These three genes could have been derived by gene triplication on the X chromosome. Interestingly, ckt1r2 was mapped to chromosome 9 and lacked introns, suggesting that ckt1r2 had been retrotransposed (Fig. 8).



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  		FIG. 8. Schematic representation of the genomic structure of cypt1, the cypt1 paralogs, and Zfy-2. Numerals indicate exons. The first methionine (Met) and termination (TER) codons are indicated. Open boxes and bars indicate homologous regions

Unexpectedly, the 5'-flanking region and exon 1 of cypt1, ckt1, and ckt1r1 and the corresponding sequence of ckt1r2 was 85–90% homologous to the promoter and exon 1a sequence of Zfy-2 (NCBI accession AH003072) (Fig. 8). In Figure 9, we show the 5'-flanking sequences of cypt1 and the paralogs, as well as the Zfy-2 promoter sequence [31]. The 5'-flanking region of the Zfy-2 and cypt1 family of genes (cypt1, NT_039700; ckt1, NT_039711; ckt1r1, NT_039719; and ckt1r2, NT_039472) contained a pyrimidine-rich initiator (Inr) sequence that encompassed the transcription start site. The Inr sequence CCAGTC conformed to the consensus eukaryotic Inr sequence YYANTYY (Y is a pyrimidine; N is any nucleotide) [32]. In addition, the 5'-flanking region contained a CAT box, which is typically located close to the transcription start site in eukaryotic promoters, and the sequence similar to the consensus cAMP response element (CRE, TGACGTCA), which is an important regulatory element in haploid germ cell-specific genes [33]. A TATA box was found only within Zfy-2 and cypt1.



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  		FIG. 9. Alignment of the Zfy-2 promoter and the 5'-flanking region of cypt1 and its paralogs. Asterisks indicate nucleotides that are identical in each of the five genes. cAMP response element (CRE)-like (CRE consensus: TGACGTCA), CAAT, and initiator (Inr, consensus: YYANYY) sequences are enclosed in boxes


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we identified a novel gene, cypt1, which encodes a cysteine-rich basic protein that is present within the PT. The full-length cDNA of cypt1 was isolated from a testis cDNA library in pAP3neo using TISP55 as a probe [15], which had been cloned from a subtracted cDNA library of mouse testis that contained only cDNAs that were expressed specifically in haploid spermatids [34]. We found that expression of cypt1 was specific to haploid testicular germ cells and was regulated precisely during the development of male germ cells. We demonstrated that CYPT1 protein was a component of the postacrosomal PT. It has been demonstrated that a monoclonal MN13 antibody labels the postacrosomal PT of the sperm head, and the epitope of this antibody is dispersed within the oocyte cytoplasm during the early stage of fertilization [7, 26]. Our observation that CYPT1 was colocalized with the MN13 epitope in the heads of mildly sonicated sperm suggested that CYPT1 functions within the postacrosomal PT. The postacrosomal PT is thought to be composed of basic proteins [35], and disulfide bonds might contribute to the rigidity of the postacrosomal PT [4]. The properties of CYPT1, including the fact that CYPT1 is basic and cysteine-rich, are consistent with the notion that CYPT1 plays a central role within the postacrosomal PT.

CYPT1 contains multiple CK2 phosphorylation sites. CK2 is expressed ubiquitously and more than 100 substrates have been described for this kinase [36]. The Csnk2a2 gene that encodes the CK2 alpha catalytic subunit is expressed predominantly during the late stages of development of spermatogenic cells, and disruption of Csnk2a2 caused male infertility with oligozoospermia and round-headed spermatozoa (globozoospermia) [37]. A recent morphological analysis of Csnk2a2-deficient spermatids revealed an abnormal dilatation of the posterior region of the NE during the nuclear elongation steps of spermatid development [38], which suggested that the structure of the postacrosomal PT that overlays the posterior NE had been disrupted. As disulfide bonds maintain the structural integrity of the PT, it is possible that cysteine-rich CYPT1 is an important target of CK2 in elongated spermatids. We also found that CYPT1 contained RS dipeptides, which are potential phosphorylation sites for SRPKs. It has been reported that SRPK1 was highly expressed in testis, and that SRPK1 phosphorylated protamine 1 and lamin B receptors [39]. This kinase may also play a crucial role in spermiogenesis, particularly in nuclear formation. We speculate that CYPT1 is likely involved in nuclear shaping during spermiogenesis and that this function is regulated by kinases.

Genomic Structures and Transcriptional Regulation of the cypt1 Gene Family

The cypt1 gene has three paralogs in the mouse genome, each of which we found to be transcribed in adult testis. Moreover, the reading frame was conserved in each gene, despite the insertion/deletion of nucleotides. These observations suggested that cypt1 and the three paralogs are functional and biologically important. The cypt1 and two of the paralogs (ckt1 and ckt1r1) were mapped to the X chromosome, and each of these genes contained two exons. The ckt1r2 was mapped to chromosome 9 and had no introns, which suggested that this gene had been retrotransposed from the X-linked gene (although it is difficult to determine which gene was the progenitor of ckt1r2 based only on nucleotide sequences). All proteins encoded by these genes have the same properties as CYPT1, i.e., they are rich in basic amino acids and contain cysteine clusters. Although it is generally accepted that the mRNA and protein produced during spermiogenesis from genes that are located on sex chromosomes are distributed to all spermatids via intercellular bridges, the copy number of cypt1 (the ancestral gene) might have increased on the X chromosome and might have been retrotransposed to an autosome. Autosomal gene expression was presumably sufficient to compensate during the late stages of spermiogenesis for the absence of the full complement of X chromosome gene products in elongated spermatids, which have a Y chromosome.

Interestingly, the 5'-flanking region and exon 1 of cypt1 and the paralogs (including the corresponding region of intronless ckt1r2) were highly homologous to the promoter and exon 1a sequences of Zfy-2. There are multiple genes that are related to Zfy-2: in mice, there are two Y-linked copies (Zfy-1 and Zfy-2), an X-linked copy (Zfx), and an autosomal copy (Zfa) that was retroposed from the X-linked gene. In a comparison of the exon/intron structure of these genes, Zfy-1 and Zfy-2 appeared to have diverged by gene duplication [40]; exon 1a and the promoter were present only in Zfy-2. It was reported that Zfy-2 was expressed predominantly in germ cells of adult testis [41, 42], which resembles the expression profile of the cypt1 gene family, but differs from the expression profile of Zfy-1, which is limited to embryonic germ cells. The 5'-flanking sequences of these testis-specific genes appear to have the same transcriptional regulation mechanism. In 1994, Zambrowicz et al. [43] characterized the Zfy-2 promoter and found that it was expressed exclusively in spermatids. They used a lacZ-linked transgene in their study of Zfy-2, but they used the 5'-flanking region of ckt1r2 to generate transgenic mice. All of these genes contained the Inr sequence. In eukaryotic promoters that lack a TATA box, the Inr element is sufficient for accurate transcription initiation [32]. The cypt1 genes and Zfy-2 promoters share several identical cis sequences that are recognition sites for transcription factors. These 5'-flanking regions usually contain cAMP-responsive element (CRE)-like sequences. The cognate transcription factor CRE modulator tau is essential for the transcription of several testis-specific genes that have a CRE sequence in the proximal promoter [33]. Because the 5'-flanking region of cypt1 and the paralogs that we examined in this study have the aforementioned features, it is reasonable to conclude that these regions are promoters. Note that the retrotransposed ckt1r2 also has the Zfy-2 promoter-like sequence in the 5'-flanking region. The existence of the original gene promoter within the retrotransposed gene suggests that the ancestral gene has the same region as an exon, but we failed to identify such an ancestral gene in the mouse genome. Perhaps some promoters of the retrotransposed gene were derived from the 5'-untranslated region of the ancestral gene [44].

In summary, our results suggest that cypt1 and three paralogous genes comprise a novel haploid germ cell-specific gene family. These genes encode postacrosomal PT proteins that are involved in nuclear shaping in spermatids, which is crucial both for spermiogenesis and fertilization. Further studies of the molecular mechanisms of nuclear shaping in spermatids should provide insight into male infertility that is characterized by globozoospermia.


   ACKNOWLEDGMENTS
 
We are grateful to Hiromi Nishimura for excellent technical assistance. We thank Drs. K. Toshimori and M. Maekawa for kindly supplying us with monoclonal antibody MN13 and valuable comments.


   FOOTNOTES
 
2 Current address: Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Back

1 Correspondence: Yoshitake Nishimune, Research Institute for Microbial Disease, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp Back

Received: 3 June 2004.

First decision: 22 June 2004.

Accepted: 22 July 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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