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BOR - Papers in Press, published online ahead of print January 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026096
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BIOLOGY OF REPRODUCTION 70, 1485–1492 (2004)
DOI: 10.1095/biolreprod.103.026096
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Rosbin: A Novel Homeobox-Like Protein Gene Expressed Exclusively in Round Spermatids

Tohru Takahashi2,3, Hiromitsu Tanaka2, Naoko Iguchi2, Kouichi Kitamura2, Yily Chen2, Mamiko Maekawa4, Hiromi Nishimura2, Hiroshi Ohta2, Yasushi Miyagawa2,3, Kiyomi Matsumiya3, Akihiko Okuyama3, and Yoshitake Nishimune1,2

Department of Science for Laboratory Animal Experimentation,2 Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan Department of Urology,3 Osaka University Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan Department of Anatomy and Developmental Biology,4 Graduate School of Medicine, Chiba University,Chiba 260-8670, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian spermiogenesis is a complex process occurring in a highly coordinated fashion within the seminiferous tubules. To elucidate the molecular mechanisms controlling haploid germ cell differentiation, we have isolated haploid germ cell- specific cDNA clones from a subtracted cDNA library of mouse testis. One of these cDNAs, Rosbin, is 3.2 kilobases (kb) long and has an open reading frame of 2385 nucleotides encoding a putative protein of 795 amino acid residues. A computer-mediated homology search revealed that it contained a domain similar to that of homeobox genes. Northern blot analysis revealed a 3.2-kb mRNA expressed exclusively in male germ cells. Transcription of the Rosbin gene was not observed in prepubertal testis but became detectable after Day 23. By Western blot analysis the protein encoded by this gene had a molecular mass of 89 kDa, expressing specifically in the testis and localized to the nucleus of stages IV–VIII haploid round spermatids, predominantly at stages VII–VIII of spermatogenesis. ROSBIN is associated with and is most likely phosphorylated by protein kinase A. We suggest that it plays an important role in transcriptional regulation in haploid germ cells.

testis, developmental biology, gene regulation, spermatid, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During mammalian spermatogenesis, spermatogonial stem cells proliferate and differentiate through a highly specialized and complicated process. This process occurs in the seminiferous tubules and can be divided into three phases: a mitotic phase, in which spermatogonia proliferate and differentiate; a meiotic prophase, in which spermatocytes undergo reduction divisions; and a differentiation phase, called spermiogenesis, in which biochemical and morphological changes take place in haploid spermatids. Then the formation of mature spermatozoa, including acrosome and flagellum formation, nuclear condensation, and cytoplasmic elimination, is complete [1]. This process of undifferentiated spermatogonia into differentiated spermatozoa is dependent on stringent temporal and stage-specific gene expression. As the differentiation of haploid cells can only be observed during this process, our interest is especially focused on the mechanism of haploid germ cell differentiation (spermiogenesis). Transcriptional regulation in spermiogenesis is divided into at least two categories [2]. The first is the chromatin remodeling, where somatic histone is replaced by protamine, resulting in a highly condensed nucleus and cessation of gene expression. The other is the binding of transcription factors to specific sequences. Several transcription factors have been demonstrated to be specifically expressed during spermiogenesis. CREM (cAMP- responsive element modulator) has been shown to play important roles in the regulation of spermiogenesis via binding to the sequence of cAMP-responsive element (CRE). Mice that are CREM deficient have reduced testis weight and their seminal fluid completely lacks mature spermatozoa. Histological analysis of CREM-deficient mice shows spermatogenesis is arrested at the early round spermatid stage [3, 4]. Thus, CREM is one of the most important transcription factors. However, some proteins specifically expressed in haploid germ cells do not contain CRE motifs in their promotor regions [5], suggesting that other regulatory mechanisms must exist.

To elucidate the molecular mechanisms of spermiogenesis, we isolated many cDNA clones specifically expressed in haploid germ cells using a subtracted haploid germ cell- specific cDNA library [6, 7]. It was generated by subtracting the mRNA from 17-day-old mouse testes, having no haploid germ cells, from the cDNA of 35-day-old mouse testes [7]. From this library, we isolated a novel gene, having both a homeobox and a transcription activation domain, which we named Rosbin. The protein expressed exclusively in haploid round spermatids and localized to the nucleus. The stage-specific expression of ROSBIN during the late round spermatid stage, when transcriptional activity is enhanced, and its localization to the nucleus suggest a role of the ROSBIN protein in gene expression regulation.


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

Experimental animals were purchased from SLC Co. Ltd. (Shizuoka, Japan). All animal experimentations were carried out in our animal facilities and the Animal Experimentation Committee at the Research Institute for Microbial Diseases, Osaka University, approved all experimental protocols.

Cloning, Sequencing, and Identification of Rosbin cDNA

Previously, a haploid germ cell-specific cDNA library was generated by subtracting the mRNA of 17-day-old testes from the cDNA of 35-day- old testes [7]. Haploid germ cell-specific cDNA clones (transcription increased in spermiogenesis, TISP) were isolated from the subtracted library, confirmed for specific expression by Northern blot analysis using testis mRNAs at the ages of 17 and 35 days old, and sequenced. Computer- assisted sequence analysis indicated that TISP41 clone encoded a protein having some basic domains and also a homeobox domain homologous to the zebrafish MSH-D protein (Swiss-Prot accession no. Q01704) [8].

To determine the complete cDNA sequence of TISP41, we recloned the cDNA by screening the adult testis cDNA library of Escherichia coli MC1061A cells [6] seeded at 2 x 105 colony-forming units on nitrocellulose filters placed on Luria broth plates. After incubation at 37°C, colonies were transferred to two nylon replica filters and lysed by sequentially soaking in the following solutions at room temperature: 5 min in 0.5 N NaOH-1.5 M NaCl, 5 min in 0.5 M Tris-HCl (pH 7.4)-1.5 M NaCl, and 5 min in 2x saline-sodium citrate (SSC; 1x SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate). After baking at 80°C for 2 h, the filters were washed and bacterial debris removed. A 32P-labeled probe was prepared by the BcaBEST random primer kit (Takara, Shiga, Japan) using an approximately 1.5-kilobase pair (kbp) EcoRI-NotI fragment of TISP41. The filters were hybridized with the probe in 4x SSC, 10x Denhardt solution, 0.1% SDS, and 100 µg/ml denatured sonicated salmon sperm DNA at 65°C for 20 h. Twenty independent positive clones were isolated by screening 4 x 105 of the colonies and were then sequenced. The inserts of all clones were similar in size (2.4 kbp) (cDNA 1: Fig. 1), but we could not locate the first methionine codon. To identify full-length TISP41, a {lambda}gt11 library of adult mouse testis made by oligo(dT) and random primers (Clontech, Tokyo, Japan) was screened with synthetic oligo nucleotides of Ros 1 (Table 1). Experimental procedures were performed according to the manufacturer's instructions. Thirteen independent clones were isolated and sequenced. The inserts of all clones were of a similar size of approximately 1.1 kbp long (cDNA 2: Fig. 1), and containing almost identical sequences, with some variation at the 3' terminal region. However, none of the clones contained a poly A sequence, but about 400 base pairs (bp) at the 3' end was identical to the 5' sequence of the 2.4-kbp clones. The 1.1-kbp clones (cDNA 2) were supposed to be connected with the first 2.4-kbp clones (cDNA 1) (Fig. 1).



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  		FIG. 1. Schematic presentation of Rosbin cDNA and its cloning procedure. A) Rapid amplification of cDNA ends (RACE) was performed using primer sets for nested PCR. After cDNA 1 was isolated from the adult mouse testis library [6], cDNA 2 was isolated from the random primer {lambda}phage library of mouse testis (Clontech) using synthetic probe Ros 1 (top). To confirm these two cDNA clones had originated from two different parts of one longer mRNA, nested reverse transcription-polymerase chain reaction (RT-PCR) was performed by using various primer sets (A–G) set outside the same 400-bp region. Expected fragment size is shown (bottom). B) PCR products were isolated by agarose gel electrophoresis. Numbers at both margins indicate position of size markers (lane, M). Lane numbers 1–6 correspond to the numbers at the left margin of expectable fragments in A


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  		TABLE 1. Synthetic oligonucleotides used for probe and primers

To confirm that both cDNAs (cDNA 1 and 2) were generated from a whole mRNA of Rosbin, the 5' rapid amplification of cDNA ends (RACE) was performed using poly(A)+ RNA of the testis. Poly(A)+ RNA was isolated by Dynabeads mRNA direct kit (Dynal Biotech, Oslo, Norway) from a C57BL/6 mouse testis in accordance with the manufacturer's instructions. Using primer A and a Rosbin-specific reverse primer (Fig. 1 and Table 1), nested polymerase chain reaction (PCR) was performed using SuperscriptII (Gibco BRL, NY) in accordance with the manufacturer's instructions. The PCR products were separated by gel electrophoresis (Fig. 1). These fragments were extracted and directly sequenced by thermal cycle sequencing. Dideoxy-chain-termination sequencing reactions were performed with fluorescent dye-labeled primers and thermal cycle sequencing kits (Applied Biosystems, Foster City, CA). The reaction products were analyzed using a Genetic Analyzer 3100 (Applied Biosystems). The DDBJ, GenBank, EMBL, Swiss-Prot, and PIR databases were searched for homology with the whole Rosbin cDNA or for the deduced amino acid sequence.

Northern Blot Analysis

The freshly removed organs of adult C57BL/6 mice were homogenized in RNAzol B (Tel-Test Inc., Friendswood, TX). The germ cells and other somatic cells of the testis were prepared as described in our previous report [9]. Total RNA samples were extracted according to the manufacturer's recommendations and quantified by optical density measurement. RNA samples in 2.2 M formaldehyde were subjected to electrophoresis in a 1.1% agarose gel that contained 0.66 M formaldehyde. The RNA bands were transferred to a nitrocellulose filter in 20x SSC. Hybridization was performed by incubating the filter with a 32P-labeled cDNA probe, prepared using the BcaBEST Random Primer Kit (Takara), at 42°C for 16 h in a hybridization solution containing 4x SSC, 5x Denhardt solution, 0.2% SDS, 12 µg/ml denatured sonicated salmon sperm DNA, and 50% formamide. The filters were washed twice in 0.3x SSC plus 0.1% SDS at 60°C. The signals were detected by an Image Analyzer (Fuji Film, Tokyo, Japan).

Antiserum Preparation

A synthetic peptide (N terminal-VFKVESRLDSDQQH, residues 772– 785) designed from the deduced amino acid sequence of the ROSBIN protein was purified (Kitayama Labesu, Nagano, Japan). Polyclonal antiserum was raised by injection of the synthetic peptide followed by booster injections at 3-wk intervals, five times in total, to Japanese white rabbits. Anti-ROSBIN antibody was affinity purified by the synthetic antigen peptide column before use.

Western Blot Analysis

Freshly prepared organs or cell fractions of adult C57BL/6 mice were homogenized on ice in a lysis buffer containing 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% NP-40, and 0.5 ml/L protease inhibitor cocktail (Sigma, St. Louis, MO). After centrifugation, the protein concentration of each supernatant was estimated using the Bradford Protein Assay (Nacalai Tesque Inc., Kyoto, Japan). Each extract containing approximately 50 µg protein was subjected to SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride membrane filters (Millipore, Bedford, MA). The filters were blocked with 5% nonfat milk and washed for 15 min with TBS-T (TBS: 50 mM Tris-HCl [pH 7.5], 150 mM NaCl; and T: 0.05% Tween-20) before being incubated with the anti-ROSBIN antibody (1:200 dilution) in TBS-T at 4°C overnight. The filters were then washed in TBS- T for 5 min, rewashed three times, and incubated at 25°C for 1 h with anti-rabbit immunoglobulins (Ig) conjugated to horseradish peroxidase (1: 1000 dilution) (Amersham Pharmacia Biotech, Tokyo, Japan). After further washing, the reactive bands were visualized by development with the POD Immunostain Kit (Wako, Osaka, Japan).

Immunohistochemistry

To prepare frozen tissue specimens, testes of adult C57BL/6 mice were put into O.T.C. compound embedding medium (TISSUE-TEK, Sakura, Tokyo, Japan) and frozen at –20°C. Frozen sections (8 µm thickness) of the testes were placed onto Micro Glass Slides (Matsunami Glass Ind. Ltd., Osaka, Japan) and fixed with 100% ethanol for 20 min at 4°C. Each section was incubated with blocking solution (Nacalai), reacted with anti- ROSBIN antibody (1:500 dilution) or preimmune rabbit serum as a control and then incubated with FITC-labeled anti-rabbit Ig (1:1000 dilution) (Amersham Pharmacia Biotech). Sections were counter-stained with 4,'6- diamidino-2-phenylindole, dihydrochloride (DAPI) (Nacalai).

Transfection of Cultured Cells with Expression Vectors and Western Blot Analysis

PCR amplification of the Rosbin cDNA coding region was performed by using sense primer RSP (Table 1) for the 5' region of the Rosbin cDNA (nucleotide residues 331–348 bp) fused to a linker (XhoI) oligonucleotide, and primer for the 3' region, RAP (Table 1), of the Rosbin cDNA nucleotide residues 2704–2721 bp fused to a linker (BamHI) oligonucleotide. Amplified products were digested with XhoI and BamHI and ligated at the XhoI and BamHI sites of the mammalian expression vector pEGFP-C1 (Clontech). The resulting clone was capable of expressing the EGFP-ROSBIN fusion protein.

The expression vector of pEGFP-Rosbin was transfected to human embryonic kidney (HEK) 293 cells with LipofectAMINE PLUS reagent (Gibco BRL) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were observed under a fluorescent microscope and then harvested for Western blot analysis. The filters were reacted with anti-ROSBIN antibody (1:200 dilution) or anti-GFP monoclonal antibody (1:300 dilution). Each filter was then incubated with peroxidase-conjugated anti-rabbit Ig (1:1000 dilution) or peroxidase-conjugated anti-rat Ig (1:1000 dilution) (Dako Cytomation Norden A/S, Glostrup, Denmark).

Immunoprecipitation of Endogenous ROSBIN Proteinand In Vitro Kinase Assay

HEK-293 cells transfected with the expression vector pEGFP-Rosbin and mouse testes were lysed with TBS-T with 0.5 ml/L protease inhibitor cocktail (Sigma), and the lysates were centrifuged at 10 000 rpm for 10 min at 4°C. The supernatants were treated with protein G-Sepharose beads prewashed with TBS-T at 4°C for 1 h to eliminate nonspecific binding materials. Preimmune normal rabbit serum or specific anti-ROSBIN antibody was added to the testicular lysate at 1:500 dilution, and anti-GFP antibody (Living Colors Full-Length A.v. Polyclonal Antibody; Clontech) was added to the lysate of transfected HEK-293 cells at 1:500 dilution, and the samples were rotated overnight at 4°C. The samples were incubated with protein G-Sepharose beads at 4°C for 1 h and centrifuged.

Prewashed protein G-Sepharose beads reacted with testis lysate were subjected to Western blot analysis. The filters were then reacted with anti- ROSBIN antibody or anti-PKA{alpha} cat (PKA) antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

The lysate of transfected HEK-293 cells incubated with prewashed protein G-Sepharose beads was washed three times with TBS-T and then two times with kinase assay buffer (40 mM HEPES [pH 7.4], 10 mM MgCl2, 3 mM MnCl2, 5 mM CaCl2, and 150 mM NaCl) and incubated at 37°C for 10 min in 40 µl of kinase assay buffer with 1 µl ATP (1 mM) and 0.5 µl cAMP-dependent protein kinase (Promega, Tokyo, Japan). The samples were subjected to SDS-PAGE and filters were reacted with anti- ROSBIN antibody (1:500 dilution), anti-GFP monoclonal antibody (1:300 dilution), or anti-phosphoserine rabbit antibody (1:500 dilution) (Zymed Laboratories Inc., South San Francisco, CA). The filters reacted with each of anti-ROSBIN, anti-PKA, or anti-phosphoserine antibody were then incubated with anti-rabbit Ig conjugated with horse-radish peroxidase (1: 1000 dilution) (Amersham Pharmacia Biotech). The filter incubated with anti-GFP monoclonal antibody was incubated with anti-rat Ig conjugated with horse-radish peroxidase (1:1000 dilution) (Dako Cytomation Norden A/S). After further washing, the reactive bands were visualized by development with the POD Immunostain Kit (Wako).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and Characterization of cDNA EncodingRosbin Protein

We have isolated a haploid germ cell-specific cDNA clone designated TISP41 from a subtracted cDNA library of mouse testis [7]. To isolate the full-length cDNA of the clone, an adult mouse testicular cDNA library of pAP3neo [6] together with the random primer {lambda} phage library (Clontech) were screened with appropriate probes described in the Materials and Methods. We isolated two clones of 1.1- kbp and 2.4-kbp insert having 400 bp of identical sequence at the 3' end of the former and at the 5' end of the latter clones (cDNA 1 and 2 in Fig. 1). Because Northern blot analysis with the 1.1-kbp fragment as a probe showed just a single band of approximately 3.2 kb, the same as the blot using the 2.4-kbp probe, the 1.1-kbp clone was supposed to be the 5' part of the complete TISP41 cDNA connected with the 2.4-kbp clone. To identify the full-length sequence and to ensure both cDNAs, 1.1 kbp and 2.4 kbp, were generated from a TISP41 cDNA, we performed 5' rapid amplification of cDNA ends (RACE) using synthetic oligo primers designed to produce products containing the same 400-bp region (Fig. 1 and Table 1) and poly(A)+ RNA isolated from the testis of a C57BL/6 mouse. As shown in Figure 1, the size of the PCR products were 1020, 515, 634, 740, 695, and 576 bp from the nested PCR reactions. Whole products were extracted from agarose gel and directly sequenced. Sequence analysis showed the two cDNA fragments were produced from a single transcribed product.

The deduced amino acid sequence of the cDNA is shown in Figure 2 (DDBJ accession no. AB101658). We presumed that the ATG located at nucleotide position 334 was the translation initiation codon of the cDNA because a stop codon was located at nucleotide position 280, 54 bases upstream from the ATG. The presumed open reading frame was from positions 334 to 2718, encoding 795 amino acid residues. A putative polyadenylation signal, AATATAA, was located at nucleotide position 2941–2947. The poly(A) tail started at position 2955. The deduced amino acid sequence had four basic regions located at amino acids 112– 116, 183–197, 252–263, and 550–553. We named this gene Rosbin (round spermatid basic protein). The amino acid sequence at 252–263, KKIKKKKKKKHR, would be a nuclear localization signal (Fig. 2A). The sequence also contained four cAMP-dependent phosphorylation sites at 88– 91, 114–117, 233–236, and 551–554. The genomic DNA of Rosbin was mapped to the F2.2 region of the mouse chromosome 3, using the NCBI BLAST the Mouse Genome program (http://www.ncbi.nlm.nih.gov/genome/seq/ MmBlast.html). It compared the alignment with Rosbin cDNA, and it consists of eight exons. By a computer-mediated homology search, we found ROSBIN had domain homology to the MSH-D homeobox of zebrafish at residues 680–739 (22% identity and 48% similarity) (Fig. 2, A and B). The amino-terminal regions of the protein had no homology to any homeobox-containing genes but showed a proline-rich domain (38% proline) at 102–164 (Fig. 2A). The proline-rich domain localized at the amino-terminal region could either function as a transcriptional regulator or interact with other proteins required for the efficient selection of a DNA target site [10, 11]. Furthermore, the deduced amino acid sequence analysis revealed a high homology with an unknown human putative protein translated from a cDNA (DDBJ accession no. AK002082-1) and we tentatively called it h-ROSBIN (Fig. 2A).



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  		FIG. 2. A) Comparison of deduced amino acid sequences between mouse and human ROSBIN. Stars under the sequences indicate identical amino acids in both mouse and human. Gaps are introduced to maximize the alignment. Shaded regions indicate basic domains (residues 112–116, 183–197, 252–263, and 550–553) and the overlined sequence is the proline-rich domain (residues 102–164). Underlined is the putative homeobox domain (residues 680–739). Open boxes indicate the cAMP-dependent protein kinase (PKA) phosphorylation sites (residues 88–91, 114–117, 233–236, and 551–554), and the shaded box (residue 772–785) indicates the cDNA region used for preparation of anti-ROSBIN antiserum. B) Comparison of the homeobox domains of ROSBIN and MSH-D of zebrafish. Each letter between the two sequences indicates the identical residues and + indicates conserved residues

Expression of Rosbin mRNA

Using the full-length Rosbin cDNA as a probe, Northern blot analysis showed that Rosbin was specifically expressed in the testis as a major transcript of 3.2 kbp but was not detectable in organs such as the brain, heart, intestine, kidney, liver, lung, muscle, ovary, or spleen (Fig. 3A). To examine the developmental changes in Rosbin transcription in the mouse testis, total testicular RNAs at the ages of 2– 5, 8, 11, 14, 17, 23, 29, and 35 days and adult older than 2–3 mo were analyzed. Rosbin mRNA was not found at the age of 17 days but was abundantly detected on and after the age of 23 days (Fig. 3B). Furthermore, it was expressed exclusively in germ cells (Fig. 3C).



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  		FIG. 3. Northern blot analyses of Rosbin gene. The expression of Rosbin mRNA was examined in various organs by using full-length Rosbin cDNA as a probe (A). Expression of Rosbin mRNA at different developmental ages of the testes. The RNA samples of mouse testes at ages 2–5, 8, 11, 14, 17, 23, 29, and 35 days and A, adults older than 2 mo were analyzed (B). Expression of Rosbin mRNA in fractionated testicular cells (T, whole testis; G, germ cell fraction; S, Sertoli cell fraction; L, Leydig cell fraction) (C). The positions of the 28S and 18S ribosomal RNAs are indicated in the right margin. Signals rehybridized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA are shown as controls

Expression and Localization of ROSBIN Protein

Western blot analysis showed a specific band of 89 kDa in the testis with anti-ROSBIN antibody (Fig. 4A). To investigate the developmental changes in translation in the mouse testis, total protein extracts of testis at the ages of 2, 3, and 4 wk were analyzed. The ROSBIN protein signal was detected in the testis extracts from the age of 3 wk (Fig. 4B). Considering that the expression of Rosbin mRNA began after the age of 17 days, Rosbin protein was begun to express on a day between the age of 17 and 21 days.



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  		FIG. 4. Western blot analyses with anti-ROSBIN antibody. Protein samples were extracted from various organs of an adult mouse (B, brain; H, heart; In, intestine; K, kidney; Li, liver; Lu, lung; M, muscle; S, spleen; T, testis; Sp, sperm) (A), and from the testes at various ages (2W, 2 wk; 3W, 3 wk; 4W, 4 wk) (B). Molecular markers are indicated at the left margin

Immunohistochemical analysis of frozen sections of an adult mouse testis with anti-ROSBIN antibody revealed that a positive signal existed in germ cell nuclei but not in Sertoli or Leydig cells. The signal was first detected in haploid round spermatids at stages IV–V, then increasing strongly through stages VII–VIII (Fig. 5) but stopped abruptly in elongating spermatids later than stages IX–X. The signal was not detectable at any other stages. These results indicate that ROSBIN protein is specifically expressed in the nuclei of round spermatids predominantly at steps 7 and 8.



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  		FIG. 5. Immunohistochemical analyses of mouse testis with anti-ROSBIN antibody. Sections of an adult mouse testis were immunostained with anti-ROSBIN antibody (A, D, and G) and the same sections were counter stained with DAPI in blue (B, E, and H), respectively, or preimmune rabbit serum (C,). Roman numerals represent stages of seminiferous tubules (B). Cross sections of seminiferous tubules at each stage were examined at higher magnification (D–I). P.S., R.S., and E.S., Pachytene spermatocyte, round spermatid, and elongated spermatid layers, respectively (A, C, D, G: FITC fluorescence; B, E, F: DAPI staining; F and I: merge). Bars = 100 µm

Ectopic Expression of ROSBIN Protein in Cultured Cells

To investigate the effects and subcellular localization of ROSBIN in cultured somatic cells, HEK-293 cells were transfected with the pEGFP-Rosbin fusion gene. Localization of ROSBIN was restricted to a nuclear foci, present in a punctate form discretely in the nucleus (Fig. 6). This distribution pattern is similar to the physiological localization of ROSBIN in haploid round spermatids. After harvesting the transfected cells, we performed Western blot analysis to confirm the expression of ROSBIN protein. A single band reacting with anti-ROSBIN antibody showed the same size as the band reacting with anti-GFP monoclonal antibody at a reasonable position for the fusion protein of ROSBIN and GFP (approximately 116 kDa).



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  		FIG. 6. Ectopic expression of EGFP-ROSBIN fusion protein in transfected cultured cells. Expression vector of pEGFP-Rosbin was transfected into HEK- 293 cells using LipofectAMINE Plus reagent (A and B). The expression vector pEGFP-C1 alone was used as a control (C and D). HEK-293 cells were observed 24 h after transfection using a Leica inverted fluorescent microscope under normal light (B and D) and with Leica fluorescein isothiocyanate filter sets (A and C). Each extract of cultured HEK-293 cells transfected with either pEGFP-Rosbin (lanes: R) or EGFP-C1 (lane: C) was subjected to Western blot analysis using anti-ROSBIN antibody or anti-GFP monoclonal antibody (E). Bar = 50µm

ROSBIN Protein Is Associated with and Phosphorylated by Protein Kinase A

As ROSBIN has four cAMP-dependent phosphorylation sites and a proline-rich domain that could be associated with other proteins, we tested whether ROSBIN can form a complex with and be phosphorylated by PKA. In an immunoprecipitation complex of testicular lysate with anti- ROSBIN antibody, the PKA catalytic domain was coprecipitated (Fig. 7A). Furthermore, an in vitro kinase assay of HEK-293 cells transfected with the expression vector of pEGFP-Rosbin showed that the fusion protein was not phosphorylated by endogenous PKA but by exogenous PKA catalytic protein, demonstrable by antiphosphoserine antibody (Fig. 7B). Both mock-transfected cells (pEGFP- C1 only) and without ATP addition showed no detectable band with antiphosphoserine antibody (data not shown). These results indicate that the ROSBIN protein could be phosphorylated by PKA at its serine residues.



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  		FIG. 7. Association of ROSBIN with protein kinase A (PKA) and in vitro phosphorylation of EGFP-ROSBIN. A) Testicular lysate of adult mouse was immunoprecipitated with anti-ROSBIN antibody, and the immune complex was subjected to Western blot analysis using anti-ROSBIN antibody or anti-PKA antibody (left margin). Lysate indicates mouse testicular lysate. Preimmune and anti-ROSBIN indicate immunoprecipitants with rabbit normal serum and anti-ROSBIN antibody. B) HEK-293 cells transfected with either mock (lane M) or EGFP-ROSBIN (lane R) were analyzed by Western blot analysis using anti-ROSBIN antibody, anti-GFP monoclonal antibody, or antiphophoserine antibody (bottom). Arrows indicate molecular markers at the left margin. The EGFP-ROSBIN band is shown by an arrowhead


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To study the mechanisms of germ cell differentiation, it is vital to isolate and characterize the genes specifically expressed in testicular germ cells. For this purpose, we isolated specific cDNAs developmentally expressed in spermatogenesis using several different methods [6, 7]. One of these was to isolate haploid-germ-cell-specific cDNA clones from a specific subtracted cDNA library. We made the library by subtracting the mRNA of a 17-day-old mouse testes from the cDNA of a 35-day-old testes and achieved the identification and characterization of various genes [12] such as Rosbin. In this article, characterization of ROSBIN showed both a proline-rich domain at the N-terminal and homeobox domain at the C-terminal region. Proline-rich regions are reported to activate transcription when linked to various DNA binding domains [10, 11]. Homeobox domains encompass about 60 amino acids, initially identified in several regulators of Drosophila embryogenesis, and are also found in the genes of many vertebrates. Homeodomain-containing proteins are known to bind to DNA and to act as sequence-specific transcription factors, which regulate the expression of other genes.

Homeobox genes, the expression of which is restricted to specific cell lineages, are of particular interest as candidates to regulate the differentiation of various tissues and cells. Some homeobox genes are reported to be expressed in testicular germ cells. TGIF subclass homeobox gene, Tex1, is specifically expressed in germ cells at the spermatid stage [13]. Pbx4, a Pbx family gene, which is involved in axial patterning and organogenesis, is expressed in the testis specifically in spermatocytes in the pachytene stage of the first meiotic prophase [14]. Paired/Pax family homeobox gene, Tox, expresses in the spermatid and spermatozoa in the adult mouse [15]. In these reports, only the germ cell mRNA expression of these homeobox genes is confirmed and their function in gene regulation is largely unknown.

To evaluate the role of homeobox genes in spermatogenesis, specific gene-deficient mice have been generated. Hox-a4, of the Hox gene family, is expressed at high levels in meiotic and postmeiotic male germ cells, as shown by Northern blot analysis and in situ hybridization [16, 17]. The homozygous knockout (KO) mutant of the Hox-a4 gene is viable and fertile [18]. Nkx6.2, originally named Gtx, has been identified as a novel homeobox gene expressed in brain glial cells and testicular germ cells [19]. The null mutant mice can reproduce normally with a typical litter size of 8–12 pups, which are not distinguishable from their heterozygous and wild-type littermates [20]. Esx1 is an X chromosome-linked homeobox gene, the expression of which is restricted to adult testis and extraembryonic tissues [21, 22]. Esx1 hemizygous mutant males were fertile, demonstrating that Esx1 is not essential for spermatogenesis [23]. In contrast, Sperm 1 (Sprm-1) mutant mice exhibit normal testicular morphology and produce normal numbers of sperm yet displayed subnormal fertility [24]. Sprm-1 is a member of the POU domain family, which is specifically expressed in developing male germ cells immediately before the first meiotic division, and its translate is expressed in haploid spermatids after the meiotic division [24, 25]. A novel protein containing a plant homeodomain motif, POG, is also involved in spermatogenesis, and in the POG-deficient mouse, meiosis is impaired [26]. As the reproduction phenotype of KO mice in various homeobox genes varies widely from normal fertility to severe infertility, the role of these genes in spermatogenesis still requires much elucidation because its regulatory functions are largely unknown.

In contrast with the homeobox genes, mutant mice lacking other genes related to transcription in haploid germ cells demonstrate a global arrest of spermiogenesis. One of these, CREM, acts as a master controller of haploid-specific genes [27]. Its expression is mostly restricted to step 7–8 round spermatids [28]. It has also been reported that components of general transcription machinery are highly expressed in round spermatids [29]. After this step, transition proteins and protamines replace histones, resulting in chromatin condensation, and overall transcription begins to cease. The ROSBIN protein was detected in the nucleus of round spermatids at a very limited number of steps of spermiogenesis, mainly steps 7 and 8 (Fig. 5), similar to the period of CREM expression. Although CREM plays an important role in the regulation of transcription in spermiogenesis, some genes specifically expressed in haploid germ cells do not have any CRE motifs in the promotor region [5]. Thus, some of the transcription in round spermatids must be stimulated by other transcription factors specifically existing in haploid germ cells. CREM in the testis is activated by a testis-specific coactivator, ACT, instead of the phosphorylation of CREM that occurs in the somatic tissue, so that transcriptional activation is PKA independent [30]. However, many transcription factors are phosphoproteins and their functions could be regulated by phosphorylation. PKA expression is ubiquitous and testicular expression is also reported [31].

In the present study, we could not demonstrate the phosphorylation of ROSBIN in the immune complex of Rosbin- endogenous PKA in testicular lysate (Fig. 7A). As ROSBIN associated with PKA and the EGFP-ROSBIN recombinant protein was phosphorylated at ROSBIN, not at EGFP by exogenous PKA (Fig. 7), the function of endogenous ROSBIN is likely to be regulated by PKA. Consequently, ROSBIN may play a role in the cAMP signaling pathway during spermatogenesis and be a candidate for gene regulation in haploid germ cells.

The prevalence of infertility among couples of a reproductive age is approximately 15%, with a roughly equal distribution between the genders [32]. Impaired spermatogenesis accounts for 90% of all male infertility, although the underlying causes are not obvious. Our study has demonstrated that germ cell-specific genes that do not affect somatic cells could be responsible for male infertility when impaired by some mutation or single nucleotide polymorphisms [33]. CREM-deficient mice demonstrated the round spermatid maturation arrest seen in human male infertility cases [3, 32]. Other genes may also be responsible for round spermatid maturation arrest [34–37]. We found a putative human homologue of Rosbin using computer-assisted homology searches (Fig. 2) and believe Rosbin can be nominated as a candidate gene for human male infertility by round spermatid maturation arrest.


   ACKNOWLEDGMENTS
 
We are grateful to Amy S. Herlihy for her technical assistance and critical reading of the manuscript. And we also thank the other members of our laboratory for their help and suggestions.


   FOOTNOTES
 
1 Correspondence: Yoshitake Nishimune, Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. FAX: 81 6 6879 8339, nishimun{at}biken.osaka-u.ac.jp Back

Received: 2 December 2003.

First decision: 15 December 2003.

Accepted: 12 January 2004.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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