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. 2011 Jan;30(1):9–16. doi: 10.1089/dna.2010.1048

Localization and Characterization of Rat Transmembrane Protein 225 Specifically Expressed in Testis

Shirui Yang 1,,*, Weiping Wang 1,,*, Chen Lei 1, Qingmei Liu 1, Fengqin Xu 1, Xiaowei Xing 2, Hao Chen 1, Jiajia Liu 1, Shiliang Wu 1, Minghua Wang 1,✉
PMCID: PMC3020359  PMID: 20979528

Abstract

Testis is the one and only location of spermatogenesis and sexual hormone production. Spermatogenesis is a complicated physiological process regulated by many genes specifically and differentially expressed in the testis. In this study, Transmembrane Protein 225 (TMEM225), which is specifically expressed in rat testis, has been identified. TMEM225 was cloned from the testis cDNA library and was mapped to chromosome 8q22 by browsing the University of California Santa Cruz genomic database. It contains an open reading frame with a length of 696 bp, encoding a protein with four putative transmembrane helices. TMEM225 mRNA expression was evaluated by reverse transcription–polymerase chain reaction and in situ hybridization. In addition, the subcellular location of TMEM225 was evaluated. The results obtained highlighted age related specific expression of TMEM225 in testis, specifically during the adult period after age of 13 months. In situ hybridization analysis indicated that TMEM225 mRNA was mainly expressed in spermatocyte cells and round spermatids. Green fluorescence protein localization analysis showed that rat TMEM225 mainly surrounded the nuclear membrane, with a minority distribution in the cytoplasm, and the distribution of TMEM225 was affected by the deletion of N-terminal transmembrane domain. As the expression phase is not related to the first wave of spermatozoon development, our data presented here suggest that TMEM225 may play an important role in sperm degeneration but not in spermatogenesis.

Introduction

Atransmembrane protein is a protein that spans the entire biological membrane one or a few times. To date, hundreds of transmembrane proteins have been identified (Aruga and Mikoshiba, 2003; Haugstetter et al., 2005; Schweneker et al., 2005). Tetraspans are a group of membrane proteins with four transmembrane domains, which span the membrane four times. Tetraspans transmembrane family members, also known as tetraspanin superfamily, have various effects on cell proliferation, motility, and adhesion in different cells (Morita et al., 1999; Christophe-Hobertus et al., 2001; Hulett et al., 2001; Mazzocca et al., 2002; Schweneker et al., 2005). For example, TM4SF protein was found to form massive tetraspanin-enriched microdomains and functions collaboratively with integrins in cell adhesion and motility (Mazzocca et al., 2002). Tight junctions are very important for barrier function in epithelial and endothelial cells, and four-transmembrane helices protein Claudin11 was found to be a tight junction protein at the human blood–testis barrier (Fink et al., 2009).

Spermatogenesis and sexual hormone production only exist in testis. Spermatogenesis is a complicated physiological process including cell division (Eberhart and Wasserman, 1995), differentiation, and interactions between cells in the seminiferous tubule (Takenaka et al., 2008). A large number of transmembrane proteins have been reported to be involved in spermatogenesis (Endo et al., 1996; Cheng et al., 2003; Hyenne et al., 2007; Sze et al., 2008). Recently, many testis-expressed transmembrane proteins have been discovered. Testis-specific expression protein TETM4 has four strong transmembrane helices and was suggested to be associated with receptor complexes on the surface of specific cells in the testis and participated in signaling events (Hulett et al., 2001). Sdmg1, a conserved transmembrane protein, was found to be associated with germ cell sex determination and germline–soma interactions in mice (Best et al., 2008). Degenerative spermatocyte, a novel gene encoding a transmembrane protein, may be required for the initiation of meiosis in spermatogenesis (Endo et al., 1996). Membrane protein Tmem184a expressed exclusively within the Sertoli cells of the developing testis cords was reported to mediate sex-specific signaling events during Sertoli cell differentiation (Svingen et al., 2007).

Despite the fact that the complete nucleotide sequence of the human genome is known, many genes encoding membrane proteins were continuously discovered. So far, some membrane proteins remain unidentified. Using bioinformatics and an experiment validation method, we have identified a novel transmembrane protein TMEM225, which is specifically expressed in rat testis. The TMEM225 protein is composed of four transmembrane helices. In the present study, reverse transcription–polymerase chain reaction (RT-PCR) in situ hybridization analysis, and green fluorescence protein (GFP) subcellular localization were employed to delineate the function of TMEM225. The results obtained highlighted age-related specific expression of TMEM225 in testis, specifically during the adult period after age of 13 months. In situ hybridization analysis indicated that TMEM225 mRNA was mainly expressed in spermatocyte cells and round spermatids. GFP localization analysis showed that rat TMEM225 mainly surrounded the nuclear membrane, with a minority distribution in the cytoplasm, and the distribution of TMEM225 was affected by the deletion of N-terminal transmembrane domain. The data presented here suggest that TMEM225 may play an important role in sperm degeneration but not in spermatogenesis.

Materials and Methods

Cloning of rat gene TMEM225

BLAST search at University of California Santa Cruz (UCSC) genome browser (http://genome.ucsc.edu) with the reported mouse unnamed protein (GenBank accession number: BAB24641) allowed us to identify a 2.6-kb homologous region in rat genome, in which no known protein was identified. According to the genomic DNA sequence in this region, primers (sense: 5′-GAT GGT AGT CTC TGG TAGA-3′; antisense: 5′-TGC AGA CCA TCA AGT CAC AG-3′) were designed and synthesized (Biocolour BioTech). PCR was performed with adult rat testis cDNA as template. The PCR conditions were as follows: 94°C for 5 min, 35 cycles of 20 s at 94°C, 30 s at 56.5°C, and 50 s at 72°C, followed by a final extension of 5 min at 72°C. The PCR product was subjected to T-A cloning and was sequenced on an ABI PRISM sequencer.

In silico analysis

To determine the mapping information, the cDNA sequence of TMEM225 was applied for genomic searching at http://genome.ucsc.edu. BLASTP tool (www.ncbi.nlm.nih.gov) was applied for identifying the orthologs of TMEM225 in different species. The SMART tool was used for domain searching (http://smart.embl-heidelbeg.de), and the Vector NTI package (Informax) for protein molecular weight prediction. PSORT (http://psort.nibb.ac.jp) was used for subcellular localization prediction. The HMMTOP server (www.enzim.hu/hmmtop) was used for the prediction of the transmembrane helices and topology. Vector NTI software was used for protein sequence alignment analysis. The GenBank accession numbers used for analysis were NP_001019530 (rat), NP_001013765 (human), XP_001137001 (chimpanzee), XP_001108697 (monkey), BAB24641 (mouse), XP_852701 (dog), NP_001073753 (cow), and XP_001501682 (horse).

Expression pattern analysis of TMEM225

The expression pattern of TMEM225 was determined by RT-PCR (Huang et al., 2004). Total RNA was extracted from eight different tissues (lung, liver, ovary, spleen, testis, heart, kidney, and skeletal muscle) with a single-step isolation method using TRIzol reagent according to the manufacturer's instructions. The cDNA was synthesized using 2 μg of total RNA, Superscript II reverse transcriptase (Gibco BRL), and Oligo (dT) 15 (Promega) as per the manufacturer's protocols. The 423-bp product was amplified on an FS-918 DNA Amplifier (Shanghai Fusheng Institute of Biotechnology) using the primer pair TMEM225-F (5′-ATA AAG TTA CCC ACA GTC C-3′) and TMEM225-R (5′-TCA TTG CTT TGC TGC TAC-3′). The PCR conditions were as follows: 94°C for 2 min, 35 cycles of 30 s at 94°C, 30 s at 52°C, and 40 s at 72°C, followed by a final extension of 5 min at 72°C. To determine age-related expression of TMEM225 in rat testis, RNA were also extracted from rat testis at different ages (1 day, 15 days, 35 days, 65 days, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, and 18 months). Using the primer pair of TMEM225-RT-A (5′-ATG ATG CGC ATT CCA AAC AGA A-3′) and TMEM225-RT-B (5′-TCA CAG AGC CCA GGT CAC ACG A-3′), PCR product size was 696 bp. The PCR conditions were as follows: 94°C for 2 min, 35 cycles of 30 s at 94°C, 30 s at 53°C, 50 s at 72°C, followed by a final extension of 5 min at 72°C. The expression of the β-actin was analyzed as a control (sense primer: 5′-GGC CCC TCT GAA CCCT AAG-3′; antisense primer: 5′-TGC CAC AGG ATT CCA TAC CC-3′). The PCR conditions were the same as for TMEM225 except that the annealing temperature was changed to 58°C. PCR product size was 500 bp.

In situ hybridization

The mRNA expression of TMEM225 in rat testis was determined by in situ hybridization technique (Xing et al., 2004). Sprague–Dawley rats (6 and 14 months) were sacrificed and rat testis tissues were fixed in ice-cold 4% paraformaldehyde for 2 h and then soaked in 40% sucrose overnight. The tissues were frozen at −20°C and 30-μm sections were cut. TMEM225 in situ hybridization kit was purchased from Boster Bioengineering. As longer probes were difficult to penetrate the cell, short oligonucleotide probes were used in this study. To strengthen the signals, three probes were used. The sequences of oligonucleotide probes were as follows: (1) 5′-CCA CAG TCC ATG GCT GGG ATG CTG TCC TCC TTT-3′; (2) 5′-GCA GTT CAC CTA CAT GAT TTC TCA AAA TAA GTG TG-3′; (3) 5′-AAC AGA TGT GCG TGC ATG AAA TTC TGT GTA CCC CA-3′. The probes were labeled with digoxigenin (Boster Bioengineering). Prehybridization was performed for 3 h. Subsequently, hybridization was performed in a solution that contained digoxigenin-labeled TMEM225 probes. The control sections were treated with the solution without TMEM225 probes. Posthybridization washes were performed at 37°C by incubation in 2 × SSC twice (each for 5 min), in 0.5 × SSC for 15 min, and in 0.2 × SSC twice (each for 15 min). Sections were incubated in blocking buffer for 30 min at 37°C and then incubated with biotin-labeled antidigoxigenin antibody at 37°C for 2 h. Slides were washed in 0.5 M PBS (pH 7.2–7.6) four times, each for 5 min. Sections were incubated in SABC at 37°C for 20 min. Slides were washed in 0.5 M PBS (pH 7.2–7.6) three times, each for 5 min. After that, sections were incubated in biotined peroxidase at 37°C for 20 min. Slides were washed in 0.5 M PBS (pH 7.2–7.6) four times, each for 5 min. The specific signals were visualized by incubation in diaminobenzidine buffer. Water was used to end the reactions. Sections were redyed with hematoxylin and photographs were taken. Negative controls were subjected to the same conditions as above.

Analysis of subcellular localization

The PCR product of the complete TMEM225 open reading frame (ORF) with XhoI and EcoRI restriction sites was subcloned into the XhoI/EcoRI sites of pEGFP-C1 (Clontech) and the recombinant green fluorescence expression vector pEGFP-TMEM225 was produced. Restriction sites had been introduced by the forward (5′-ATC TCG AGC AAT GAT GCG CAT TCC-3′) and reverse (5′-ATG AAT TCA GTC ACA GAG CCC AGG-3′) primers. The N-terminal 35 amino acids–deleted mutant pEGFP-D35-TMEM225 was also constructed by PCR using the forward (5′-ACC TCG AGA ATT GAT TCC CAA AGTG-3′) and reverse (5′-TGA ATT CAG TCA CAG AGC CCA GG-3′) primers. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin at 37°C in a 5% humidified CO2 atmosphere. Cells were cultured directly on glass coverslips in six-well plates at a density of approximately 2×105 cells per well and incubated for 24 h. The incubation was continued for another 3 h in the presence of the Superfect Reagent (Qiagen), 1.7 μg pEGFP-TMEM225 or pEGFP-D35-TMEM225, and pEGFP-C1 green fluorescence protein expression vector as a control. The medium was replaced by 10% charcoal-treated fetal calf serum (DCC; Hyclone). The cells were incubated for 48 h and then washed three times with PBS (pH 7.4). The HeLa cells were fixed in 3% paraformaldehyde for 20 min and then permeabilized with 0.5% Triton X-100 in PBS for 5 min. After rinsing with PBS, DAPI (4′,6-diamidino-2-phenylindole; 2.5 μg/ml; Invitrogen) in PBS was used to stain the nucleus for 30 min and visualized with a microscope equipped with fluorescence optics (Leica DM R2).

Results

Rat TMEM225 gene and its genomic organization

Mouse unnamed protein (GenBank accession number: BAB24641) was found to be transcribed from the testis, so the rat testis cDNA was used as a template for PCR amplification. A single band with a size of about 696 bp was excised from the agarose gel, cloned into pMD18-T vector (TaKaRa), and then subjected to sequencing. The sequence was submitted to GenBank with the accession no. AY634367 and with the name PMP22CD, but its homologs in human and mouse were renamed as TMEM225 because it was not related to the claudin family. So we also used the gene name TMEM225 for rat PMP22CD. TMEM225 cDNA encodes a protein with 231 amino acids (Fig. 1). The molecular weight and isoelectric point are predicted to be 26.7 kDa and 9.45, respectively, using Vector NTI software. It is composed of four exons and three introns (Table 1), the open reading frame is from 119 to 814 nucleotides, the ATG start codon (nucleotides 119–121) is preceded by an in-frame stop codon TAG, and there is not any typical polyadenylation signal.

FIG. 1.

FIG. 1.

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Nucleotide sequence and deduced protein sequence of the rat TMEM225 gene. TMEM225 encodes a polypeptide of 231 amino acids; amino acids are identified by their one-letter code; nucleotides are numbered at the left side of each line. TMEM225, transmembrane 225; *, the termination codon TGA; TAG, in-frame stop codon.

Table 1.

Genomic Structure of Transmembrane 225 Gene

3′ Splice acceptor Exon Size (bp) 5′ Splice donor Intron Size (bp)
cDNA end CGACGCTCCG 1 299 TGGCCTGAAGgtcagaacta 1 684
ttcccaacagAGAGCCTGGA 2 147 TTCTTCACAGgtagcttcct 2 270
tgaacactagGTTGCCTTTT 3 135 CTTACCTGTGgtaagtatcc 3 695
ctgtccctagGTATCTTCAG 4 329 GGAAAGATTC aaaaaaaaa PolyA  
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Exon sizes are given in bp and the exonic and intronic sequences at the splice junction are shown in capital and lowercase letters, respectively. The exon–intron splicing signals gt and ag are in bold.

In silico analysis of TMEM225 gene

TMEM225 was mapped to rat chromosome 8q22 by browsing the UCSC genomic database (http://genome.ucsc.edu). Another interesting finding was that the gene TMEM225 and 41 olfactory receptor genes (Olr1301 to Olr1341) are located closely to each other and cluster together in the same chromosome loci rat 8q22. The use of putative transmembrane domain prediction software (HMMTOP) revealed that the TMEM225 protein is composed of four alpha-helical transmembrane domains (TM-1, TM-2, TM-3, and TM-4) of 20, 23, 20, and 20 amino acids, respectively, N- and C-terminal cytoplasmic domains of 10 and 71 amino acids, respectively, two intracellular loops of 39 and 20 amino acids, and a short extracellular loop of eight amino acids (Fig. 2). The HMMTOP analysis also revealed that the N-terminus is outside of the membrane, the first loop of TMEM225 is longer than the second one, and the TMEM225 contains a very short N-terminus tail, but a more than 71-residue-long C-terminus. A BLASTP search of published protein databases of NCBI for sequences similar to that of TMEM225 indicated that it had orthologs in human, chimpanzee, monkey, dog, mouse, cow, and horse. Multiple alignment analysis was made by Vector NTI. Figure 3 showed that TMEM225 was highly conservative in different species: there were six serine residues and seven leucine residues highly conserved in all the species, and five amino acids block PRSIV and five amino acids block VTWAL were highly conserved in its C-terminus. Rat TMEM225 shares homology with the orthologs in human (43% identity and 64% similarity), chimpanzee (44% identity and 64% similarity), monkey (43% identity and 63% similarity), dog (45% identity and 65% similarity), mouse (83% identity and 91% similarity), cow (41% identity and 61% similarity), and horse (40% identity and 62% similarity).

FIG. 2.

FIG. 2.

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Predicted structure of TMEM225 in the membrane. The structure was drawn based on the prediction of HMMTOP. TMEM225 shows four transmembrane domains with both the N- and C-termini extracellularly and a long C-terminal domain.

FIG. 3.

FIG. 3.

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The sequence alignment of rat TMEM225 protein and its orthologs. The sequences used for alignment include those of rat (NP_001019530), human (NP_001013765), chimpanzee (XP_001137001), monkey (XP_001108697), mouse (BAB24641), dog (XP_852701), cow (NP_001073753), and horse (XP_001501682).

Age-dependent specific expression of TMEM225 in rat testis

RT-PCR was used to determine the expression pattern of the gene. To investigate the tissue distribution of rat TMEM225, RNA from eight different tissues including lung, liver, heart, testis, skeletal muscle, kidney, and ovary were extracted and reverse transcribed into cDNA and then were analyzed by PCR. The tissue distribution pattern of TMEM225 mRNA was shown in Figure 4. Among the tissues analyzed, TMEM225 expression was detected only in the testis. It suggested that TMEM225 was specifically expressed in testis. To determine whether the expression of TMEM225 was age dependent, testis from rats of different ages were analyzed by RT-PCR. Figure 5 shows that TMEM225 began to be expressed in adult rat testis around 13 months of age.

FIG. 4.

FIG. 4.

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Expression pattern of TMEM225 in eight rat tissues.

FIG. 5.

FIG. 5.

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Analysis of age-dependent expression of TMEM225 in rat testis by RT-PCR. M: 100-bp DNA ladder; lanes 1–17: 18-month-, 15-month-, 14-month-, 13-month-, 12-month-, 11-month-, 10-month-, 9-month-, 8-month-, 7-month-, 6-month-, 5-month-, 4-month-, 65-day-, 35-day-, 15-day-, 1-day-old rat, respectively. The expression of β-actin was analyzed as a control.

Analysis of TMEM225 mRNA expression in rat testis by in situ hybridization

To further investigate the cell distribution of TMEM225 mRNA in testis, testis from rats aged 6 and 14 months were analyzed by in situ hybridization. In Figure 6, the presence of TMEM225 mRNA is indicated by brown staining of cytoplasm of spermatocyte cells and round spermatids, whereas no obvious positive signals were detected in negative control sections. The expression of TMEM225 mRNA was only detected in rats aged 14 months but not in 6-month-old rats (data not shown).

FIG. 6.

FIG. 6.

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In situ hybridization of TMEM225 mRNA in 14-month-old rat testis tissue. The TMEM225 mRNA was detected in spermatocyte cells and round spermatids of 14-month-old rat testis, using oligonucleotide probes. The presence of TMEM225 mRNA was revealed by brown staining (A, B). No significant brown signals were detected in the seminiferous tubules of controls (C, D). The cell nucleus was stained purple with hematoxylin. (A) Fourteen-month-old rat testis under low-power lens; (B) 14-month-old rat testis under high-power lens; (C, D) negative control under low- and high-power lens, respectively. Color images available online at www.liebertonline.com/dna.

TMEM225 protein expression and its subcellular location

To investigate the subcellular location of our interested protein, the PSORT prediction tool and signalP3.0 server were used. The subcellular location prediction results showed that TMEM225 was localized in the cytoplasm, and there was a high potentiality of localization in the endoplasmic reticulum. The signal sequence prediction results displayed that the 1–35 amino acids have the high possibility to belong to the signal anchor (data not shown). To verify the subcellular location and the function of N-terminal residues in the location of our interested protein, wild pEGFP-TMEM225 and truncated pEGFP-D35-TMEM225 were transfected into HeLa cells. As shown in Figure 7, when pEGFP-TMEM225 were overexpressed in HeLa cells, we found that the strongest green fluorescence concentrated around the nuclear membrane, and we also observed granular green fluorescence scattering in the cytoplasm and did not detect any fluorescence signal in the cell membrane. In contrast, we observed that the green fluorescence was distributed throughout the cell in the control group, which was transfected with pEGFP-C1. The green fluorescence did not concentrate around the nuclear membrane anymore, when the mutant pEGFP-D35-TMEM225 overexpressed in HeLa cells, and the fluorescence was dispersed in the cytoplasm.

FIG. 7.

FIG. 7.

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Subcellular localization of TMEM225 protein in HeLa cells. (A–C) Cells transfected with pEGFP-C1. (D–F) Cells transfected with pEGFP-TMEM225. (G–I) Cells transfected with pEGFP-D35-TMEM225. (A, D, G) The nucleus of cells were stained with DAPI. (C) Colocalization of A and B. (F) Colocalization of D and E. (I) Colocalization of G and H. Color images available online at www.liebertonline.com/dna.

Discussion

In the present study, we report the isolation and characterization of a novel rat gene TMEM225, which is mapped to chromosome 8q22. It contains an ORF with a length of 696 bp, encoding a protein with four putative transmembrane helices, which are likely to adopt a membrane topology with both the N- and C-termini extracellularly. On the basis of the transmembrane domain prediction, the TMEM225 protein can be divided into the following domains: four transmembrane domains (TM-1, TM-2, TM-3, and TM-4) of 20, 23, 20, and 20 amino acids, respectively, N- and C-terminal cytoplasmic domains of 10 and 71 amino acids, respectively, two intracellular loops of 39 and 20 amino acids, and a short extracellular loop of 8 amino acids (Fig. 2).

Previous studies have shown that most tetra transmembrane proteins locate at the cell membrane or endoplasmic membrane (Namiki et al., 1988; Christophe-Hobertus et al., 2001; Haugstetter et al., 2005; Schweneker et al., 2005). We also determined the localization of TMEM225 in the cell; however, it is not located at the cell membrane. On the contrary, TMEM225 protein is granularly scattered in the cytoplasm and is mainly concentrated around the nuclear membrane (Fig. 7D–F). As the outer nuclear membrane is continuous with the membrane system of the endoplasmic reticulum, TMEM225 protein localization is similar to the proteins targeting into the endoplasmic reticulum. The cellular distribution of TMEM225 was significantly changed by the deletion of the N-terminal transmembrane domain (Fig. 7G–I).

TMEM225 is located at chromosome 8q22. A very interesting finding is that 41 olfactory receptor genes (Olr1301 to Olr1341) are also clustered together at this chromosomal locus. TMEM225 is a four-transmembrane protein. It is surprising that the olfactory receptor protein is also a membrane protein with seven transmembrane helices (Sengupta et al., 1996; Dwyer et al., 1998). Whether TMEM225 and olfactory receptor have a relationship in evolution remains to be investigated. Whether TMEM225 plays a role as a receptor similar to the olfactory receptor in the nuclear membrane remains to be identified in our further studies.

The testis is the location of spermatogenesis and sexual hormone production (Lacy and Petitt, 1970). Spermatogenesis is a complicated physiological process including cell division, differentiation, and interactions between cells in the seminiferous tubule (Wu et al., 2001; Mazzocca et al., 2002; Ohta et al., 2003; Hyenne et al., 2007; Svingen et al., 2007). Some intra- and intercellular events are unique to the testis. These unique functions are likely resulted from the expression of specific genes in the testis. Because spermatogenesis is age dependent, these genes are expressed differentially at various stages of development. Our study results indicated that the transmembrane protein TMEM225 is specifically expressed in the testis, which was supported by the result of bioinformatic analysis showing that all the ESTs of TMEM225 were transcribed from the testis, and the expression is also age dependent, specifically after adulthood (Figs. 4 and 5). Further, the results of in situ hybridization (Fig. 6A, B) showed that TMEM225 is mainly expressed in spermatocyte cells and round spermatids of seminiferous tubules in adult rat testis. There are two phases in which the testis grow substantially, namely, in embryonic and pubertal age. TMEM225 expression is after puberty stage, at which the testes have already been matured, and the expression phase does not match the testis development stage, which suggests that TMEM225 is not related to the development of testis. The first spermatogenesis wave of male rat was at 44 days after birth. But TMEM225 mRNA is only detected after the rat is over 1 year old. This suggests that it may not be required for spermatogenesis. The data presented here suggest that TMEM225 may play an important role in sperm degeneration but not in spermatogenesis.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (no. 30700826) and the Medicine Development Foundation of Soochow University (no. EE134709).

Disclosure Statement

No competing financial interests exist.

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