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Testis |
Department of Obstetrics and Gynaecology,2
Department of Biochemistry,3
Department of Surgery,4 The University of Hong Kong, Hong Kong, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VCY2 (variable charge, Y chromosome 2; alias BPY2) locates in the frequently deleted AZFc region. It is a testis-specific protein with no active X homologue [7]. The functional role of the gene in spermatogenesis is unknown, but the genomic structure of VCY2 has been characterized [8]. VCY2 consists of eight exons, with the first ATG codon located within exon 4 and the stop codon within exon 8. Only one functional copy of VCY2 is found in the frequently deleted DAZ gene cluster and multiple partial nonfunctional copies, consisting of 5'-untranslated exons scattered along Yq [8]. VCY2 encodes a 13.9-kDa protein with 106 amino acids and a calculated overall isoelectric point (pI) of 10.9. It is also a highly positively charged protein because of an abundance of the basic amino acid residues arginine and lysine.
The function of VCY2 protein is still not known, and no functional conserved motif or domains are found in VCY2 by Pfam database search. The identification of VCY2-interacting protein will provide an important clue regarding the potential function of VCY2. The objective of the present study therefore was to investigate the likely function of VCY2 using a yeast two-hybrid system. We have recently reported the interaction of VCY2 and UBE3A [9]. In the present study, we identified a second VCY2-interacting protein, designated VCY2IP-1, that has substantial homology to two human microtubule-associated proteins (MAPs), MAP1B and MAP1A. These results may suggest the role of VCY2 in the cytoskeletal network via interaction with VCY2IP-1.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To prepare the full-length VCY2 cDNA for yeast two-hybrid analysis and to determine the localization of VCY2IP-1 in human testicular tissues with normal spermatogenesis, fresh testicular tissues from whole adult human testis were obtained from a total of three unrelated men undergoing orchidectomy as hormonal treatment for prostate cancer. The operations were performed in 2002 at the Department of Surgery, The University of Hong Kong. Informed consent was obtained from the patients and the study protocol was approved by the Ethics Committee of the University of Hong Kong. All these tissues revealed morphologically normal spermatogenesis with normal tubule cellularity and late spermatids in all tubular cross-sections. All tissue specimens were divided into two pieces: One was used for RNA extraction for reverse transcription-polymerase chain reaction (RT-PCR), and one was fixed overnight in 10% neutral buffered formalin, washed in 70% ethanol, and embedded in paraffin for in situ hybridization. Human testis mRNA was isolated using Quickprep mRNA extraction kit (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's specifications.
Yeast Two-Hybrid Screen
All vectors, yeast strains, reagents, and methods were derived from the MATCHMAKER Two-Hybrid System 3 (Clontech, Palo Alto, CA). The bacterial strain Escherichia coli KC8 (from Dr. K.M. Yao) was used for propagation of plasmid constructs. The yeast strains Saccharomyces cerevisiae AH109 and Y187 (Clontech) were used as hosts in the two-hybrid assay. AH109 contains two nutritional reporter genes for adenine and histidine. Both AH109 and Y187 contain the LacZ reporter gene. VCY2 cDNA (383 base pairs [bp], nucleotides [nt] 321–703) encoding the full-length VCY2 gene product was generated from human testis mRNA by RT-PCR with ThermoScript reverse transcriptase (Invitrogen). The VCY2 fragment was then amplified by PCR with Pfu Turbo polymerase (Stratagene, La Jolla, CA) using the forward primer 321 (5'-atagaattcctaatgatgacgcttgtc-3') with a EcoRI site and reverse primer 703 (5'-cacgatggatgttatctatt-3'). The amplified products were digested with EcoRI and cloned in-frame into the EcoRI site of pGBKT7. The correct insertion of the VCY2 was confirmed by restriction analysis and verified by ABI dye terminator sequencing using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). The resulting plasmid (designated pGBKT7-VCY2) contains the VCY2 coding sequence in frame with the DNA-binding domain of GAL4 protein and was used for yeast two-hybrid assay. Absence of autonomous activation of reporter genes in yeast was verified by bait before library screening. Expression of the Gal4 VCY2 fusion was verified by Western blot analysis with anti-myc polyclonal antibody (Clontech). AH109 yeast cells were sequentially transformed with pGBKT7-VCY2 and with a human testis cDNA library containing 3.5 x 106 independent clones (oligo[dT] and random-primed cDNAs in the pACT2 vector). Briefly, after transformation of bait and library, cells were plated on synthetic dropout (SD)-leu-trp medium at high density, selecting just for the presence of both proteins. Putatively positive colonies were selected based on the histidine and adenine prototrophy and transactivation of 
-galactosidase activity of interacting bait and prey proteins. Secondary and tertiary screens were performed in a similar manner to rule out fortuitous interactions. To validate the screening, -galactosidase activity of the putatively positive clones was compared with that of controls. Two controls were included for yeast two-hybrid assay: pGBKT7-p53 (encoding a fusion of the DNA-binding domain with murine p53 protein) and pGADT7-T (encoding a fusion of the DNA-activation domain with SV40 large T antigen), which are known to interact and were used as a positive control. pGBKT7-Lam (encoding a fusion of the DNA-binding domain with human lamin C) and pGADT7-T was used as a negative control.
Plasmid DNA was prepared from positive-interacting clones by transformation in E. coli KC8. The resulting positive-interacting clones were sorted into groups with identical insert according to the restriction pattern with HaeIII and confirmed in-frame with GAL4 by DNA sequencing with T7 sequencing forward primer and 3' AD sequencing reverse primer (Clontech). Representatives of these groups were purified and cotransformed into yeast with the original VCY2 bait to verify specific activation of the HIS3 and lacZ reporter genes.
Confirmation of Specificity of the VCY2/VCY2IP-1 Two-Hybrid Interaction
Yeast mating
The specificity of protein-protein interaction was further tested by yeast mating according to the manufacturer's instructions. Briefly, pGBKT7-VCY2, pGBKT7-Lam, and pGBKT7 vector only were transfected into yeast strain Y187 (MAT) and grown in medium lacking tryptophan; pACT2-VCY2IP-1 and pACT2 vectors alone were transformed into yeast strain AH109 and grown in medium lacking leucine. The transfected yeast strain AH109 (MATa) was then mixed with Y187 (MAT) cells and grown in medium lacking tryptophan and leucine. After growing for 5 days, the cells were replica plated in medium lacking adenine, histidine, tryptophan, and leucine with X--Gal. Transfected Y1
7 with pGBKT7 vector alone or pGBKT7-Lam was used as a negative control for a fortuitous interaction with transfected AH109 with pACT2 vector alone or pACT2-VCY2IP-1. Lamin C neither forms complexes nor interacts with most other proteins. pGBKT7-53 and pGADT7-T were used as a control for positive interaction.
In vitro coimmunoprecipitation The cDNA inserts from the pGBKT7-VCY2 and pACT2-VCY2IP-1 constructs were amplified by PCR. The forward primer contains a nonannealing epitope tag (myc for VCY2 and hemagglutinin [HA] for VCY2IP-1) and T7 promoter sequences at the 5' end. The amplified sequences were used to produce [35S]methionine-labeled proteins by in vitro transcription-translation using a TNT rabbit reticulocyte lysate system (Promega, Madison, WI). The proteins were mixed and immunoprecipitated by the addition of anti-myc antibody in the presence of protein G-agarose (Invitrogen). In the negative control, the myc-tagged, radiolabeled VCY2 protein was mixed with HA-tagged, radiolabeled Max protein and immunoprecipitated by the addition of anti-myc antibody. All the immunoprecipitation products were separated on a 15% polyacrylamide gel. The gel was subsequently soaked in Amplify Fluorographic Reagent (Amersham Biosciences, Little Chalfont, UK), dried, and exposed to Biomax x-ray film (Kodak, Cambridge, UK) overnight.
Full-Length VCY2IP-1 cDNA The full-length VCY2IP-1 cDNA was analyzed by RT-PCR using human testis mRNA and by PCR amplification using four primer pairs based on the sequence of two overlapping clones, MHC1 and BC006358, and two predicted exonic sequences located in the chromosome 19 working-draft sequence segment. The primer region and the PCR product sizes are summarized in Figure 3. The PCR was performed using the following primer pairs: for VCY2IP-1N1 (nt 148–316), 5'-gcgtctgcaaccttgatgaa-3' (forward primer) and 5'-ggttccggagctcatcatac-3' (reverse primer); for VCY2IP-1N2 (nt 1405–2403), 5'-gggcgagccgagagcaaaga-3' (forward primer) and 5'-cacgggatccgagtcagaca-3' (reverse primer); for VCY2IP-1N3 (nt 2647–3094), 5'-gctgctgccaaaaccaagggg-3' (forward primer) and 5'-ccgggcgtgcgtctctgcgta-3' (reverse primer); for VCY2IP-1N4 (nt 1–781), 5'-aagatggcggcggtggctgg-3' (forward primer) and 5'-gccattgacggcgaagaag-3' (reverse primer). The sequence of these PCR-amplified fragments was verified by ABI dye terminator sequencing using ABI PRISM 310 Genetic Analyzer (Applied Biosystems).
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Chromosome Assignment
A human monochromosomal somatic cell hybrid DNA panel was purchased from UK Human Genome Mapping Project Resource Centre (HGMP-RC; Cambridge, UK) for PCR amplification using primer pair VCY2IP-1N2. The PCR was performed (PTC-200; MJ Research, Waltham, MA) using the following program: 94°C for 1 min; 35 cycles of 94°C for 40 sec, 59°C for 50 sec, and 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide.
Northern Blot Analysis of VCY1IP-1
Northern blot analysis was performed on two different human multiple-tissue Northern blots. To determine the full-length VCY2IP-1, a human multiple-tissue Northern blot (MTN IV; Clontech) was probed with three PCR products amplified from human testis cDNA with three primer pairs: VCY2IP-1N1, VCY2IP-1N2, and VCY2IP-1N3. The G3PDH cDNA control probe was provided by Clontech. The predominant expression of VCY2IP-1 in testis was further confirmed by a second human multiple-tissue Northern blot (OriGene Technologies, Rockville, MD) using a probe generated from a primer set VCY2IP-IN3. All probes were randomly labeled with [32P]dCTP using Rediprime II kit (Amersham). All probes were hybridized using Expresshyb hybridization solution (Clontech) at 68°C overnight. The blot was washed twice in 2x SSC (1x: 0.15 M sodium chloride and 0.015 M sodium citrate)/0.1% SDS at 65°C for 5 min and exposed to Biomax x-ray film. The intensity of individual bands was quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Sperm Preparation for RT-PCR
All semen samples were collected from men visiting our subfertility clinics, and sperm preparation was performed as previously described [10]. Informed consent was obtained from the patients and the study protocol was approved by the Ethics Committee of the University of Hong Kong. Ribonucleic acid of human spermatozoa was extracted by Quickprep mRNA extraction kit (Invitrogen). First-strand cDNA synthesis was performed according to the manufacturer's instructions (Invitrogen) using oligo(dT)20 primer and ThermoScript reverse transcriptase. The primer pairs for VCY2 amplification were as follows: forward primer, 5'-gcgtctgcaaccttgatgaa-3'; reverse primer, 5'-ggttccggagctcatcatac-3'. Primer pairs for VCY2IP-1 were VCY2IP-1N1 and VCY2IP-1N3 as described in the previous section. Water was used as a blank control. The PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide.
In Situ Hybridization to Human Testis Sections
To generate riboprobes for in situ hybridization, VCY21N2 primer pairs as described above were used to amplify a 998-bp VCY2IP-1 cDNA fragment. This cDNA fragment was then cloned into a pGEM-T Easy vector (Promega) containing T7 and SP6 sequences for in vitro transcription. The [35S]uridine triphosphate (Amersham) was incorporated into the riboprobes by either T7 (sense) or SP6 (antisense) RNA polymerase (Promega). All sections were cut at a thickness of 5 µm, mounted and deparaffinized, rehydrated, and treated with proteinase K. Final probe concentration was normalized for 2 x 105 cpm/µl. After hybridization, the sections were washed under high-stringency conditions to remove nonspecific hybridization. The slides were overlaid with autoradiography emulsion, exposed for 1 wk at 4°C, developed photographically, and lightly stained with hematoxylin and eosin.
Bioinformatic Tools
A BLAST search (http://www.ncbi.nlm.nih.gov/blast/) was used to identify clones containing fragments identical with or homologous to our VCY2IP-1 gene sequence. Chromosomal mapping localization of VCY2IP-1 was analyzed with the HGMP UCSC Genome Browser (http://genome.cse.ucsc.edu/). Multiple-sequence alignment of VCY2IP-1, MAP1A, and MAP1B was performed using the ClustalW web server (http://www.ebi.ac.uk/clustalw/). The following softwares and databases were used for the protein analysis of VCY2 and VCY2IP-1: Pfam for motif and domain analysis (http://www.sanger.ac.uk/Software/Pfam/), and PROTSCALE for molecular weight analysis (http://tw.expasy.org/cgi-bin/protScale.pl/).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A bait vector encoding full-length VCY2 was used in yeast two-hybrid screens of a human testis cDNA library. Twenty-six positive-interacting clones were isolated and characterized, 16 of which encoded the same 948-bp cDNA insert. This VCY2-interacting clone (MHC1) was named VCY2IP-1 (VCY2-interacting protein-1). The specificity of the interaction was further confirmed by the absence of -galactosidase activity after cotransformation of pGBKT7-VCY2 with the negative controls, indicating that the original positive result was specific for and dependent on the presence of VCY2.
Confirmation of VCY2 and VCY2IP-1 Interaction by Yeast Mating
The specificity of the interaction between VCY2IP-1 and VCY2 was further confirmed by testing interactions between specific proteins during yeast mating using two different yeast strains. As shown in Figure 1A, blue diploid colonies were grown when the Y187 yeast strain containing pGBKT7-VCY2 mated with the AH109 yeast strain containing pACT2-VCY2IP-1. No diploid colonies were observed in all the negative controls, indicating the specificity of the protein interaction in the yeast-mating assay and the necessity of VCY2 and VCY2IP-1 proteins for the interaction (Fig. 1).
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VCY2IP-1 Interacts with VCY2 In Vitro by Coimmunoprecipitation
To validate the yeast two-hybrid screening result, the interaction between VCY2 and VCY2IP-1 was further confirmed by in vitro coimmunoprecipitation. Coimmunoprecipitation was demonstrated in a mixture of in vitro-transcribed and -translated, 35S-labeled, epitope-tagged VCY2-myc and VCY2IP-1-HA using anti-myc antibody (Fig. 2). As shown in Figure 2, VCY2 was specifically associated with VCY2IP-1 but not with Max-HA (negative control). The results of this coimmunoprecipitation analysis confirmed that the association between VCY2 and VCY2IP-1 is specific and not an artifact of the yeast two-hybrid system.
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VCY2IP-1 Sequence Analysis
Having established that VCY2 interacts with VCY2IP-1, we next sought to determine the sequence of VCY2IP-1. By sequence analysis, VCY2IP-1 has a 948-bp cDNA insert with a translation terminator codon at the 3' end but no ATG triplet Kozak consensus at the 5' end of VCY2IP-1 cDNA [11]. BLASTX analysis with putative protein sequence derived from the VCY2IP-1 cDNA showed more than one uncharacterized protein identical to our translated VCY2IP-1. Among these proteins, AAH06358.1, which is found in human brain, has the longest cDNA sequence (BC006358), encoding 672 amino acids. To determine whether VCY2IP-1 matched to BC006358, two primer sets (VCY2IP-1-1N1 and VCY2-IP-1N2) derived from sequence overlapping MHC1 and BC006358 were used for amplification with human testis mRNA (Fig. 3). The two amplified fragments produced a single band of the expected size. By sequence analysis, these fragments were fully matched to the sequence of MHC1 and BC006358, indicating that these fragments may come from the same transcript. By human genome database searches using the BLASTN algorithm, two putative exons on chromosome 19 were predicted in the upstream sequence of BC006358. This indicated that BC006358 is an incomplete cDNA clone. To determine the upstream sequence of VCY2IP-1 at the 5' end, two primer sets (VCY2IP1-1N3 and VCY2IP-1-1N4) derived from sequence overlapping BC006358 and the two predicted exonic sequences were used for PCR amplification with human testis mRNA (Fig. 3). All amplified fragments produced a single band of the expected size, and their sequences were matched to BC006358 and the two predicted exonic sequences. To further confirm whether these fragments belonged to the same transcript, Northern blot analysis on a human tissue blot (MTN IV) was performed using the VCY2-1N1, VCY2-1N2, and VCY2-1N3 amplified fragments as radiolabeled probes. We found that all these probes gave a single, intense band of 3.2 kilobases (kb) in human testis (Fig. 4, A–C), indicating that all three amplified fragments belong to the same VCY2IP-1 transcript. The full-length VCY2IP-1 cDNA sequence was deposited into the GenBank/EMBL/DDBJ database (GenBank accession no. AJ440784) and the Human Gene Nomenclature Database. The nucleotide context of the first ATG triplet conforms well to the Kozak consensus. The principal open reading frame consisted of 3263 bp and encoded a predicted protein of 1059 amino acids, with a relative molecular mass of 112 kDa and an isoelectric point (pI) of 6.9. The open reading frame was confirmed by GENSCAN [12].
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Chromosomal Mapping of the Human VCY2IP-1 The chromosomal location of the VCY2IP-1 gene was confirmed by PCR amplification using a panel of human monochromosomal somatic cell hybrid DNAs and VCY2IP-1-1N1 primer set. A genomic fragment of VCY2IP-1 was amplified only in chromosome 19, whereas none of the other panel samples, including the hamster and mouse as negative controls, produced fragments of the expected size (data not shown). This result was also supported by a contig NT_011288.7, a working-draft sequence segment of human chromosome 19 that is fully matched to the 948-bp VCY2IP-1 cDNA and mapped to chromosomal region 19p13.11 in the human genome database.
Human Genomic Structure of VCY2IP-1 The exon-intron boundaries and intron sizes as predicted by the human genome database are shown in Table 1. The VCY2IP-1 gene contains seven exons and six introns. The full-length human VCY2IP-1 cDNA and intron-exon boundaries are shown in Figure 5. The boundaries conform to the expected GT and AG sequences [13].
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Sequences Homologous to the VCY2IP-1 Sequence
BLAST database searches were performed to examine the VCY2IP-1 sequence (GenBank accession no. AJ440784) for relationships with other proteins. VCY2IP-1 likely is a member of the large family of microtubule-associated protein (MAPs), because it exhibits a significant sequence homology with two structural human MAP1 proteins, MAP1A (GenBank accession no. P78559) and MAP1B (GenBank accession no. P46821). The full-length VCY2IP-1 (amino acids 1–1059) revealed highly conserved open reading frames 59.3% similar (55.7% identical) to the full-length MAP1B (amino acids 1–2459) and 41.9% similar (31.3% identical) to the full-length MAP1A (amino acids. 1–2805). As shown in Figure 6, the first 510-amino acid stretch (residues 1–510) of VCY2IP-1 revealed highly conserved open reading frames with 58.7% similarity (47.9% identity) and 62.1% similarity (52.2% identity) to the NH2-terminus region of MAP1B (residues 14–524) and MAP1A (residues 1–303), respectively. As shown in Figure 6B, the 217-amino acid stretch (residues 842–1059) of VCY2IP-1 revealed highly conserved open reading frames with 58.6% similarity (45.8% identity) to the microtubule-binding domain of the light-chain LC1 in the COOH-terminus of MAP1B (residues 2218–2459). It also revealed 48.0% similarity (36.5% identity) to the light-chain LC2 in the COOH-terminus of amino acids 2578–2805 of MAP1A. However, it is interesting to note that part of the heavy chain in the N-terminal region of MAP1B and MAP1A is missing in VCY2IP-1 (Fig. 6B). Motif search by PROSITE databases revealed that both VCY2IP-1 and MAP1B have an N-glycosylation site and similar phosphorylation sites, including cAMP- and cGMP-dependent protein kinase, protein kinase C, and casein kinase II phosphorylation sites. Homology analysis also showed that VCY2IP-1 is highly conserved and related to MAP1A and MAP1B sequences in different species, including mouse (MAP1A GenBank accession no. Q9QYR6, MAP1B accession no. P14873), rat (MAP1A accession no. P34926, MAP1B accession no. P15205), and Torpedo californica (ENP2 accession no. AAA49279). The first 510 amino acids in the VCY2IP-1 are highly conserved within the NH2-terminus of MAP1B and MAP1A in human, mouse, and rat and in the electromotor neuron-associated protein (ENP) of T. californica. In the C-terminal portion of VCY2IP-1, 286 residues (amino acids 773–1059) are highly conserved within the COOH-terminus of MAP1B and MAP1A in human, mouse, and rat (data not shown).
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Multiple-Tissue Distribution of VCY2IP-1 mRNA
The tissue distribution of the VCY2IP-1 transcript was determined by Northern blot analysis using two multiple-tissue blots. VCY2IP-1 was expressed as a single band of approximately 3.2 kb in all human tissues with variable intensity except in muscle (Fig. 4, A–C and F). The highest expression level of VCY2IP-1 was detected in human testis in both multiple-tissue blots (Fig. 4, A–C and F). To demonstrate the presence of intact mRNA and to quantify the expression of VCY2IP-1 transcript in each tissue, the same membrane was stripped and probed with glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probe. A G3PDH signal was observed in all tissues at different intensities (Fig. 4, D and G).
Localization of VCY2IP-1 mRNA in Human Testicular Sections
In situ hybridization was performed to determine the distribution of VCY2IP-1 expression in human testis (Fig. 7). On testicular sections hybridized with a VCY2IP-1 antisense probe (complementary to endogenous tissue mRNA), silver grains were found more abundantly in spermatocytes and spermatids (Fig. 7, A and B). Fewer silver grains were observed in spermatogonia (Fig. 7B). Only some background staining was observed on testis sections hybridized with the negative-control sense probe (Fig. 7, C and D). These results indicated that the VCY2IP-1 is transcribed in germ cells.
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Expression of VCY2 and VCY2IP-1 in Human Ejaculated Spermatozoa
The expression of VCY2 and VCY2IP-1 transcripts on human spermatozoa was further investigated by RT-PCR analysis. Contamination by other somatic cells was excluded by the swim-up spermatozoa preparation as described previously [10]. We showed the expression of VCY2 and VCY2IP-1 transcripts in human ejaculated spermatozoa (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the biological function of VCY2IP-1 is not known, the demonstration of extensive homology with MAP1A and MAP1B has implications for the possible function of VCY2 in the cytoskeletal network. The conservation of amino acid sequences between VCY2IP-1, MAP1B, and MAP1A indicates that these proteins may have arisen from a common ancestral MAP1 gene. The MAPs share a capacity to interact with the COOH-terminus tubulin domain, to stabilize microtubules, and to link them with other cytoskeletal polymers [14, 15]. The extensive homology of the microtubule-binding domain in the COOH region of MAP1B to VCY2IP-1 suggests that VCY2IP-1 may also bind to microtubules. Phosphorylation sites in MAP1B and MAP1A have been shown to be important for regulating the binding of microtubules [16, 17] and to affect the in vitro binding of MAP1B with microfilaments [18]. The identification of similar phosphorylation sites in VCY2IP-1 might indicate a crucial regulatory event for the interaction between VCY2IP-1 and microtubules, although additional experiments will be required to define the exact phosphorylation status of VCY2IP-1.
The testis is one of the most abundant sources of microtubule networks. Different types of MAPs have been shown to be involved in different networks. These include MAP1 and MAP2 in germ cells [19, 20], tau in spermatid manchette [21], motor cytoplasmic dynein in axoneme [22], and MAP4 in Sertoli cells [23]. The MAP1 has been extensively characterized as a brain-specific protein with the capacity to bind microtubules [24]. Although the function of MAP1 has not been defined in human testis, both MAP1A and MAP1B proteins have been identified in the synaptonemal complex of mouse spermatocyte nucleus [19]. The distribution of MAP1 during meiosis and spermiogenesis and its localization in the nuclear matrix suggest participation of the molecules in the organization and dynamics of the synaptonemal complex during meiotic prophase.
The involvement of VCY2IP-1 in spermatogenesis is indicated by several lines of evidence. Multiple-tissue distribution of VCY2IP-1 showed that it was most abundantly expressed in testis but only weakly, or not at all, in other tissues. In situ hybridization of sections from human testis indicated that VCY2IP-1 is expressed most highly in germ cells, and RT-PCR showed the presence of VCY2IP-1 transcripts in human ejaculated spermatozoa. Our published data have recently reported the expression of VCY2 in germ cell nuclei in testicular sections by immunohistochemical analysis [25]. Based on these data, we propose that VCY2 and VCY2IP-1 have an important role throughout germ cell development, although the functional relevance of the VCY2 and VCY2IP-1 interaction remains elusive.
We previously reported the interaction of VCY2 and UBE3A and the presence of UBE3A transcripts in human ejaculated spermatozoa [9]. We proposed that the interaction of VCY2 and VCY2IP-1 in the microtubule network might be regulated by UBE3A through the interaction of VCY2 and other proteins in the microtubule network. Consistent with this hypothesis, a close relationship between ubiquitin and/or ubiquitin-protein conjugates and the microtubule network has been reported [26, 27]. More specifically, ubiquitin protein ligase-like protein has been implicated in the cytoskeletal regulation during cell-cycle progression [28].
In summary, the present study has provided novel information regarding the possible role of VCY2. Based on the yeast two-hybrid data and the high homology of VCY2IP-1 with MAP, we propose that VCY2 has a role in the microtubule network via interaction with VCY2IP-1. Further studies will be necessary to elucidate both the physiological role of interaction during germ cell development and the possible functions of VCY2 and VCY2IP-1 in the microtubule network.
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FOOTNOTES |
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Received: 21 April 2003.
First decision: 16 May 2003.
Accepted: 29 September 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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