Molecular Human Reproduction, Vol. 9, No. 1, 9-17,
January 2003
© 2003 European Society of Human Reproduction and Embryology
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Identification and characterization of a novel human testis-specific Golgi protein, NYD-SP12
Submitted on July 18, 2002; accepted on October 24, 2002
1 Laboratory of Reproductive Medicine, Center of Human Functional Genomics, Nanjing Medical University, Nanjing, 210029, People’s Republic of China and 2 Department of Life Sciences, Indiana State University, Terre Haute, IN 47809, USA 3 To whom correspondence should be addressed. e-mail: shajh{at}njmu.edu.cn
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ABSTRACT |
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A novel human testis-specific gene, NYD-SP12, was identified by hybridizing human adult or fetal testes cDNA samples with a human cDNA microarray containing 9216 clones. mRNA expression level of NYD-SP12 was 30-fold higher in human adult testes than fetal testes. Similarly, semi-quantitative RT–PCR revealed a differential expression pattern of an NYD-SP12 homologous gene in mouse adult and infant testes. PCR and hybridization analysis of NYD-SP12 mRNA from multiple human tissues indicated the expression of NYD-SP12 exclusively in the testis. In-situ hybridization revealed that the expression of this gene was confined to spermatogenic epithelium and was not found in interstitial cells. NYD-SP12 transcript was not detected in patients with spermatogenic arrest and Sertoli cell-only syndrome. NYD-SP12 cDNA (GenBank accession number: AF345909) consisted of 2070 bp. The predicted 1707 bp open-reading fragment encoded a 569 amino acid protein that was 77% identical to a mouse homologue. Furthermore, computerized SMART and Motif analysis revealed that the protein contained a Structural Classification Of Proteins (SCOP) domain in the C-terminus and a cluster of phosphorylation sites for PKC, CK and cAMP/cGMP-dependent protein kinase. Interestingly, the EGFP-NYD-SP12 fusion protein was localized to the Golgi apparatus. In conclusion, the results suggest that NYD-SP12 is involved in spermatogenesis, and that NYD-SP12-encoded protein might function in the Golgi apparatus.
Key words: Golgi apparatus/infertility/NYD-SP12/spermatogenesis/testis
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Introduction |
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Spermatogenesis is a complex multistep differentiation process leading to the development of highly specialized haploid male gametes. Abnormality of any single step in spermatogenesis could cause male infertility. The threat of this problem to humankind has increased significantly in the last century. Over the past 50 years, average sperm counts have declined by
50%, from 113x106/ml in 1940 to 66x106/ml in 1990 (Carlsen et al., 1992). A sperm count <20–25x106/ml would inevitably result in sterility. Before solutions to the problem can be found, a complete understanding of the physiological mechanisms for spermatogenesis is needed. To date, spermatogenesis has been extensively studied but molecular details of this complex multistep differentiation remain largely elusive. Spermatogenesis consists of three principle phases: (i) the formation of spermatogonia from germline stem cells followed by mitotic divisions to produce primary spermatocytes, (ii) meiosis, by which diploid spermatocytes develop into haploid spermatids, and (iii) spermiogenesis, production of mature sperm from round spermatids (Baarends and Grootegoed, 1999). These phases are regulated mainly by gene activation/shut-down. Some of the genes are expressed only in the testes because meiosis does not occur in any somatic cells. Investigation into the testis-specific genes is expected to lead to a broader and better understanding of spermatogenesis.
A few testis-specific genes have previously been identified. One example of such genes is male germ cell-associated kinase (Mak). Mak encodes a protein kinase related to CDC2 kinase. The kinase is expressed mainly in the late pachytene and dramatically decreases in postmeiotic haploid cells, suggesting its role in meiosis (Jinno et al., 1993). A second example of a testis-specific gene is the one encoding Golga male-enhanced antigen-2 (mea2), a mouse Golgi matrix protein. Expression of Golga/mea2 was specifically enhanced in the testes. Transgenic mice with truncated Golga/mea2 lost fertility (Matsukuma et al., 1999). Other testis-specific genes include those encoding nuclear protamines, the acrosomal enzyme acrosine and the cell-surface protein fertilin (Snell and White, 1996; Myles and Primakoff, 1997). In addition to the above genes, more testis-specific genes may exist and serve unique functions in the testes.
The present studies aimed to identify novel testis-specific genes based on three criteria. First, the genes would be expressed specifically in the testes. Second, expression level of the genes would correlate with testicular development. Third, expression of the genes would be altered in male infertility patients. Our data demonstrated that one novel gene met these criteria and was named as NYD-SP12. NYD-SP12 and the encoded protein were further characterized in the present studies.
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Materials and methods |
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cDNA microarray hybridization and molecular cloning
Spermatogenesis-related genes were screened by hybridizing adult or fetal testes samples with cDNA microarray containing 9216 human clones, as previously described (Sha et al., 2002). The differentially expressed genes in adult and fetal testes were purified using mini-preps (QIAprep Spin Miniprep Kit; Qiagen, Hilden, Germany). These genes were then sequenced by ABI377 automatic sequencer (Applied Biosystems, USA). DNA sequences of the differentially expressed genes were entered in the data base for the search of homologous proteins using BLAST and SMART programs. The nucleotide and the putative protein structure were also analysed using GenRunner program.
Chromosome mapping
The chromosomal mapping of a spermatogenesis-related gene, NYD-SP12, was carried out using the TNG4 radiation hybrid (RH) panel (Research Genetics, USA) according to the manufacturer’s instructions. Human genomic DNA was amplified using PCR with a forward primer 5'-CACATGTTGCC TCAGACAG-3' and a reverse primer 5'-GCCTCACTGTATCTTCTCTGG-3' which spanned a region of 178 bp in NYD-SP12. Thirty-five cycles of PCR (94°C for 30s, and 58°C for 30s and 72°C for 1 min) were performed on a 1 µl template of each hybrid in a 50 µl reaction. The PCR result was submitted to the Stanford Human Genome Center (http://www-shgc.stanford.edu) for chromosomal location of the NYD-SP12 gene in relation to the respective chromosomal framework markers.
Analysis of homologous mouse testis gene transcription using semi-quantitative fluorescent RT–PCR
Due to the difficulty in obtaining human testes tissues at specific ages, we studied the transcription of the NYD-SP12 homologue in mice. A literature search in NCBI BLAST-n identified the mouse homologue, AK014869. The following experiments were designed to determine if the expression of the homologous gene AK014869 was correlated with spermatogenesis in mice. Total RNA was isolated from the testes of 7-day old (n = 100), 28-day old (n = 50), and 50-day old (n = 50) mice using TRIzol Reagent (Gibcol) according to the manufacturer’s instructions. Reverse transcription was carried out in 15 µl reaction mixture. The total RNA (1 µl), random primers (1 µl, 0.2 µg/ml; Sangon company, Shangai, China) and diethyl pyrocarbonate (DEPC) treated water (7 µl) were mixed and incubated at 70°C for 5 min. Then, 3 µl M-MLV RT 5xbuffer, 0.75 µl dNTP (20 mmom/l), 0.35 µl RNasin (50 IU/µl), 1 µl M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) and 1 µl DEPC water were added and performed at 37°C for 1 h, after which the enzyme was inactivated by heating the reaction mixture to 95°C for 5 min. Primer sequences were determined using GenRunner software according to the homologous sequences between mouse and human. The primers were: P1: 5'-ACAATGGGGAAGCGAATCTTGC-3'; P2: 5'-TGCCTCCTGTTGACTGAT GT-3'. The desired fragment for mouse cDNA was 258 bp. The 20 µl PCR reaction mixture contained 10 mmol/l Tris pH 8.3, 10 mmol/l KCl, 1.5 mmol/l MgCl2, 0.2 mmol/l dNTP, 0.5 IU Taq polymerase (Promega), 2 µl 1/1600 SYBR Green (Gene Company Ltd) and 1 µl cDNA temple. PCR was carried out using PE-5700 (Perkin–Elmer, USA) for 35 cycles. Each cycle was performed at 94°C for 30 s, 55°C for 30 s and 72°C for 1 min respectively. The expression level of the mouse NYD-SP12 homologous gene was originally expressed as the number of cycles before entering the exponential growth in the PCR reaction (CSP12). To minimize the artifact that could be caused by the use of an unequal amount of testis cDNA from mice of different ages, the expression level of ß-actin [number of cycles before entering the exponential growth in the B-actin (Cactin) PCR reaction] in each testis was measured using the same method. For each PCR experiment, triplicates of each sample were amplified and the average of CSP12 or Cactin of the triplicates was used in the analysis. PCR negative controls for each sample contained aliquots of non-reverse-transcribed RNA. Results from at least three experiments were analysed using Stata 7.0 software and P < 0.01 was considered statistically significant.
Analysis of NYD-SP12 tissue distribution by PCR and hybridization
Expression of NYD-SP12 in various human tissues was determined using Multiple Tissue cDNA kit (MTC; Clontech, CA, USA), according to the manufacturer’s instructions. Briefly, human NYD-SP12 cDNA and human ß-actin cDNA were amplified by PCR with 5 pmol/µl of the two primer pairs P3/P4 and P5/P6 respectively. Human ß-actin was used as a control. The sequences of P3 and P4 for human NYD-SP12 cDNA were 5'-ACAATGGGGAA GCGAATCTTGC-3' and 5'-GATGAACTGAGGGGAATACCACC-3' respectively. The sequences for human ß-actin cDNA were:. P5: 5'-CGGTTGGCCTTGGGGTTCAGGGGG-3'; P6: 5'-ATCGTGGGGGCGCCC CAGGCACCA-3'. The resulting PCR products were electrophoresed in 1.5% (w/v) agarose gels, transferred onto HybondTM-N+ nylon membranes (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) and detected by hybridization with the DIG-labelled probes. Prehybridization was performed in a solution containing 10% dextran sulphate, 1xDenhardt’s solution, 0.1% sodium dodecyl sulphate (SDS), 6xstandard saline citrate (SSC) and single strand salmon sperm DNA at 58°C for 30 min. One of each DIG-labelled tissue-specific probe was added to the mixture and hybridization was allowed to proceed for 4 h. After hybridization, membrane was washed in 1xSSC, 0.1% SDS for 2x30 min at 42°C and in 0.2xSSC, 0.1% SDS for 2x20 min at 65°C. The membrane was then blocked, incubated with AP-conjugated anti-DIG antibodies, washed, and incubated with a chemiluminescent AP substrate according to the manufacturer’s protocols (Boehringer Mannheim, Roche, Germany). The chemiluminescent bands were visualized by autoradiography. The possibility of amplifying NYD-SP12 genomic DNA in the experiment was minimized by designing the primers with the sequences in exon 8 and exon 11. The PCR product of genomic DNA and RNA could be distinguished easily according to their sizes (cDNA of NYD-SP12).
In-situ hybridization to mouse testes section
After PCR amplification using the primers P1 and P2, the PCR product was purified and inserted into PinPoint Xa-1 vector (Promega) by ligating the PCR fragment to upstream of the SP6 polymerase promoter. T4 ligation reaction was done according to the manufacturer’s protocol (Promega). Antisense and sense constructs were determined by sequencing and linearized by HindIII digestion. DIG-labelled antisense and sense riboprobes were synthesized with SP6 polymerase using DIG-RNA labelling Mix (Roche), respectively. The probes were precipitated using ethanol at –20°C overnight and resuspended in DEPC water for hybridization.
Mouse testes at various differentiation stages were sliced and immersed in 4% paraformaldehyde for 1 h. The tissues were then placed in fresh 4% paraformaldehyde at 4°C overnight, dehydrated in a graded series of ethanols, cleared in xylene, and embedded in paraffin. The embedded tissues were sliced into 2.5 µm sections and placed on slides pretreated with polylysine. The sections were deparaffinized in xylene, rehydrated in ethanol, and washed with DEPC water and phosphate-buffered saline (PBS). After being treated with proteinase K (20 µg/ml) for 10 min at 37°C, sections were post-fixed by 4% paraformaldehyde for 5 min, then washed twice with PBS. The sections were prehybridized in hybridization buffer (DIG Easy Hyb; Roche) without probe at 60°C for 1 h to block non-specific binding sites. Hybridization was then initiated by adding the antisense or sense probes at an optimal concentration (100 ng/ml) for 18 h at 60°C in a humidity-regulated chamber. After hybridization, the sections were washed twice in 45% formamide/4xSSC at 60°C for 10 min, in 2xSSC, 1xSSC and 0.5xSSC at 60°C for 20 min each, and in 0.01 mol/l PBS at room temperature for 10 min. Immunological detection was carried out with AP-conjugated anti-DIG antibodies and visualized with nitroblue tetrazolium (250 µg/ml) and 5-bromo-4-chloro-3-indolyl phosphatase (225 µg/ml) according to the manufacturer’s instructions (DIG Nucleic Acid Detection Kit; Roche).
NYD-SP12 transcription analysis in azoospermic patients
Tissue samples were obtained from the testes of 11 azoospermic male patients aged 21–46 years via testicular biopsy. Patients gave informed consent and ethical approval was obtained Nanjing Medical University, People’s Republic of China. Total RNA (3.5 µg/µl) was extracted from the testes samples using TRIzol Reagent (Gibco). The same procedures of reverse transcription and PCR were performed as described above to detect NYD-SP12 mRNA.
Construction and subcellular localization of pEGFP-NYD-SP12 fusion protein
A plasmid containing pEGFP and NYD-SP12 cDNA was constructed according to the following protocols. The NYD-SP12 open reading fragment (ORF, nucleotides 131–1840) was amplified from pTriplEx2-NYD-SP12 vector by PCR with the forward primer 5'-GGGGGAATTCCTC GAGATGGATGCAGGAAGCAGTAG-3' (containing XhoI site) and the reverse primer 5'-GGGGGGATCCTCACAGTACTAGGTGGGCATTG-3' (containing BamHI site). The amplified NYD-SP12 PCR product was purified and digested with XhoI and BamHI. These enzymes were also used to digest the plasmid pGEFP-C3 from Clontech. The XhoI–BamHI fragment in pGEFP-C3 plasmid was then replaced with the corresponding fragment from the NYD-SP12 PCR product. The identity of the insert ORF was confirmed by DNA sequencing.
The constructed pEGFP-NYD-SP12 cDNA was transfected into JAR, CHO and COS-7 cells using Qiagen transfection kit. Briefly, cells were seeded at a density of 4x105 cells/well in a 6-well plate on the day before transfection and cultured overnight. Monolayer JAR cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco BRL) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS). CHO cells were cultured in DMEM/Ham’s F-12 medium (v/v=1/1, Gibco BRL) containing 10% FBS. COS-7 cells were grown in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS. All cells were incubated in a humidified 5% CO2/95% air mixture at 37°C. Before transfection, the cells were washed with fresh cell growth medium. The constructed plasmid (2.5 µg) and 10 µl FolyFect Transfection Reagent (Qiagen) were mixed for 15 min, then added to each well. The cells were incubated with the transfection reagent for 24 h. As a control, pEGFP-C3 vector without insert was transfected into cells in separate wells simultaneously under the same condition. In addition, JAR cells were treated with 2 µg/ml BODIPY FL C5-ceramide (Molecular Probes, Eugene, Oregon, USA) for 25–30 min at 37°C to stain Golgi apparatus according to the manufacturer’s protocol. The GFP fusion protein and stained Golgi apparatus were visualized by Laser Confocal Microscope (Zeiss). About one-tenth of cells were transfected reproducibly under these conditions.
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Results |
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Hybridization with cDNA microarray identified a novel spermatogenesis-related gene, NYD-SP12
Hybridization of cDNA microarray containing 9216 human clones with adult and embryo human testis probes identified a spermatogenesis-related clone, NYD-SP12. This gene was highly expressed in adult and not in embryo testes. The relative hybridization signal intensities of the gene in adult and embryo testes were 74.72 and 12.00 respectively, 6-fold stronger in adult than in embryo testes (Figure 1). The complete nucleotide sequence of NYD-SP12, subcloned into plasmid pTriplEx2, has been accepted by GenBank with the accession number AF345909. Similar age-dependent expression of a mouse homologous gene was observed in postnatal day 7 and day 50 (adult) mouse testes. The hybridization signal intensity in the mouse testes was 24.84 and 5.88 respectively (data not shown).
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Computer-assisted determination of NYD-SP12 chromosome location and the structure of NYD-SP12-encoded protein
NYD-SP12 cDNA consisted of 2070 nucleotides spanning eleven exons, which contained an ORF of 1707 bp. The ORF encodes a protein of 569 amino acids with an expected molecular weight of 65 kDa and a deduced pI of 9.16 (Figure 2). BLAST search revealed a high homology of NYD-SP12 to a mouse cDNA AK014869. NYD-SP12 and AK014869 are 88% identical in nucleotide sequence (Figure 3A). The encoded proteins by NYD-SP12 and AK014869 are 77% identical in amino acid sequence (Figure 3B). RH mapping revealed that NYD-SP12 gene had two STS (SHGC-154504, SHGC-100646) included in a clone RP11-440C18, which was located on 3q26.32. The results from RH mapping confirmed those from a genomic search using BLAST.
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The search using BLAST indicated that NYD-SP12-encoded protein contained homologous amino acid sequences (466–563) in the C-terminus to those found in a mouse protein, male-enhanced antigen-2 (Mea2) (Figure 2). The search through SMART showed that NYD-SP12-encoded protein also contained a SCOP domain (amino acids 169–280), which belonged to the TPR-like (tetratricopeptide repeat) superfamily sharing common coiled-coil structures (Figure 2). Computer-assisted sequence analysis by GenRunner showed that NYD-SP12-encoded protein contained three glycosylation sites (26th, 165th, 446th), and a cluster of phosphorylation sites, including nine protein kinase C (PKC) phosphorylation sites (5th, 27th, 28th, 93th, 401th, 402th, 422th, 547th, 552th), six casein kinase II phosphorylation sites (101th, 201th, 228th, 319th, 418th, 495th) and one cAMP-dependent phosphorylation site (29th).
Expression of mouse NYD-SP12 homologous gene depended on testicular development
Semi-quantitative fluorescent PCR indicated that the mouse NYD-SP12 homologous gene was expressed differentially depending on spermatogenic stages. The expression level was quantified according to the number of cycles before entering the exponential phase in the PCR reaction. This number (C SP12) is inversely proportional to the expression level of the mouse NYD-SP12 homologous gene. C SP12 for 7, 28 and 50 day old mice was 27.97 ± 1.50, 18.47 ± 1.00 and 19.44 ± 1.06 respectively, while C actin was not significantly different among the three age groups (Figure 4). Expression levels of the mouse NYD-SP12 homologous gene in 28 and 50 day old mouse testes was significantly higher than in 7 day old mouse testes (P < 0.01). The result revealed a correlation between NYD-SP12 expression and testicular development.
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NYD-SP12 was specifically expressed in the testes
Expression pattern of NYD-SP12 was also examined by PCR and membrane hybridization in 16 human tissues including testis, pancreas, kidney, colon, spleen, white blood cells, prostate, ovary, thymus, heart, small intestine, lung, placenta, liver, brain and muscle. Except for a weak signal in the pancreas and kidney, the gene was specifically expressed in testis (Figure 5). The amount and integrity of the RNA used for this membrane hybridization were verified by hybridizing with DIG-labelled human ß-actin cDNA probe.
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In-situ hybridization revealed cellular localization of the mouse homologous NYD-SP12 mRNA
In-situ hybridization with a DIG-labelled antisense riboprobe detected the mouse homologous NYD-SP12 mRNA in 28 and 50 day old, but not 7 day old, mouse testes (Figure 6a–c respectively). Moreover, the mouse homologous NYD-SP12 mRNA were detected primarily in the spermatocyte. Little signal was detected in spermatogonia (Figure 6b), and no NYD-SP12 mRNA was found in Leydig cells (Figure 6d). Negative-control using sense probe confirmed the specific expression of NYD-SP12 mRNA (Figure 6e).
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Abnormal expression of NYD-SP12 mRNA in patients with spermatogenesis arrest
A total of 11 patients with azoospermia was included in the present study. RT–PCR studies found no detectable NYD-SP12 mRNA in patients with Sertoli cell-only syndrome (1–5 in Figure 7) or patients with spermatogenesis arrest (6 and 7 in Figure 7).
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pEGFP-NYD-SP12 fusion protein was localized to Golgi apparatus
Laser confocal microscopy localized pEGFP-NYD-SP12 fusion protein to Golgi apparatus 24 h after transfection. NYD-SP12 chimeras appeared to be localized exclusively in the perinuclear region (Figure 8a–c). In contrast, the pEGFP-C3 protein as a control was distributed homogeneously in the cytoplasm (Figure 8f–h). To further identify the perinuclear compartment where pEGFP-NYD-SP12 fusion protein was localized, the transfected JAR cells were stained with a fluorescent Golgi-specific probe, BODIPY FL C5-ceramide (Figure 8d). The fluorescent Golgi-specific probes and pEGFP-NYD-SP12 fusion proteins were co-localized to the perinuclear region (Figure 8e). The result demonstrated that pEGFP-NYD-SP12 fusion protein was localized specifically to Golgi apparatus.
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Discussion
The present study has identified a novel human testicular gene, NYD-SP12. Further characterization of NYD-SP12 provided information about its nucleotide sequence, chromosomal location, tissue distribution of mRNA, age-dependent expression, structure of the encoded protein, subcellular localization of the encoded protein, and the abnormal expression in patients. These results strongly suggest that NYD-SP12 is involved in spermatogenesis.
The link between NYD-SP12 and spermatogenesis was first identified by a close correlation between the expression level of NYD-SP12 and the testicular development. Hybridization of human cDNA microarray with adult and fetal testes samples indicated that NYD-SP12 was up-regulated in adult testes (Figure 1). Although a coincidence cannot be excluded, activation of the NYD-SP12 gene during testicular development is likely to be required for spermatogenesis, which is supported by the following evidence.
Consistent with its potential role in spermatogenesis, NYD-SP12 was expressed highly and specifically in human testes. Among 15 major non-testis human tissues tested in the present study, NYD-SP12 was not detectable in 13 tissues, and only weak expression was seen in the pancreas and kidney. An interpretation of the link of NYD-SP12 to both the pancreas and the testes will be discussed later. The high tissue specificity of NYD-SP12 expression pattern largely implied its involvement in the unique functions of the testes. Although the functions of NYD-SP12 remain unknown at present, its potential role in spermatogenesis is predicted to be important.
The importance of NYD-SP12 in spermatogenesis was further suggested by a close correlation between the silence of NYD-SP12 and spermatogenesis arrest. All the patients with Sertoli cell-only syndrome or spermatogenesis arrest at spermatocyte did not express NYD-SP12 (Figure 7). This correlation, together with the age-dependent differentiation and tissue-specific expression, provided compelling evidence for a crucial role of NYD-SP12 in testes development and spermatogenesis.
NYD-SP12 was linked not only to human spermatogenesis but also to mouse testicular development. The mouse NYD-SP12 homologous gene was identified by searching the genebank. The homologous gene had properties similar to those of human NYD-SP12. Expression level of this gene increased significantly in adult (28 and 50 day old) mouse testes compared with infant mouse (7 day old). The age-dependent differential expression was demonstrated by both semi-quantitative fluorescent PCR (Figure 4) and in-situ hybridization studies (Figure 6), the latter revealing that the mouse NYD-SP12 homologous gene was expressed only in spermatogenic cells. Interstitial cells in mouse testes did not contain detectable mRNA of the homologous gene. All of the data from mouse samples indicated the involvement of the mouse NYD-SP12 homologous gene in spermatogenesis.
Although all of the above results indicated a close relationship between NYD-SP12 and spermatogenesis, the exact function of NYD-SP12-encoded protein remains to be clarified. Our present study demonstrated that NYD-SP12-encoded protein was localized to Golgi apparatus (Figure 8). In these studies, a GFP-NYD-SP12 fusion protein was constructed and visualized in three different cell lines. All of the transfected cells displayed the fusion protein in a perinuclear region, which was later identified as the Golgi network. According to computer-assisted prediction, NYD-SP12-encoded protein may contain structures that are characteristic of a large number of recently identified Golgi proteins. These proteins all have coiled-coil structure and have been classifed as a family of protein. The family includes golgin-230/245/256, golgin-97, golgin-160/mea2/GCP170, and giatin/macrogolgin (Fritzler et al., 1993, 1995; Linstedt and Hauri, 1993; Erlich et al., 1996; Griffith et al., 1997). NYD-SP12 protein was predicted to have a SCOP domain belonging to the TPR-like superfamily. The SCOP domain could possibly form a coiled-coil structure. The C-terminus of NYD-SP12 was also homologous to the C-terminus of Mea2, a protein located in the cytoplasmic motif of mouse Golgi membrane (Kondo and Suton, 1997). These results, though insufficient, suggested that NYD-SP12-encoded protein functions in the Golgi apparatus.
A second clue that may be helpful to identify the functions of NYD-SP12 was the weak expression of NYD-SP12 in the pancreas (Figure 5). The pancreas and the testes share a common property, that is, producing digestive enzymes. The pancreatic exocrine glands produce amylase, lipase, DNase and protease for the digestion of food in the intestines. In sperm, a large quantity of enzymes exists in the sperm acrosome. These enzymes are normally shielded before being deposited in the female reproductive tract. Once in the female reproductive tract, the acrosomal enzymes are exposed and able to digest the corona radiata and zona pellucida of an oocyte to facilitate the penetration of sperm into the egg (Thorne-Tjomsland et al., 1988). Recent studies by others suggest that the Golgi apparatus could play a crucial role in the formation of acrosomes. At early stages of spermatogenesis (Golgi and cap phase), the Golgi apparatus is actively engaged in the formation of the acrosomal vesicle, producing and delivering the proteins and membranes needed for its enlargement and differentiation (Burgos and Gutierrez 1986; Westbrook-Case et al., 1995; Moreno et al., 2000). A few Golgi proteins, such as golgin-84, GKAP42 (42 kDa cGMP-dependent protein kinase anchoring protein) and GCP-60, were specifically or abundantly expressed in the testis (Bascom et al., 1999; Yuasa et al., 2000; Sohda et al., 2001). Transgenic male mice bearing a truncated gene of the Golgi protein Golga3/Mea2 (human homologue: golgin-160/GOLGA3) became sterile due to spermatogenesis block. These transgenic mice apparently lacked spermatids and sperm. The pachytene spermatocytes that expressed a low level of truncated Mea2 underwent apoptotic degeneration in homozygous testes (Banu et al., 2002). Based on the present observation and previous findings, we speculate that NYD-SP12 might be involved in the sorting and modification of acrosomal enzymes in the Golgi apparatus.
Initial characterization of NYD-SP12 in the present studies also revealed chromosomal location of the gene, molecular weight and pI of the encoded protein, and potential phosphorylation sites in the protein. The information will be useful in future attempts to determine the functions of the protein.
Spermatogenesis involves fast multi-stage differentiation events. Some of them may be shared by all fast-growing tissues/cells such as haemocytoblast in the red bone marrow, stem cells in the embryo, and cancerous cells in a tumour. Any novel findings about the gene activation and other molecular details of spermatogenesis will possibly lead to a better understanding of the mechanisms for the differentiation of other fast-growing tissues/cells. Thus, the significance of the present findings may not be limited to a search for spermatogenesis-related genes.
In conclusion, the results strongly suggested that the novel gene NYD-SP12 could play important roles in spermatogenesis. The results were also consistent with the speculation that NYD-SP12-encoded protein may be involved in the sorting and modification of acrosomal enzymes in the Golgi apparatus. Further study is needed to address its specific role in Golgi apparatus-regulated spermatogenesis.
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Acknowledgment |
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This research was supported by China National 973 fund, No. G1999055901.
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