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Testis |
a Laboratory of Reproductive Medicine, Center of Human Functional Genomics, Nanjing Medical University, Nanjing, Jiangsu Province 210029, People's Republic of China
b Department of Life Sciences, Indiana State University, Terre Haute, Indiana 47809-0001
c Department of Cell Biology, Institute of Basic Medical Sciences, CAMS & PUMC, Beijing 100005, People's Republic of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, gametogenesis, gene regulation, sperm maturation, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two percent of human males are infertile because of severe defects in sperm production [12]. The causes of spermatogenic anomalies have often been ascribed to infection, immunological factors, anatomic malformation, or chemical insult [13]. Relatively little research has focused on possible genetic aetiologies. A significant number of clinically severe oligozoospermia or azoospermia cases are thought to be the result of genetic disorders, including microdeletion in the Y chromosome and other chromosome and meiotic abnormalities [14, 15]. In the clinical cases, spermatogenic arrest is an interruption of germ cell differentiation that may result in either oligozoospermia or azoospermia and can be diagnosed via testicular biopsy [16]. Although spermatogenesis must require many gene products, mutation or absence of the gene expressed at different development levels of spermatogenesis may lead to spermatogenic arrest and infertility [17]. Identification of new genes specifically involved in spermatogenesis and analysis of the phenotypes could provide both insight into this developmental process and a more rational basis for treatment of male infertility.
Using human testis cDNA microarrays, in situ hybridization (ISH), and other techniques, we identified and characterized a novel human gene, NYD-SP16, which is highly expressed in human testis and contains one transmembrane domain. The development-dependent and functional expression and the subcellular localization indicate its potential role in development of testis and spermatogenesis. Its nucleotide sequence is available from GenBank under accession number AY027526.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protocols for human testis cDNA microarray construction, adult and embryo testis cDNA probe preparation, hybridization, and signal analysis have been described in detail recently [18, 19]. The cDNA microarray was derived from the Human Testis 5'-STRETCH PLUS cDNA library (HL5503U; Clontech, Palo Alto, CA). This library reportedly contains 1 million clones, representing the cDNA population involved in the testis. The insert cDNAs came from 25 Caucasian men (20–65 yr old), and the average length was 3.4 kilobases. According to the Clontech manual PT3003-1, 12 000 
TriplEx2 clones were picked randomly and converted to p TriplEx2 plasmid clones. The inserts were amplified from the plasmid clones by polymerase chain reaction (PCR) using 5'-CCATTGTGTTGGTACCCGGGAATTCG-3' as a forward primer and 5'-ATAAGCTTGCTCGAGTCTAGAGTCGAC-3' as a reverse primer. Among the 12 000 clones, 9216 PCR products were randomly selected to spot on a 8- x 12-cm Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, U.K.) by an automatic array (BioRobotics, Cambridge, U.K.) and crosslinked by ultraviolet light. Eight housekeeping genes were used as positive controls. TriplEx2 phage DNA and pUC18 plasmid DNA without insert cDNA were used as negative controls.
The total testes mRNA from deceased human adults and accidentally aborted 6-mo fetuses was extracted according to the Trizol RNA isolation protocol (Gibco BRL, Grand Island, NY) separately. The Poly(A)+ mRNA was purified using an poly(dT) resin affinity column (Qiagen, Hilden, Germany). The cDNA probes were prepared by incorporation of [
-33P]deoxyadenosine 5' triphosphate, 33P-labeled dATP (NEN Life Science, Boston, MA) in a reverse transcription reaction following the manufacturer's instruction.
After prehybridization, the microarray was hybridized with the 33P-labeled adult and embryo testis cDNA probes in denatured hybridization solution. The membranes were washed stringently, exposed to a phosphor screen, and scanned by a FLA-3000A plate/Fluorescent image analyzer (Fuji Photo Film, Tokyo, Japan). The radioactive signal intensity of each spot was linearly scanned and read using the Array Gauge software (Fuji). After subtracting the background, the signal intensity of the corresponding spots from the adult and the fetus were compared. When the intensity in adult and fetus differed by >3-fold, the clones were considered differentially expressed.
Sequencing and Analysis of Clones
After microarray hybridization, the differentially expressed cDNA plasmids were extracted and purified in mini-preps (QIAprep Spin Miniprep Kit; Qiagen). The full length of the insert was sequenced in an autosequencer (ABI model 377, Perkin-Elmer, Norwalk, CT) at Huada Gene Center (Beijing, China). The sequences were then compared with those in a database (www.ncbi.nlm.nih.gov) to determine the sequence homology with other species and the gene locus on the human chromosome. Meanwhile, the nucleotides and the deduced protein were also analyzed by Gene Runner, SMART (http://www.smart.embl-heidelberg.de/), and TMHMM (http://www.cbs.dtu.dk/services/tmhmm/) programs. This screening process reveals the differentially expressed genes in adults and embryos. In addition, novel genes that did not match any known human genes in GenBank could be isolated. NYD-SP16 was one of the novel genes whose complete nucleotide sequence was accepted by GenBank.
Multiple Tissue Distribution of NYD-SP16 mRNA
To determine the tissue distribution of NYD-SP16, primers specific to 5' end of NYD-SP16 overlapping an intron were used to amplify cDNAs of 16 human tissues using the Human MTC Panel I and II kit (Clontech). The forward primer (5'-AGCAGGGGCAAACTATACACGG-3') was located at 1019–1040 base pairs (bp) and the reverse primer (5'-ACCTGGCTCCTGCCTGTTTACC-3') was located at 1498–1519 bp of the cDNA. The PCR products of 16 tissues were resolved by electrophorese on 1% agarose gel and transferred to Hybond-N+ nylon membrane. The NYD-SP16 cDNA probe was labeled with the same primers used for the PCR. The template was NYD-SP16 clone plasmid, and digoxigenin (Dig)-labeled dNTPs were used (Dig DNA Labeling Mix; Roche Diagnostics, Indianapolis, IN). The reaction solution contained 2 µl 10x reaction buffer, 1.5 µl 25 mmol/L MgCl2, 1.5 µl Dig-dNTPs (1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM Dig-11-dUTP, alkali-labile), 0.5 U Taq DNA polymerase, 12 µl distilled water, 1 µl template, and 1 µl of each primer. The reaction protocol was as follow: 35 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 60 sec. The membrane was hybridized with Dig-labeled probe, incubated with anti-dig antibody conjugated with alkaline phosphatase (1:10 000 dilution) and the chromogenic substrate disodium 3-(4-methoxyspiro{1, 2-dioxetane-3,2'-{5'-chloro) tricydo[3.3.1.13,7] decan}-4-yl) phenylphosphate CSPD (Roche), and exposed to x-ray film. The integrity of tissue cDNA was tested using the primers of human ß-actin in parallel as above.
Semiquantitative PCR Analysis of Mouse Homologous Gene AK006554 in Mouse Testis at Different Stages
Because of the obstacles involved in obtaining human testis tissue at specific ages, we studied the expression of the mouse homologue of human NYD-SP16. A search of the NCBI Blast-nr database identified a mouse testis cDNA AK006554 with high homology to NYD-SP16. To evaluate AK006554 expression level in mouse testes at different stages, fluorescent semiquantitative PCR was performed with mouse-specific primers and SYBR Green (Molecular Probes, Eugene, OR). Total RNA of Week 1, Week 4, and Week 7 mouse testes was extracted using Trizol reagent and was reverse transcribed to cDNA. Primers were specific to mouse sequence: sense 5'-ACCTTACAGGTCGTTGCCAGAG-3' and antisense 5'-TTTCCCTACCCTGTCCCTTCTT-3'. PCR solution contained 1 µl of template, 2 µl 10x buffer, 1.5 µl 25 mmol/L MgCl2, 1.5 µl 20 mmol/L dNTPs, 0.15 µl Taq polymerase, 10.9 µl H2O, 1 µl SYBR Green (1:1600 dilution), and 1 µl of each primer. The PCR was performed in a quantitative PCR amplifier (PE5700; Perkin-Elmer, Norwalk, CT) under the same conditions as described above. To obtain relatively accurate results, each template of different mouse testes was processed in three tubes in the same PCR mixture. Mouse ß-actin was run in parallel with the same template under the same conditions. Cak and Cactin, the number of cycles of AK006554 and ß-actin amplification that were completed before entering the exponential growth phase of the PCR, was monitored in real time by amplifier. To minimize the artifact caused by unequal amount of testis cDNA from mice of different stages, the expression of AK006554 was defined as the ratio of Cak:Cactin. Difference among groups was determined by SPSS 10.0 software. Differences at P < 0.05 were considered significant.
ISH of Mouse Testis
Tissue section preparation The testes from 7-wk-old mice were fixed in 3% paraformaldehyde, embedded in paraffin, and cut into 20-µm sections.
Mouse ISH probe preparation The cDNA hybridization probe was Dig-labeled AK006554 cDNA. The same PCR labeling method and the same mouse-specific primers were used to amplify the adult mouse testis cDNA using PCR in the presence of Dig-labeled dNTPs (Roche). PCR conditions was the same as described above.
ISH After removal of paraffin, sections were treated by proteolytic digestion for 30 min at 37°C with 10 µg/ml proteinase K dissolved in 50 mM EDTA and 0.1M Tris-HCl, followed by two rinses of 5 min each in 0.01 M PBS. Sections were prehybridized for 1 h at 60°C and then incubated with Dig-labeled probe overnight at 37°C, followed by a stringency wash. Hybridization signal was detected with alkaline phosphatase-conjugated anti-Dig and visualized with nitroblue tetrazolium/5-bromocresyl-3-indolylphosphate chromogen (Roche) under a light microscope. The stages of mouse seminiferous tubules were determined as described by Russell et al. [20]. Some tissue sections were processed without probe to serve as negative controls.
Expression Analysis of Mouse Homologue in Mouse Spermatogenic Cells
To confirm mouse homologous expression in spermatogenic cells, PCR was performed with cDNAs of different mouse spermatogenic cells.
Isolation of the mouse spermatogenic cells By utilizing differential sedimentation velocity at unit gravity of cells that differ in volume, homogeneous populations of six kinds of spermatogenic cells were isolated successfully from the Bal B/c mouse testes throughout the development period, as described previously [21–23]. The optimum days or weeks for the recovery of specific cell types are as follows: Day 6, primitive type A spermatogonia; Day 9, type B spermatogonia; Day 14, leptotene primary spermatocytes; Day 21, pachytene spermatocytes; Week 5, round spermatids; Week 8, elongating spermatids. The identity and purity of the isolated germ cell populations were verified by light and electron microscopy, based on distinctive morphological features characteristic of the respective cell types [24].
Complementary DNA preparation and PCR analysis Total RNA of six kinds of cells was isolated using Trizol reagent. Different cDNAs were obtained by reverse transcription as described above and were used as templates for PCRs. The above mouse-specific primers were used, and the housekeeping gene GAPDH was run in parallel as a control under the same procedure. Mouse testis cDNA template was used as a positive PCR control for two primers. The reaction protocol was as follow: 32 cycles of 94°C for 45 sec, 55°C for 45 sec, and 72°C for 60 sec. Amplified products (8 µl) were electrophoresed on a 1% agarose gel containing ethidium bromide and were visualized under ultraviolet illumination.
Subcellular Localization of NYD-SP16 Protein Based on Green Fluorescent Protein Fusion Product
Plasmid construction of the pEGFP-N2 fusion protein pEGFP-N2-NYD-SP16 fusion protein was constructed by inserting the PCR-derived open reading frame (ORF) (nucleotides 106–867) of NYD-SP16 into the EcoRI/XhoI sites of the pEGFP-N2 vector (Clontech). The identity of the insert ORF of 762 bp was confirmed by DNA sequencing.
Lipofection of recombination vector to human choriocarcinoma JAR cell line Human choriocarcinoma JAR cells were maintained in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% fetal calf serum (SI JI Qing, Hangzhou, P.R. China). The transfection of the plasmid into JAR cells was performed according to the manufacturer's manual (PolyFect Transfection Reagent; Qiagen). As a control, pEGFP-N2 vector without insert was transfected into JAR cells at the same time under the same conditions. The fusion protein expression was examined using a confocal laser microscope (Zeiss, Göttingen, Germany).
NYD-SP16 Expression in the Testes of Patients with Male Infertility
Testicular tissues were obtained via biopsy from 11 patients with male infertility at the First Affiliated Hospital of Nanjing Medical University (Nanjing, P.R. China). The clinical diagnoses based on testicular biopsy were Sertoli-cell-only syndrome (SCOS) in two patients and spermatogenic arrest at different stages in nine patients. Total RNA (about 3.5 µg/µl) was extracted using Trizol reagent and then reverse transcribed to cDNA with avian myelobastosis virus reverse transcriptase. All the following procedures, including amplification, transferation, and hybridization, were performed as described above. The same NYD-SP16 and ß-actin cDNA probes were used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In hybridization studies with the human testis cDNA microarray, 95% of adult human testis mRNA and 96% of embryo testis mRNA gave a positive signal. The 5' sequence of the differentially expressed clones was compared with the NCBI database (Blast-nr), and 42 novel genes were isolated. Among these new genes, NYD-SP16 was more highly expressed in adult testis than in embryo testis. The hybridization signal intensity was 28.16 in adults and 3.78 in embryos, with an expression level in the adult about 6.44-fold higher than that in the embryo (Fig. 1). The full nucleotide and amino acid sequences of NYD-SP16 are displayed in Figure 2. The 1595-bp NYD-SP16 cDNA contains a complete ORF of 762 bp with a methionine start codon at position 106 and a TGA stop codon at position 868. The first start codon is preceded by an in-frame stop codon TAG at position 82, suggesting that ATG at position 106 is the start codon for NYD-SP16 protein. In 3' untranslated regions, however, a typical putative polyadenylation signal, AATAAA, is not present. Instead, an atypical polyadenylation signal, AGTAAA, which is one kind of mutation of AATAAA [25], was found 15 bp upstream from the poly(A)+ tail. Multiple sequence comparison among different species by the program BLAST identified a homologous cDNA in adult mouse testis, AK006554. Two regions (492–860 bp and 117–386 bp) in NYD-SP16 were 85% and 82% homologous, respectively, to the two regions (483–851 bp and 108–377 bp) in AK006554. Further, NYD-SP16 protein shares 73% amino acid sequence identity with that of mouse homologue protein BAB24651 encoded by AK006554 (Fig. 3). These data indicate high levels of homology between NYD-SP16 and the mouse homologue at both the nucleotide and protein level; therefore, NYD-SP16 is a human-mouse homologous gene.
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Locus of NYD-SP16 on the Human Chromosome
A search of GenBank contig maps assigned NYD-SP16 to human chromosome 5q14. NT007059, one clone on human chromosome 5q14 (length 1 036 632 bp), contains the genomic sequence of NYD-SP16. NYD-SP16 is spliced by nine exons (with lengths of 88–298 bp) and eight introns (with lengths of 452–25 375 bp). NYD-SP16 encompasses 29 689 bp of genomic DNA (from bp 355 822 to 425 511) of NT007059.
Model of the Protein Encoded by NYD-SP16
The ORF encodes a 254-amino acid protein with a predicted molecular mass of 
29 kDa and an isoelectric point of 9.51. SMART and TMHMM 2.0 analysis revealed that one region of predominantly hydrophobic amino acid at position 143–165 functions as a transmembrane domain, suggesting that NYD-SP16 may be a transmembrane protein (Fig. 2). No other motifs were identified in the predicted protein sequence of NYD-SP16.
Tissue Distribution of NYD-SP16 mRNA
The expression profile of NYD-SP16 in human tissues was studied using multiple-tissue PCRs combined with membrane hybridization. Of the 16 tissues examined, NYD-SP16 was transcribed highly in testes and pancreas. Low levels were found in the heart, lungs, and brain. Very low expression was detected in the placenta. NYD-SP16 transcription was absent in skeletal muscles, liver, kidneys, thymus, small intestines, colons, spleen, leukocytes, prostate gland, and ovaries (Fig. 4).
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Development-Dependent Expression in Mouse Testis
Because of the limited availability of human testis samples, development-dependent expression of NYD-SP16 mRNA was conducted using its mouse homologue and semiquantitative PCR. The number of cycles completed before entering the exponential growth, recorded by amplifier PE5700 for AK006554, were 24.64 ± 0.4358, 20.8 ± 0.3905, and 19.14 ± 0.2594 in Week 1, Week 4, and Week 7 mouse tissue samples, respectively. The cycles completed for ß-actin (control) for the three times were 18.62 ± 0.3568, 18.30 ± 0.2865, and 18.28 ± 0.3270, respectively. The expression level in the mouse testis was indicated by the Cak:Cactin ratio: the lower the Cak:Cactin value, the higher the expression level in the testes of mice. The Cak:Cactin at ages 1, 4, and 7 wk was 1.323 ± 0.029, 1.137 ± 0.031, and 1.047 ± 0.034, respectively. The difference among the three times was significant (P < 0.01, ANOVA). Further comparison using the Student-Newman-Keuls test revealed significant differences between each pair of times (P < 0.01). These results suggest that expression of NYD-SP16 increases from Week 1 to Week 4 to Week 7, indicating possible involvement in testicular development (Fig. 5).
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ISH of Mouse Testis
Based on the high level of homology between human NYD-SP16 and its mouse homologue, ISH was conducted to identify the type of testicular cells in which NYD-SP16 gene was expressed using mouse Week 7 paraffin-embedded testis sections. ISH results indicated the presence of positive signal in the cytoplasm of spermatogenic cells at every stage, i.e., spermatogonium, primary spermatocyte, spermatid, and mature sperm in the 7-wk-old mouse testis (Fig. 6). Elevated levels of signal were first observed weakly in spermatogonium, and expression increased in cells at more mature stages of spermatogenesis (Fig. 7). No signal was observed in the Leydig cells. The extent of expression as determined by examining tubule staining at different stages is summarized in Figure 7.
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Expression Analysis of Mouse Homologue in Mouse Spermatogenic Cells
The expression pattern of NYD-SP16 mouse homologue was analyzed by reverse transcription PCR. Result showed that the expression of mouse homologue was very weak in primitive type A spermatogonia and then increased from type B spermatogonia to elongating spermatids, indicating that the expression level was increased during spermatogenesis (Fig. 8). The expression pattern in spermatogenic cells was consistent with the results obtained by ISH and semiquantitative PCR in different stages of mouse testis, suggesting that expression of the NYD-SP16 mouse homologue is development dependent.
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Expression and Subcellular Localization of NYD-SP16 GFP Fusion Protein
The deduced structure of NYD-SP16 protein appears to contain a transmembrane domain. The subcellular localization of the fusion protein was examined by transient transfection in human choriocarcinoma JAR cells. Fusion protein was exclusively expressed in the cytomembrane of the JAR cells, whereas the single EGFP protein, as a control, was evenly distributed throughout the whole cell without any compartmentalization (Fig. 9). The result of expression localization is consistent with the predicted transmembrane domain, suggesting that NYD-SP16 is a membrane protein.
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Abnormal Expression of NYD-SP16 in the Testes of Patients with Male Infertility
Membrane hybridization results indicated that NYD-SP16 was not expressed in the testes of patient with SCOS. NYD-SP16 expression in patients with spermatogenic arrest varied. In patients with arrest at the spermatogonium and primary spermatocyte stages, NYD-SP16 expression was not detected. In patients with arrest at the spermatid stage, NYD-SP16 expression level was weak or absent, and in patients that had spermatogenic cells of every stage, NYD-SP16 expression level was high. These results indicate a trend of increasing expression in more mature spermatogenic cells. The expression of ß-actin was comparable in the testes of all the patients (Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To isolate new genes expressed in testis, many approaches have been developed based on differential expression of genes, such as suppression subtractive hybridization [26, 27], differential display technology [28–30], and cDNA microarrays [31]. Of these methods, the use of cDNA microarrays for a genome-wide approach to functional characterization of a large numbers of genes and identification of their expression profiles has increased rapidly. This approach is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, and tissue-specific genes, such as those of the testis [19, 32, 33]. We employed a human testis expression microarray containing 9216 clones from a human testis cDNA library. After microarray hybridization, 731 differentially expressed cDNA clones were identified, and 42 novel genes were cloned and accepted by GenBank [19]. Among these new genes, NYD-SP16, which consists of 1595 bp with an ORF of 762 bp, was initially characterized. A database search revealed high nucleotide and protein sequence homology between NYD-SP16 and a mouse homologue, suggesting that NYD-SP16 is a human-mouse homologous gene. Because of this homology between human and mouse and the limitations and difficulties encountered obtaining human testis tissue, we tested NYD-SP16 homologue expression in mouse testes of different stages and carried out mouse ISH and PCR.
The relationship between NYD-SP16 and spermatogenesis is indicated by several lines of evidence. Multiple tissue distribution of NYD-SP16 showed that it was strongly expressed only in testis and pancreas and was weakly or not expressed in 14 other organs (Fig. 4). Activation of this gene seems required for the function of the testis, i.e., spermatogenesis. Hybridization of the cDNA microarray with probes from adult and embryo testes indicated NYD-SP16 was expressed more highly in human adult testes than in embryo testes, which do not produce sperm (Fig. 1). Similarly, semiquantitative PCR analysis of cDNAs isolated from mouse testes of different development stages revealed that the amount of mRNA expressed in mouse testis increased significantly from Week 1 to Week 4 to Week 7 (Fig. 5). Previous studies revealed that there are only Sertoli cells and spermatogenous cells in the seminiferous tubules of 1-wk-old mice and 6-mo-old human embryos, in which spermatogenesis has not started. However, seminiferous tubules of both 4-wk-old mice and human adults contain not only Sertoli cells and spermatogenous cells but also various spermatogenic cells. At Week 4, mice have reached puberty, spermatocytes begin to undergo meiosis, and round spermatids are generated, but mice of this age are still unable to produce mature sperm; at Week 7, mice can produce mature sperm. Strong expression of NYD-SP16 homologue in adult mice and not in newborn mice was confirmed by microarray hybridization and is correlated with the function of testis, which is spermatogenesis. Results from ISH of adult mouse testes also showed that elevated levels of NYD-SP16 mRNA were first observed weakly in spermatogonium, and expression increased through spermatogenesis (Figs. 6 and 7). This observation was further confirmed by PCR analysis of mouse spermatogenic cells. Electrophoresis results revealed very weak expression in primitive type A spermatogonia and increased expression levels from type B spermatogonia to elongating spermatids, consistent with the results of ISH (Fig. 8). These results imply that the expression of NYD-SP16 is developmentally regulated. NYD-SP16 was expressed in spermatogenic cells but not interstitial cells in normal human testes. ISH result revealed positive signal in spermatogenic cells but not in the Leydig cells. The lack of NYD-SP16 expression in the interstitial cells was further confirmed by blot hybridization in patients with SCOS. These patients have no spermatogenic cells but do have interstitial cells. Thus, NYD-SP16 expression was not detected in these patients (Fig. 10). This result linked the NYD-SP16 specifically to the functions of spermatogenic cells other than interstitial cells of the testis. All of these findings indicated that the expression of NYD-SP16 is developmentally dependent, suggesting that NYD-SP16 may play a role in testicular development and spermatogenesis.
Although there is a close relationship between NYD-SP16 and spermatogenesis, the exact function of NYD-SP16 protein remains elusive. The programs SMART and TMHMM 2.0 predicted that NYD-SP16 protein contains one transmembrane domain, suggesting that this protein probably is located in the plasma membrane. The N-terminal (1–142 amino acids) faces the extracellular space, the middle portion of the protein (143–165 amino acids) transverses the plasma membrane, and the C-terminal (166–254 amino acids) of the protein is presumably exposed to the cytoplasm (Fig. 2). To test this hypothesis, the subcellular localization of the green fluorescent fusion protein by transfection in human choriocarcinoma JAR cells was examined. The NYD-SP16 fusion protein, pEGFP-N2-NYD-SP16, was exclusively expressed in the cytomembrane, supporting the structural hypothesis (Fig. 9). Because NYD-SP16 is a transmembrane protein, it may serve as a channel, transporter, receptor, or membrane-bound enzyme. The enzyme possibility is supported by the expression of the same gene in the pancreas. The pancreas mainly produces digestive enzymes, including amylase, lipase, DNase, and protease. In sperm, many enzymes exists in the sperm acrosome. These enzymes are normally shielded by a layer of cholesterol before the sperm are deposited in the female reproductive tract. Once capacitated in female reproductive tract, the acrosomal enzymes are exposed and able to digest the corona radiata of the oocyte to facilitate the penetration of sperm into the egg. The above hypothesis that NYD-SP16 encodes an enzyme must be tested by determining enzymatic activities of the protein.
The present study also revealed abnormal expression of NYD-SP16 in infertile men. Among the patients with spermatogenic arrest, most of them had no or insufficient expression of NYD-SP16 in the testes. Only the patients with every stage of spermatogenic cells fully expressed NYD-SP16 (Fig. 10). The trend of increasing expression with the presence of the more mature spermatogenic cells in testis is consistent with the development-dependent characteristics of NYD-SP16. The role of NYD-SP16 in male infertility could be examined further using point- or null mutation in a mouse model.
The present study has provided evidence implicating the possible involvement of NYD-SP16 in spermatogenesis. Its development-dependent expression, cellular localization, and protein characteristics point to a possible role for NYD-SP16 in spermatogenesis. Further research is required to determine the physical function of NYD-SP16 protein in spermatogenesis and its relationship with clinical male infertility.
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FOOTNOTES |
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2 Correspondence: Jia Hao Sha, Key Laboratory of Reproductive Medicine of Jiangsu Province, 140 Hanzhong Road, Nanjing Medical University, Nanjing, Jiangsu Province 210029, P.R. China. FAX: 86 25 6662908; shajh{at}njmu.edu.cn
Received: 7 February 2002.
First decision: 28 February 2002.
Accepted: 30 July 2002.
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