Caveolae-dependent endocytosis is required for class A macrophage scavenger receptor-mediated apoptosis in macrophages.

SR-A (class A macrophage scavenger receptor) is a transmembrane receptor that can bind many different ligands, including modified lipoproteins that are relevant to the development of vascular diseases. However, the precise endocytic pathways of SR-A/mediated ligands internalization are not fully characterized. In this study, we show that the SR-A/ligand complex can be endocytosed by both clathrin- and caveolae-dependent pathways. Internalizations of SR-A-lipoprotein (such as acLDL) complexes primarily go through clathrin-dependent endocytosis. In contrast, macrophage apoptosis triggered by SR-A-fucoidan internalization requires caveolae-dependent endocytosis. The caveolae-dependent process activates p38 kinase and JNK signaling, whereas the clathrin-mediated endocytosis elicits ERK signaling. Our results suggest that different SR-A endocytic pathways have distinct functional consequences due to the activation of different signaling cascades in macrophages.

Integrity of the blood-testis barrier in healthy men after suppression of spermatogenesis with testosterone and levonorgestrel.

STUDY QUESTION: Do exogenous male hormonal contraceptives that suppress intratesticular testosterone and spermatogenesis interfere with the blood-testis barrier integrity in men? SUMMARY ANSWER: When spermatogenesis was suppressed by testosterone alone or combined with levonorgestrel (LNG) treatment in men, the structural appearance of Sertoli cell tight junctions remained intact in the human testis. WHAT IS ALREADY KNOWN: Testosterone promotes the integrity of the blood-testis barrier. Intratesticular androgen deprivation induced by exogenous testosterone plus a progestin to suppress spermatogenesis in a contraceptive regimen may disturb the structural and functional integrity of the blood-testis barrier. STUDY DESIGN, SIZE AND DURATION: Testicular biopsies were obtained from a sub-study of a randomized clinical trial of 36 healthy Chinese men who were treated for 18 weeks and followed for at least a 12-week recovery period. PARTICIPANTS/MATERIAL, SETTING, METHODS: Healthy Chinese male volunteers (27-48 years) were randomized to two treatment groups (n = 18/group) for 18 weeks: (1) testosterone undecanoate (TU) 1000 mg i.m. injection followed by a 500 mg injection every 6 weeks and (2) TU + LNG 250 µg orally daily. Blood samples were obtained from all participants before and during treatment and at the end of the recovery phase. Open testicular biopsies for this study were obtained from four men before treatment and from four men in each of the TU and TU + LNG groups at 2 and 9 weeks of treatment. The presence of antisperm antibodies was checked in the archived serum samples of the subjects at baseline, during treatment and at the end of the recovery period. Stored testicular biopsy samples from cynomolgus monkeys treated with either sub-cutaneous testosterone or placebo for 12 weeks were used for additional protein expression studies. MAIN RESULTS AND ROLE OF THE CHANCE: Expression of blood-testis barrier associated proteins quantified by immunohistochemistry (claudin 3, claudin 11, junctional adhesion molecule-A, zonula occludens-1) remained unchanged despite a significant decrease in the numbers of pachytene spermatocytes and round spermatids in the seminiferous tubules at 9 weeks in the TU + LNG group. This was confirmed by immunoblots showing a lack of quantitative change in these tight junction proteins in monkeys after testosterone treatment. There were no increases in serum antisperm antibodies in the volunteers during the study. LIMITATIONS/REASONS FOR CAUTION: The duration of the study was short and the long-term effects of male hormonal contraceptive treatments on the integrity of the blood-testis barrier remain to be determined. WIDER IMPLICATIONS OF THE FINDINGS: This study supports the safety of male hormonal contraceptive treatment and does not corroborate the previous findings of disturbed immunological integrity of the blood-testis barrier from animal studies such as androgen receptor knockout mice and exogenous hormonal treatment in rats. STUDY FUNDING/COMPETING INTEREST: The study was supported by grants from the Contraceptive Research and Development Program and the Mellon Foundation (MFG-02-64, MFG-03-67), Endocrine, Metabolism and Nutrition Training Grant (T32 DK007571), the Clinical and Translational Science Institute at Los Angeles Biomedical and Harbor-UCLA Medical Center (UL1RR033176 and UL1TR000124) and the Los Angeles Biomedical Research Institute Summer High School Student Program.

[Expression of annexin A7 in spermatogonial stem cells].

OBJECTIVE: To study the expression of Annexin A7 in the mouse testis, especially in different types of spermatogonia. METHODS: We prepared Annexin A7 recombinant protein using prokaryotic expression, adsorbed the Annexin A7 antibody with it after identified by mass spectrometry, and detected the expression of Annexin A7 by Western-blot and immunohistochemistry. RESULTS: Annexin A7 was expressed in a development-dependent manner in the spermatogonia of the prepubertal mice and in the type-A single (As) and type-A paired (Apr) spermatogonia of adult mice. These results were confirmed by the co-localization of Annexin A7 and Stra8, a known determinant of differentiated spermatogonial stem cells (SSCs). CONCLUSION: Annexin A7 is the internal factor of As and Apr spermatogonia, which might be involved in the biological functions of SSCs.

[Location of Dctn1 in the mouse testis and sperm and its role in spermiogenesis].

OBJECTIVE: To investigate the role of dynactin 1 (Dctn1) in the process of mouse spermiogenesis. METHODS: Western blot and indirect immunofluorescence were used to analyze the expression and location of Dctn1 in the mouse testis and spermatozoa. The highest efficiency of small interference RNA (siRNA) was verified by GC2-spd cell line in vitro and in vivo studies, respectively. Dctn1 siRNA mixed with the indicator (0.4% trypan blue) was injected into the seminiferous tubules of 3-week-old ICR mice through rete testis microinjection, and negative control siRNA injected into the control testes. The normal group included 3-week-old ICR mice that did not receive any treatment. Spermatozoa were collected from the cauda epididymis 3 weeks after siRNA injection for morphological analysis. RESULTS: Dctn1 was mainly localized in the tail of spermatozoa. After interference, the sperm tail abnormality in the Dctn1 siRNA group was (23.57 +/- 0.55)%, significantly higher than (12.35 +/- 2.29)% in the control (P < 0.01, n = 3), and it was (3.37 +/- 0.69)% in the normal group. CONCLUSION: Dctn1 plays an important role in mouse spermiogenesis, and mainly affects the formation of the tail of spermatozoa.

[Expression of carbonic anhydrase II in human testes and spermatozoa and its clinical significance].

OBJECTIVE: To investigate the expression of carbonic anhydrase II (CA2) in human testes and spermatozoa, and to compare the expressions of CA2 in ejaculated spermatozoa between normozoospermic and asthenozoospermic men. METHODS: The localization of CA2 in human testes was observed by immunohistochemistry, and that in human sperm by immunofluorescence. Western blot was used to detect the expression of CA2 in the semen samples obtained from 16 normozoospermic and 16 asthenozoospermic volunteers. RESULTS: The CA2 protein was shown to be localized in the tail of elongating spermatids by immunohistochemistry and in the flagellum of human sperm by immunofluorescence. Western blot revealed an obviously increased expression of CA2 in the spermatozoa of asthenozoospermic patients, with statistically significant difference from the normozoospermic group (1.84 +/- 0.32 vs 1.41 +/- 0.26, P < 0.05). CONCLUSION: The CA2 protein is expressed in the spermatogenic stage of elongating spermatids in human testes and localized in the sperm tail. The expression of CA2 is significantly increased in the spermatozoa of asthenozoospermic men, which might be responsible for low sperm motility.

Role of translation by mitochondrial-type ribosomes during sperm capacitation: an analysis based on a proteomic approach.

Mammalian spermatozoa contain a complex population of mRNAs, some of which have been demonstrated to be translated de novo by mitochondrial-type ribosomes using D-chloramphenicol (CP), a specific inhibitor of mitochondrial translation. However, little is known about the functions of these mRNAs in mature sperm. In the present study, differential proteomic approaches were applied to study sperm protein profiles translated by mitochondrial-type ribosomes using the inhibitor CP and 44 proteins were identified with lower expression in CP-treated sperm in comparison to capacitated sperm (ratio >or= 1.5, p<0.05). Results of Western blot and real-time PCR suggest that four proteins were translated by mitochondrial-type ribosomes. Bioinformatics analysis indicated that 26 of 44 proteins were involved in some critical processes correlated to sperm-egg interaction event. In addition, Mups, whose functions in reproduction have never been studied, were chosen for further study. Our results showed that Mups proteins were localized to the acrosome and flagellum of precapacitated sperm, and were also expressed in the equatorial segment of capacitated sperm. The depletion of Mups using neutralizing antibodies significantly inhibited capacitation in a dose-dependent manner, subsequently inhibited acrosome reaction and sperm-egg fusion. In summary, mitochondrial translation during capacitation can store proteins beneficial for sperm-egg interaction.

The proteomic analysis of human neonatal umbilical cord serum by mass spectrometry.

AIM: To investigate the proteome composition and function of human neonatal arterial umbilical cord. METHODS: Serum proteomic analyses were performed on samples from both males and females by using a combination of techniques: (1) removal of six high-abundance proteins, (2) tryptic digestion of low-abundance proteins, (3) separation of peptide mixtures by reverse-phase high-performance liquid chromatography (RP-HPLC), and (4) peptide identification using electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: A total of 837 non-redundant proteins were identified, with 213 male-specific and 239 female-specific proteins. Among them, 319 proteins were identified by at least 2 distinct peptides. The subcellular localization, function, and pathway involvement for each of the identified proteins were analyzed. A comparison of this neonatal proteome to that of adult serum proteome revealed novel biomarkers, such as alpha-fetoprotein and periostin that were specific to newborn infants. CONCLUSION: These data will contribute to a better understanding of the composition of umbilical cord serum and aid the discovery of novel biomarkers for the prenatal diagnosis of fetal abnormalities.

NYD-SP27, a novel intrinsic decapacitation factor in sperm.

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.

The protein profile of mouse mature cumulus-oocyte complex.

In mammals, the cumulus-oocyte complex (COC) is the main component of ovarian follicles. Bi-directional communication between oocytes and surrounding cumulus granulosa cells is essential for the development of oocytes and ovarian follicles. In this study, we performed proteomic profiling of mouse mature COC, using two-dimensional gel electrophoresis and mass spectrometry. A total of 259 protein spots were identified, which correspond to 156 individual proteins. We also discovered some protein families, which may play important roles in ovarian follicular development. Immunostaining was conducted to determine the subcellular localization of specific proteins from selected protein families of interest, and to examine their expression patterns during follicle development. These data provide valuable information for future studies to identify proteins involved in ovarian follicular development and related reproductive abnormalities.

Characterization of Spindlin1 isoform2 in mouse testis.

AIM: To investigate the expression of Spindlin 1 (Spin 1) isoform2 and assess its function in mouse testis. METHODS: First, reverse-transcription polymerase chain reaction (RT-PCR) was used to determine whether Spin1 isoform2 is present in mouse testis. Then the expression patterns of the isoform between newborn and adult mice testes were compared by immunoblot analysis. Finally, the diversity of its localization in mice testes at different ages (days 0, 7, 14, 21, 28 and 60) was observed by immunohistochemistry. The localization of the protein in mouse sperm was also investigated by immunofluorescence. RESULTS: The RT-PCR results show that Spin1 isoform2 is present in mouse testis. As shown by immunoblot analysis, the isoform was more highly expressed in adult testes compared with newborn testes. Interestingly, Spin1 isoform2 did not show up in the cytoplasm of primary spermatocytes until day 14. Also, the protein exists at the tail of the mouse sperm. CONCLUSION: Spin1 isoform2 is a protein expressed highly in adult testis, which might be involved in spermatogenesis and could be necessary for normal sperm motility.

transient scrotal hyperthermia and levonorgestrel enhance testosterone-induced spermatogenesis suppression in men through increased germ cell apoptosis.

CONTEXT: In rodents and monkeys, a combination of hormonal and physical agents accelerates germ cell death. OBJECTIVE: A "proof of concept" study was performed to investigate whether addition of heat exposure or a progestin to an androgen induces germ cell death and more complete and rapid spermatogenesis suppression. DESIGN AND SETTINGS: A randomized clinical trial was performed at academic medical centers. PARTICIPANTS: We treated four groups of healthy male volunteers (18 per group) for 18 wk: 1) testosterone undecanoate (TU) 1000 mg im (first dose), followed by 500 mg im every 6 wk; 2) submersion of scrota at 43 C in water for 30 min/d for 6 consecutive days; 3) TU plus heat; and 4) TU plus oral levonorgestrel (LNG) 250 microg/d. MAIN OUTCOME MEASURES: Semen parameters, testicular histology, and germ cell apoptosis were the main outcome measures. RESULTS: Heat alone and TU plus heat suppressed sperm counts more than TU alone by wk 6. By wk 9, recovery began in the heat only group, whereas spermatogenesis remained suppressed in the TU plus heat group. Oral LNG plus TU suppressed spermatogenesis earlier and more severely than TU alone. At wk 2, significantly greater germ cell apoptosis occurred in heat and heat plus TU subjects, but not in subjects without heat treatment, compared with pretreatment subjects. By 9 wk, markedly smaller seminiferous tubule diameters and fewer spermatocytes and spermatids were noted in all 12 biopsies from men receiving TU, TU plus LNG, with most dramatic differences for the TU plus heat group, whereas no differences from pretreatment biopsies were observed in men who received heat treatment only. CONCLUSIONS: Heat causes a rapid and transient suppression of spermatogenesis. TU plus heat resulted in low-sperm output that was maintained by continuous treatment with TU. Addition of an oral progestin accelerated spermatogenesis suppression by TU alone. Increased germ cell apoptosis contributed to suppression of spermatogenesis.

Cloning and characterization of a novel sperm tail protein, NYD-SP28.

In this study, a gene coding a novel human sperm tail protein named NYD-SP28 was cloned and characterized using a complementary DNA (cDNA) microarray. Its expression was 3.5 times higher in human testis than in fetal testis, and very high in human spermatozoa. The full length of NYD-SP28 cDNA was 1798 bp and encoded a 484-amino-acid protein. Motif analysis revealed that the protein contained a cluster of phosphorylation sites, N-glycosylation sites and N-myristoylation sites. Immunohistochemical analysis of normal human testes showed that NYD-SP28 was expressed in the cytoplasm of spermatogenic cells but not in interstitial cells. The EGFP-NYD-SP28 fusion protein was also localized in the cytoplasm of transfected 7721 cells. In human spermatozoa, NYD-SP28 immunoreactivity was detected in entire sperm tail. Using the two-dimensional (2-D) gel electrophoresis and immunoblotting technique, NYD-SP28 was found to be post-translationally modified during sperm capacitation. In conclusion, these results suggest that NYD-SP28 is a new human sperm tail protein and might play an important role during sperm capacitation.

A two-dimensional electrophoresis reference map of human ovary.

The ovary plays a central role in oogenesis and gonadal hormone secretion. Proteomic analysis is a valuable approach for gaining an increased understanding of the molecular nature of the ovary. In this work, two-dimensional electrophoresis for protein separation followed by matrix-assisted laser desorption/ionization mass spectrometry and database searches, identified 231 protein spots corresponding to 138 individual proteins that were found in gels representing both the follicular and luteal phases. The data were used to construct a database online (http://reprod.njmu.edu.cn/2d). The identified proteins were functionally classified into seven groups: (1) cell signaling/communication, (2) cell division, (3) gene/protein expression, (4) metabolism, (5) cell structure and motility, (6) cell/organism defense, and (7) unclassified. Among the proteins identified, 47% had not been previously reported in the human ovary. In addition, a number of disease-related proteins were identified in this protein map, including some cancer- and polycystic ovarian syndrome-related proteins. Two proteins with phosphorylation were verified by Western blot analysis. Comparison of protein abundance between follicular and luteal stages produced seven protein spots that had been identified in our database. This study provides a preliminary reference map of normal human ovary that will form a basis for comparative studies on normal and pathological conditions of the human ovary and may serve as a potential tool for clinical diagnosis, therapeutics, and prognosis.

Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells.

AIM: To study polyethylenimine (PEI)-mediated in vivo gene transfection into testis cells and preliminary functional research of spermatogenic cell-specific gene NYD-SP12 using this method. METHODS: PEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcellular localization in spermatogenic cells at different stages were analyzed with fluorescent microscopy and propidium iodide staining. RESULTS: With the mediation of PEI, the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells (especially in primary spermatocytes). Transfection into Sertoli cells was not observed. The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis. CONCLUSION: PEI can efficiently mediate gene transfer into spermatocytes. Thus, it might be useful for the functional research of spermatogenic-cell specific genes such as the NYD-SP12 gene. In our study, the NYD-SP12 protein was visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the detailed function of NYD-SP12 in spermatocytes.

[Experimental study on the effects of jujingwan on oligospermia].

OBJECTIVE: To investigate the effects of Jujingwan on the spermatozoal ultrastructure and apoptosis of germ cells in oligospermia patients. METHODS: We treated 50 oligospermia patients with Jujingwan and observed the spermatozoal ultrastructure, the apoptosis of germ cells and the changes in the DNA ploidy proportion of spermatogenic cells by electron microscopy and FCM before the treatment and 3, 6, 9 and 12 months after it. RESULTS: Jujingwan increased sperm acrosome base density 6 months after the treatment and remarkably improved the integrity of acrosome membrane 12 months after it, with no obvious pathological changes in the nuclei and tails. Three months after the treatment, cell debris and apoptotic cells decreased significantly as compared with pre-treatment (P < 0. 05) , and very significantly 12 months after the treatment (P <0. 01). The proportion of haploid spermatozoa increased very significantly (P <0.01) , and the lost primary spermatocytes decreased significantly (P <0. 05) compared with pre-treatment. CONCLUSION: Jujingwan can increase the density of sperm acrosome base and improve the pathological changes of acrosome membrane in oligospermia patients; it can improve the activity of acrosome enzyme and the integrity of acrosome membrane, decrease the apoptosis rate of germ cells and sperm and increase the percentage of haploid spermatozoa; it can also reduce the percentage of apoptotic bodies and diploid sperm cells. It is indicated that Jujingwan can inhibit the apoptosis of germ cells and sperm and improve spermatogenesis in oligospermia patients.

Distinct temporal requirements for autophagy and the proteasome in yeast meiosis.

Meiosis is a special type of cellular renovation that involves 2 successive cell divisions and a single round of DNA replication. Two major degradation systems, the autophagy-lysosome and the ubiquitin-proteasome, are involved in meiosis, but their roles have yet to be elucidated. Here we show that autophagy mainly affects the initiation of meiosis but not the nuclear division. Autophagy works not only by serving as a dynamic recycling system but also by eliminating some negative meiotic regulators such as Ego4 (Ynr034w-a). In a quantitative proteomics study, the proteasome was found to be significantly upregulated during meiotic divisions. We found that proteasomal activity is essential to the 2 successive meiotic nuclear divisions but not for the initiation of meiosis. Our study defines the roles of autophagy and the proteasome in meiosis: Autophagy mainly affects the initiation of meiosis, whereas the proteasome mainly affects the 2 successive meiotic divisions.

Deficiency of Mkrn2 causes abnormal spermiogenesis and spermiation, and impairs male fertility.

Although recent studies have shed insights on some of the potential causes of male infertility, new underlining molecular mechanisms still remain to be elucidated. Makorin-2 (Mkrn2) is an evolutionarily conserved gene whose biological functions are not fully known. We developed an Mrkn2 knockout mouse model to study the role of this gene, and found that deletion of Mkrn2 in mice led to male infertility. Mkrn2 knockout mice produced abnormal sperms characterized by low number, poor motility, and aberrant morphology. Disruption of Mkrn2 also caused failure of sperm release (spermiation failure) and misarrangement of ectoplasmic specialization (ES) in testes, thus impairing spermiogenesis and spermiation. To understand the molecular mechanism, we found that expression of Odf2, a vital protein in spermatogenesis, was significantly decreased. In addition, we found that expression levels of Odf2 were decreased in Mkrn2 knockout mice. We also found that MKRN2 was prominently expressed in the sperm of normal men, but was significantly reduced in infertile men. This result indicates that our finding is clinically relevant. The results of our study provided insights into a new mechanism of male infertility caused by the MKRN2 downregulation.

Molecular cloning and expression of a novel alternative splice variant of BRDT gene.

In this study, a human adult testis cDNA microarray was constructed and hybridized with (33)P-labeled human adult testis, embryo testis and sperm cDNA probes, respectively. A novel alternative splice variant of BRDT gene, named BRDT-NY, presumably involved in testicular function was cloned. It was expressed 3.96-fold more in human adult than embryo testis and also expressed in human spermatozoa. Similarly, RT-PCR revealed a differential expression pattern of this gene in human adult testes and fetal testes. The full length of BRDT-NY was 3438 bp and contained a 2883 bp open reading frame, encoding a 960-amino-acid protein. Sequence analysis showed that it has two bromodomains in N-terminal of the protein. Multiple tissue RT-PCR results showed that BRDT-NY was exclusively expressed in testis. mRNA expression of BRDT-NY gene was deleted in some azoospermic patients' testes. These experiments suggested that BRDT-NY gene may have an important role in the process of spermatogenesis and may be correlated with male infertility.

Identification and characterization of the BGR-like gene with a potential role in human testicular development/spermatogenesis.

AIM: To investigate the roles of the BGR-like gene in testicular development/spermatogenesis. METHODS: A human testis cDNA microarray was hybridized with probes from human adult testes and embryo testes. The differentially expressed clones were sequenced and analyzed. Expression of the BGR-like gene was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: A new gene exhibiting 50-fold difference in expression level between adult and fetal human testes was cloned and named the BGR-like gene. The cDNA consisted of 2500 nucleotides and had an open reading frame of 1437 nucleotides encoding a putative protein of 497 amino acid residues. Homologous comparison showed that the BGR-like gene was a new alternative splicing variant of the BGR gene and had sequence homology with the bubblegum gene of human, mouse, rat and Drosophila. Protein motif analysis of the BGR-like gene revealed that it contained a conserved adenosine monophosphate (AMP)-binding domain and a fatty acyl-CoA synthetase signature motif which existed in all acyl-CoA synthetases. The BGR-like gene transcript was imperceptibly expressed in human fetal testes, highly in human adult testes and moderately in elderly testes and human Leydig cells. RT-PCR-based tissue distribution experiments showed that the BGR-like gene was exclusively expressed in testes and was a testes-specific isoform of the BGR gene. A BGR-like gene transcript was not detected in some azoospermic testes. CONCLUSION: The BGR-like gene may play an important role in spermatogenesis/testicular development and may be correlated with male infertility.

Identification of a novel testis-specific gene and its potential roles in testis development/spermatogenesis.

AIM: To identify and characterize a novel gene with potential roles in testis development and spermatogenesis. METHODS: A cDNA microarray was constructed from a human testis large insert cDNA library and hybridized with probes of human or mouse adult and fetal testes. Differentially expressed genes were isolated and sequenced. RT-PCR was used to test the tissue distribution of the genes of interest and in situ hybridization was performed to localize the gene expression in the mouse testis. A range of bioinformatical programs including Gene Runner, SMART, NCBI Blast and Emboss CpGPlot were used to characterize the new gene's feature. RESULTS: A novel testis-specific gene, NYD-SP5, was differentially expressed in fetal and adult testes. The deduced protein structure of NYD-SP5 was found to contain an IQ motif (a short calmodulin-binding motif containing conserved Ile and Gln residues), a Carbamate kinase-like domain, a Zn-dependent exopeptidase domain and a lactate dehydrogenase (LDH) C-terminal-like domain. RT-PCR analysis revealed that NYD-SP5 was predominantly expressed in the testis but not in other 15 tissues examined. In situ hybridization and RT-PCR examinations revealed that the expression of NYD-SP5 was confined in the male germ cell but not present in the somatic cell in the testes. CONCLUSION: NYD-SP5 is a newly found testis-specific gene with potential roles in testis development and spermatogenesis through a calmodulin-activated enzyme.