Expression of annexin A7 in spermatogonial stem cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the expression of Annexin A7 in the mouse testis, especially in different types of spermatogonia.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>Annexin A7 is the internal factor of As and Apr spermatogonia, which might be involved in the biological functions of SSCs.</p>

Location of Dctn1 in the mouse testis and sperm and its role in spermiogenesis / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the role of dynactin 1 (Dctn1) in the process of mouse spermiogenesis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Dctn1 plays an important role in mouse spermiogenesis, and mainly affects the formation of the tail of spermatozoa.</p>

46, XX testicular disorder of sex development: report of 2 cases and review of the literature / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the phenotype, pathogenesis and molecular biological features of 46, XX testicular disorder of sex development.</p><p><b>METHODS</b>We obtained the history of 2 patients with 46, XX testicular disorder of sex development, examined the cavitas pelvis by type-B ultrasonography, analyzed the karyotype of the chromosome, and detected the genes SRY, YRRM1, DYS240 and DAZ by PCR amplification.</p><p><b>RESULTS</b>Microrchidia, azoospermia and maldevelopment of secondary sex characteristics were observed in both of the patients, but ultrasonography revealed no female internal genitals. Their chromosome gender was karyotyped as 46, XX, with the SRY gene positive in both, but the YRRM1 gene positive in only one of the cases.</p><p><b>CONCLUSION</b>Chromosome karyotyping and detection of the SRY gene for patients with abnormal sex development can give us an insight into the genetic pathogenesis and provide us with scientific evidence for the diagnosis and treatment of the condition.</p>

Expression of carbonic anhydrase II in human testes and spermatozoa and its clinical significance / 中华男科学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

NYD-SP27, a novel intrinsic decapacitation factor in sperm / 亚洲男科学杂志(英文版)

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.

Characterization of Spindlin1 isoform2 in mouse testis / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate the expression of Spindlin 1 (Spin 1) isoform2 and assess its function in mouse testis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Spin1 isoform2 is a protein expressed highly in adult testis, which might be involved in spermatogenesis and could be necessary for normal sperm motility.</p>

Update of the researches on sperm proteome / 中华男科学杂志

Proteomics and polemic techniques are among the most valuable approaches in the research of life science in this new century, marking the beginning of a post-genome era. Two-dimensional electrophoresis and mass spectrometry, applied as key techniques in proteomic research, have given rise to new research strategies and improved the efficiency of researchers in exploring the unknown field. Its introduction into the exploration of spermatozoal proteins has given us so many pleasant surprises. This review presents some essential information about proteomics and two-dimensional electrophoresis and mass spectrometry, with a brief introduction of the recent progress in the researches on human sperm proteome, capacitation-related sperm proteins, sperm-egg interaction-related proteins, and sperm-immunity which has made great senses in the clinical problems.

Effects of Jujingwan on nitric oxide and superoxide dismutase in seminal plasma of asthenospermia patients / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the possible action mechanism of Jujingwan on asthenospermia.</p><p><b>METHODS</b>Semen routine analyses and determination of nitric oxide (NO) concentration and superoxide dismutase (SOD) activity in seminal plasma were performed in 34 cases of asthenospermia. The changes of NO concentration and SOD activity before and after the treatment with Jujingwan were observed.</p><p><b>RESULTS</b>Compared with pre-treatment, there was no significant change in NO concentration, and the activity of SOD decreased significantly after the treatment ([95.97 +/- 20.75] microg/L vs [6.14 +/- 19.99] microg/L). There was negative correlation between NO concentration and SOD activity before the treatment (r = -0.246, P < 0.05).</p><p><b>CONCLUSION</b>Jujingwan can significantly improve sperm viability in patients with asthenospermia. However, the excellent effects of Jujingwan are not displayed in the changes of NO concentration and SOD activity.</p>

Experimental study on the effects of jujingwan on oligospermia / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Jujingwan on the spermatozoal ultrastructure and apoptosis of germ cells in oligospermia patients.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells / 亚洲男科学杂志(英文版)

<p><b>AIM</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Identification and characterization of the BGR-like gene with a potential role in human testicular development/spermatogenesis / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate the roles of the BGR-like gene in testicular development/spermatogenesis.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The BGR-like gene may play an important role in spermatogenesis/testicular development and may be correlated with male infertility.</p>

Identification of a novel testis-specific gene and its potential roles in testis development/spermatogenesis / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To identify and characterize a novel gene with potential roles in testis development and spermatogenesis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>NYD-SP5 is a newly found testis-specific gene with potential roles in testis development and spermatogenesis through a calmodulin-activated enzyme.</p>

Expression of a novel beta adaptin subunit mRNA splice variant in human testes / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To identify a novel isoform of adaptin 2 beta subunit (named Ap2beta-NY) and to investigate its relationship with testicular development and spermatogenesis.</p><p><b>METHODS</b>Using a human testis cDNA microarray, a clone (Ap2beta-NY), which was strongly expressed in adult testes but weakly expressed in embryo testes, was sequenced and analyzed. Using polymerase chain reaction (PCR), the tissue distribution and expression time pattern of Ap2beta-NY were determined.</p><p><b>RESULTS</b>Ap2beta-NY was identified and has been deposited in the GenBank (AY341427). The expression level of Ap2beta-NY in the adult testis was about 3-fold higher than that in the embryo testis. PCR analysis using multi-tissue cDNA indicated that Ap2beta-NY was highly expressed in the testis, spleen, thymus, prostate, ovary, blood leukocyte and brain, but not in the heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. In addition, Ap2beta-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome, which implied that, in the testis, Ap2beta-NY was restrictively expressed in germ cells.</p><p><b>CONCLUSION</b>Ap2beta-NY is an isoform of Ap2beta and may be involved in regulating the process of spermatogenesis and testis development.</p>

Expression of a novel dipeptidyl peptidase 8 (DPP8) transcript variant, DPP8-v3, in human testis / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate the role of a novel dipeptidyl peptidase 8 transcript variant (DPP8-v3) gene in testis development and/or spermatogenesis.</p><p><b>METHODS</b>A human testis cDNA microarray was hybridized with mRNA of human adult and fetal testes. Differentially expressed clones were sequenced and characterized and their expression was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Southern-blot analysis.</p><p><b>RESULTS</b>A new transcript variant of the human dipeptidyl peptidase (DPP8), exhibiting a 5-fold higher expression level in human adult than that in fetal testes, was cloned and was named DPP8 variant 3 (DPP8-v3). The full-length sequence of DPP8-v3 was 3,030 bp, encoding a protein of 898 amino acids.</p><p><b>CONCLUSION</b>DPP8-v3 is a novel human DPP8 transcript variant highly expressed in the adult testis. Similar to DPPIV, DPP8-v3 may play a key role in the immunoregulation of testes and accordingly may influence spermatogenesis and male fertility.</p>

Expression of a novel alternative transcript of the novel retinal pigment epithelial cell gene NORPEG in human testes / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To identify a novel alternative transcript of the novel retinal pigment epithelial cell gene (NORPEG) expressed in the human testis.</p><p><b>METHODS</b>A human testis cDNA microarray was established and hybridized with cDNA probes from human fetal testes, adult testes and human spermatozoa. Differentially expressed clones were sequenced and analyzed. One of these clones was a short transcript of NORPEG which we proceeded to analyze by RT-PCR.</p><p><b>RESULTS</b>The novel short alternative transcript of NORPEG was isolated and named sNORPEG. It was 3486 bp in length and contained a 2952-bp open reading frame, encoding a 110.4-kDa protein of 983 amino acids. Amino acid sequence analysis showed that the sNORPEG protein contains six ankyrin repeats and two coiled-coil domains. It shares a high homology with the NORPEG and ankycorbin proteins in both its sequence and motifs. Blasting the human genome database localized sNORPEG to human chromosome 5p13.2-13.3. Expression profiles showed that sNORPEG was expressed in human fetal testes, adult testes and spermatozoa. Moreover, sNORPEG was found to be ubiquitously expressed in human tissues.</p><p><b>CONCLUSION</b>sNORPEG is expressed in different developmental stages of the testis and encodes a protein that may have roles in human testis development and spermatogenesis.</p>

Expression of a novel pyridoxal kinase mRNA splice variant, PKH-T, in human testis / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To identify the genes specifically expressed in human adult and fetal testes and spermatozoa.</p><p><b>METHODS</b>A human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template. The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank.</p><p><b>RESULTS</b>A novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1 069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene, HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different developmental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients.</p><p><b>CONCLUSION</b>PKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility.</p>

Application of two-dimensional electrophoresis and mass spectrometry in the research of testicular proteins / 中华男科学杂志

<p><b>OBJECTIVES</b>To evaluate the application of two-dimensional electrophoresis and mass spectrometry in the research of proteins expressed in testis.</p><p><b>METHODS</b>Protein from adult ICR mouse testes was extracted by two-dimensional electrophoresis. Two protein spots were cut from the gel, then the proteins were digested in-gel by enzyme and the generated peptides were measured by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The proteins were identified through database searching.</p><p><b>RESULTS</b>Two spots in Commasie Brilliant Blue-stained gel were identified as serum albumin and protein disulfide isomerase by database searching.</p><p><b>CONCLUSIONS</b>This rapid high resolution and efficient method is a very powerful way to analyze testicular proteins.</p>

Expression of a novel bHLH-Zip gene in human testis / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To identify specifically expressed genes in the adult and fetal testes.</p><p><b>METHODS</b>A human testis cDNA microarray was established. Then the mRNA of adult and fetal testis was purified and probes were prepared by a reverse transcription reaction with the testis mRNA as template. The microarray was hybridized with probes of adult and fetal testes. The nucleic sequences of differentially expressed genes were determined and homologies were searched in the databases of the GenBank.</p><p><b>RESULTS</b>When hybridized with adult or fetal testis probes, the positive clones were 96.8 % and 95.4 %, respectively. Among these genes, one was a new testis-specific gene, which was named TSP1. TSP1 was highly expressed in human adult testis. The cDNA of TSP1 was 1,484 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AF333098). TSP1 was also determined as Interim Gen Symbol (Unigene, No. Hs.98266). Protein analysis showed that TSP1 contained two functional domains: an N-terminal basic helix-loop-helix (bHLH) and a C-terminal leucine zipper (Zip). Homologous analysis showed that the 430 amino acid sequences deduced from the 1293 bp open reading frame (ORF) had a homology with the human gene FLJ2509 (AK098575). TSP1 had also a sequence homology with Spz 1 protein of mouse. Expression profiles showed that TSP1 was specifically and strongly expressed in the testis.</p><p><b>CONCLUSION</b>TSP1 is a gene highly expressed in adult testis. It may play an important role in spermatogenesis in the humans.</p>

Study on the relationship of balanced translocation t(1;12) with spermatogenesis / 中华男科学杂志

<p><b>OBJECTIVES</b>To investigate the relationship between chromosome balanced translocation t(1;12) (q24;q24) and spermatogenesis in infertile twin brothers.</p><p><b>METHODS</b>For twin brothers, karotype were determined. The levels of testosterone, FSH and LH were detected. YRRM1, DAZ and DYS240 were analyzed. In younger brother a biopsy was taken from testis.</p><p><b>RESULTS</b>Chromosome analysis for both twin brothers revealed a karotype of 46, XY, t(1;12) (q24;q24). Sperm count was less than 1.0 x 10(6)/ml. There was no deletion for YRRM1, DAZ and DYS240 gene on Y chromosome. Photomicrograph of seminiferous tubules showed the arrest of spermatogenesis.</p><p><b>CONCLUSIONS</b>Chromosome balanced translocation t(1;12) (q24;q24) may be the cause of the spermatogenesis arrest in infertile twin brothers. Gene in the point of translocation may be related to spermatogenesis.</p>