OBJECTIVESTo study the expression and the significance of telomerase gene hTERT in testes of infertile male.

METHODSBy using in situ hybridization(ISH) techniques, the expression of telomerase gene hTERT mRNA in testes of 47 infertile male and 10 normal testicular tissues were observed.

RESULTSIn male testes, there was a positive correlation between the expression of hTERT and the quantity and density of germ cells(spermatogonia, spermatocyte, spermatid). The expression of hTERT in some germinal cell of maturation arrest patients were not significantly different with those of normal.

CONCLUSIONSOur results suggest that the deficiency of telomerase might be a factor for germinal cell maturation arrest and there might be some other etiological factors in these patients. Our study provides experimental groundwork for the gene therapy of male infertility.

Humans ; Infertility, Male ; enzymology ; Male ; Spermatids ; Spermatocytes ; Spermatogenesis ; Spermatogonia ; Telomerase ; deficiency ; genetics ; metabolism ; Testis ; enzymology ; physiology

OBJECTIVETo prepare a specific polyclonal antibody against full-length SUN5 for detecting the expression of SUN5 in human germ cells.

METHODSBioinformatic methods were used to compare the full-length SUN5 and its variant SUN5β, and a short peptide was designed based on the differential region to prepare SUN5 antibody. The prepared antibody was used to detect the expression of SUN5 in Ntera-2 cells and in human germ cells by Western blotting and immunofluorescence assay.

RESULTSThe short peptide was correctly synthesized and SUN5 antibody was obtained and purified. Western blotting showed that the prepared antibody was capable of recognizing full-length SUN5 in Ntera-2 cells, and SUN5 expression was localized on the nuclear membrane and in the cytoplasm as shown by immunofluorescence assay. Using this antibody, we detected SUN5 expression in the spermatocytes, round spermatids and sperms in human germ cells.

CONCLUSIONWe successfully prepared SUN5-specific antibody. SUN5 is expressed in the spermatocytes, round spermatids and sperms in human germ cells, suggesting its important role in spermatogenesis.

Antibodies ; chemistry ; Blotting, Western ; Cytoplasm ; metabolism ; Fluorescent Antibody Technique ; Humans ; Male ; Nuclear Envelope ; metabolism ; Proteins ; immunology ; metabolism ; Spermatids ; metabolism ; Spermatocytes ; metabolism ; Spermatogenesis ; Spermatozoa ; metabolism

OBJECTIVETo investigate the expression of transient receptor potential melastatin (TRPM) and transient receptor potential vanilloid (TRPV) channel family genes in rat spermatogenic cells.

METHODSRat spermatogenic cells were isolated by a mechanical procedure and the total RNA was extracted using TRIzol reagent. TRPM and TRPV channel family genes were amplified by RT-PCR and the presence of the target genes was detected by agarose gel electrophoresis. The relative gene expression levels were measured by real-time quantitative RT-PCR.

RESULTSTRPV5, TRPM3, TRPM4 and TRPM7 mRNAs were expressed in rat spermatogenic cells, but TRPV1, TRPV2, TRPV3, TRPV4, TRPV6, TRPM1, TRPM2, TRPM5, TRPM6, TRPM7 and TRPM8 mRNAs were not detected. The relative expressions of TRPM and TRPV mRNA were determined by quantitative real-time RT-PCR. TRPM7 expression was the highest among all the TRPM subtypes in rat spermatogenic cells, at a level equivalent to (0.0430-/+0.0034)% of beta-actin expression. TRPM3 and TRPM4 were also highly expressed, but their expression levels were only approximately 56% and 63% of that of TRPM7, respectively. For the TRPV subfamily, only TRPV5 mRNA was abundantly expressed at the level of (0.0157-/+0.0029)% relative to that of beta-actin.

CONCLUSIONTRPV5, TRPM3, TRPM4 and TRPM7 mRNAs were coexpressed in spermatogenic cells in rats, among which TRPM4 and TRPM7 mRNA were expressed at high levels. TRPM4 and TRPM7 channels may be involved in the regulation of growth, differentiation and maturation of rat spermatogenic cells and are associated with the generation of the sperms.

Animals ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spermatocytes ; cytology ; metabolism ; Spermatogonia ; cytology ; metabolism ; TRPM Cation Channels ; genetics ; metabolism ; TRPV Cation Channels ; genetics ; metabolism

A gene that could be potentially involved in spermatogenesis was identified and characterized by using suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE) with total RNA from type A spermatogonia and pachytene spermatocytes of rat. This gene consists of 3 433 base pairs (bp) with a complete open reading frame (ORF) of 3 171 bp and encodes a putative protein containing 1057 amino acids. The nucleotide sequence displays a 93% identity to mouse ubiquitin-activating enzyme E1, Chr Y 1 (Ube1y1) and an 82% identity to human ubiquitin-activating enzyme E1 (UBE1). The putative protein of this gene contains an ubiquitin-activating enzyme signature 1 and an ubiquitin-activating enzyme active site, which are also existed in mouse ubiquitin-activating enzyme E1, human ubiquitin-activating enzyme E1 et al. So we named this gene as Rattus norvegicus ubiquitin-activating enzyme E1 (Ube1). The sequence of Ube1 was submitted to GenBank and the accession number is EF690356. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that Ube1 was specifically expressed in testis, while its expression was not detected in heart, brain, spleen, lung, liver, muscle, kidney and ovary. Comparison of the expression of Ube1 in different developmental stages of testis and Sertoli cells (real-time PCR) indicated that Ube1 was expressed more highly in spermatogonia than in spermatocytes, spermatids and Sertoli cells. In conclusion, Ube1 is a gene encoding rat ubiquitin-activating enzyme E1 and specifically expressed in testis, which might play a key role in ubiquitin system and influence spermatogenesis.
Animals ; DNA, Complementary ; genetics ; Male ; Rats ; Real-Time Polymerase Chain Reaction ; Spermatids ; metabolism ; Spermatocytes ; metabolism ; Spermatogenesis ; genetics ; Spermatogonia ; metabolism ; Testis ; metabolism ; Ubiquitin-Activating Enzymes ; genetics ; metabolism

Mammalian testis exhibits remarkably high transcriptome complexity, and spermatogenesis undergoes two periods of transcriptional cessation. These make the RNA-binding proteins (RBPs) the utmost importance during male germ cell development. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RBPs implicated in many steps of RNA processing; however, their roles in spermatogenesis are largely unknown. Here, we investigated the expression pattern of 12 hnRNP family members in mouse testes and found that most detected members are highly expressed in the testis. Furthermore, we found that most of the detected hnRNP proteins (hnRNPD, hnRNPK, hnRNPQ, hnRNPU, and hnRNPUL1) display the highest signals in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells, whereas hnRNPE1 exclusively concentrates in the manchette of elongating spermatids. The expression of these hnRNP proteins showed both similarities and specificity, suggesting their diverse roles in spermatogenesis.
Mice ; Male ; Animals ; Heterogeneous-Nuclear Ribonucleoproteins/metabolism* ; Spermatogenesis/genetics* ; Testis/metabolism* ; Spermatids/metabolism* ; Sertoli Cells ; Spermatocytes/metabolism* ; RNA-Binding Proteins/metabolism* ; Mammals

This article reviews the specific expression of many proto-oncogenes during male germ cell development. The normal expression of proto-oncogenes plays an important role in the regulation of spermatogonial mitosis, spermatocyte meiosis as well as spermiogenesis and sperm maturation.
Animals ; Gene Expression Regulation ; Male ; Mice ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; Proto-Oncogenes ; genetics ; Spermatocytes ; metabolism ; Transcription, Genetic

By means of in situ hybridization and immunohistochemistry, the protein localization and gene expression of cyclin B1 in spermatogenic cells were characterized during the spermatogenesis of rabbits. The results showed that the cyclin B1 mRNA in rabbit spermatogenic epithelium was expressed dominantly in primary spermatocytes. The expression was observed in round spermatids with a gradual decline in the process of metamorphosis of the spermatids, but not in elongated spermatids and sperms. Cyclin B1 protein was expressed in mitotic spermatogonia and meiotic spermatocytes and was observed predominantly in round and elongated spermatids. These results indicate that the expression of cyclin B1 mRNA and localization of cyclin B1 protein are dependent on the developmental stages of spermatogenic cells.
Animals ; Cyclin B1 ; genetics ; metabolism ; Gene Expression Regulation, Developmental ; Male ; RNA, Messenger ; genetics ; Rabbits ; Spermatids ; metabolism ; Spermatocytes ; cytology ; metabolism ; Spermatogenesis ; genetics ; Spermatogonia ; metabolism

There has been no study aimed at directly determiningof the periods of Sertoli cell proliferation in birds evendomestic fowl. The aims of this study were to observe thecessation of post-hatching mitotic proliferation of Sertolicells in domestic fowl, and to determine the volumedensity of Sertoli and germ cells during this period. Atotal of 50 Leghorn chicks were used in this study. Thetestes sections of the animals were immunostained withBrdU to observe the proliferation of cells from one to 10weeks of age. The volume density of the Sertoli and germcells were determined using the standard point countingmethod. The volume density of the germ cell nuclei wasinitially less than that of the Sertoli cells but the volumedensity converged by week 6, and remained relativelyconstant until the commencement of meiosis. Clearlabeling of Sertoli and germ cells was observed from week1 to week 7. The only those cells still labeled after 8 weekswere germ cells, indicating that Sertoli cell proliferationhad ceased. Therefore, it is recommended that anyresearch into the testes of domestic fowl should considerthe cessation of Sertoli cell proliferation by approximately8 weeks.
Animals ; Bromodeoxyuridine/metabolism ; Cell Differentiation/physiology ; Cell Growth Processes/physiology ; Chickens/*physiology ; Histocytochemistry/veterinary ; Male ; Mitosis/physiology ; Sertoli Cells/*cytology/metabolism ; Spermatocytes/cytology ; Testis/*cytology/metabolism

OBJECTIVETo analyze defective homologous chromosomal recombination in Han Chinese azoospermic patients.

METHODSTesticular biopsy samples from 7 healthy controls and 7 Han Chinese azoospermic patients including 2 obstructive azoospermia (OA group) and 5 non-obstructive azoospermia (NOA group) were analyzed. Immunofluorescence staining was performed to categorize early stage cells at meiosis prophase and to analyze chromosome pairing and recombination of pachytene spermatocyte. Newly developed meiotic proteins antibodies (anti-SCP3, anti-synaptonemal complex proteins 3, anti-MLH1, anti-Mut-L Homolog 1, anti-CREST, chromosome centromere antibody) were used to identify synaptonemal complex (anti-SCP3), recombination sites (anti-MLH1) and centromere (anti-CREST), respectively. Staging of spermatocyte was determined according to SCP3 formation progression. Qualitative data were compared by a Chi-square test, and ANOVA was used to analyze quantitative data.

RESULTSRespectively, 2346 and 2932 spermatocytes were categorized in the controls and azoospermic patients. The proportions of zygotene cells in both OA group and NOA group were significantly higher than that of the control group. Investigation of 1967 pachytene cells from the controls and 354 pachytene cells from azoospermic patients indicated that the mean MLH1 foci per pachytene cell of NOA group was statistically lower than that of the controls. Compared with the controls, incomplete synaptonemal complexes cells (containing gap and/or split) were significantly increased in the NOA group.

CONCLUSIONDelayed meiosis prophase is relatively common in azoospermic patients, and changes in quantity and distribution of recombination foci may be the cause for spermatogenesis arrest in Han Chinese population.

Adult ; Asian Continental Ancestry Group ; Azoospermia ; genetics ; metabolism ; pathology ; Humans ; Male ; Meiosis ; genetics ; Middle Aged ; Recombination, Genetic ; Spermatocytes ; metabolism ; Synaptonemal Complex ; genetics ; Young Adult

AIMTo immunolocalize the c-mos gene product and to investigate its spatial and temporal expression in mouse testis during postnatal development.

METHODSSemi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization techniques were used to examine c-mos mRNA and indirect immunofluorescence was used to localize c-Mos protein in mouse testis on postnatal days 14, 21, 25, 28, 30, 35, 49 and 70.

RESULTSc-mos mRNA remained low on postnatal days 14-21, increased abruptly from day 25 and peaked on day 30. Its levels decreased a little on day 35 and became almost stable thereafter until day 70. c-mos mRNA was localized in the nucleus and cytoplasm of the spermatocytes and round spermatids. The nuclear staining was much stronger than the cytoplasmic staining. Using a polyclonal anti-c-Mos antibody, Western blotting detected a single band at 43 kDa in testis lysate. c-Mos protein was exclusively localized to the elongating spermatids and was first detected on postnatal day 30. The number of c-Mos-positive spermatids increased progressively till day 49 and stabilized thereafter.

CONCLUSIONThe c-mos gene displays a spatial and temporal expression pattern in the mouse testis during postnatal development at both the mRNA and protein level. This suggests that c-mos might play important roles in spermatogenesis.

Animals ; Fluorescent Antibody Technique, Indirect ; Gene Expression ; Genes, mos ; genetics ; Male ; Mice ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatocytes ; growth & development ; metabolism ; Spermatogenesis ; genetics ; Testis ; metabolism