Role of LM23 in cell proliferation and apoptosis and its expression during the testis development.

LM23, a gene expressed specifically in the testis in a stage-specific manner, has a diverse range of functions that are important in both the life and death of spermatogenic cells. The aim of this study was to further investigate the expression of LM23 in the developing rat testis and the biological function of LM23 in proliferation and antiapoptosis in vitro. Semiquantitative reverse transcription (RT)-PCR and real-time PCR were used to examine the expression of LM23 in testis at different developmental stages. The results suggested that LM23 mRNA levels in the testis increased progressively after birth. The role of LM23 in proliferation was analyzed with cell counting kit-8 (CCK8), colony-forming efficiency (CFE) and flow cytometry assays. The results indicated that ectopic expression of LM23 in 293T cells significantly promoted cell proliferation by increasing cell numbers in S phase. Several methods were used, including CCK8, annexin V and propidium iodide staining and western blotting, to determine the role of LM23 in apoptosis. The results showed that LM23 played a protective role in H2O2-induced apoptosis of 293T cells, mediated at least in part through the Akt/PI3K signal pathway. Taken together, these results provide new insights into the role of LM23 in the development of the testes and spermatogenesis.

[Regulation of gene expression during spermatogenesis at transcriptional level].

Mammalian spermatogenesis is a highly complex cell division and differentiation process occurring in the seminiferous tubules of the testis. This processes are regulated at both transcriptional and post-transcriptional levels, any mistake in this process can lead to infertility. Unveiling the molecular mechanisms of spermatogenesis has important implications for exploring novel contraceptive approach and treatment of infertility. This review addresses recent progress towards understanding the regulation of androgen, estrogen and their receptors, transcription factors and chromatin-associated factors for spermatogenesis at transcriptional level.

LM23 is a novel member of the Speedy/Ringo family at the crossroads of life and death of spermatogenic cell.

LM23 is a gene specifically expressed in the testis of Rattus norvegicus, as previously reported by our laboratory. The aim of the study is to further investigate the biological function of LM23. Several bioinformatic tools were utilized, including PROSITE and BLAST. To determine the subcellullar localization of LM23, a polyclonal antibody specific for LM23 was generated via the immunization of rabbits. The LM23 gene was cloned from rat testis tissue, and LM23 protein was expressed in Escherichia coli. The biological function of LM23 was analyzed with microarray analysis and immunohistochemistry, using a rat model of LM23 gene knockdown. The results suggested that LM23 belongs to the Speedy/Ringo family. LM23 regulated the G1/S and G2/M transitions of the cell cycle during spermatogenesis. Downregulation of the LM23 gene during spermatogenesis could lead to the activation of both the Fas-FasL pathway and the mitochondrial pathway. These novel findings indicate that LM23 has a diverse array of functions that are important in both the life and death of the spermatogenic cell.

A novel missense mutation of the ATP2C1 gene in a Chinese patient with Hailey-Hailey disease.

Benign familial chronic pemphigus (Hailey-Hailey disease, HHD; MIM 169600) is a rare autosomal dominant hereditary disorder characterized by pruritic vesicles, painful erosions and scaly erythematous plaques at the sites of friction and flexures. Mutations in ATP2C1, which encoding the human secretory pathway Ca²(+)/Mn²(+)-ATPase protein 1 (hSPCA1), have been identified as the pathogenic gene of HHD. We found a novel, distinct, heterozygous mutation during study of a Chinese patient with HHD. We identified a C→T transition at nucleotide 1235 (p.Thr352IIe), in exon 13 of ATP2C1. This observation would be useful for genetic counseling and prenatal diagnosis for affected families and in expanding the repertoire of ATP2C1 mutations underlying HHD.

LM23 is essential for spermatogenesis in Rattus norvegicus.

LM23 is a gene with testis-specific expression in Rattus norvegicus. To reveal the function of LM23 in the testis, we used lentivirus-mediated RNA interference (RNAi) to knock down LM23 expression in a tissue-specific manner in vivo. A lentiviral vector expressing a short hairpin RNA (shRNA) targeting LM23 was microinjected into the efferent ducts of R. norvegicus testes. The expression of LM23 in the treated testes was significantly knocked down compared with controls. These LM23-shRNA testes contained germ cells arrested at the spermatocyte stage, and showed increased apoptosis and disregulation of some meiotic genes. The results demonstrate the validity of the RNAi approach for targeting LM23 and reveal that LM23 expression in the testis is crucial for meiosis during spermatogenesis in R. norvegicus.

Identification and characterization of a spermatogenesis-related gene Ube1 in rat testis.

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.

[Sperm mtDNA content and mtDNA4977bp deletion in normal and leukocytospermia men].

OBJECTIVE: To determine the sperm mtDNA content, mtDNA4977bp deletion and ROS in the seminal plasma of normal and leukocytospermia men, and to investigate the correlation of the changes of sperm mtDNA with the increase of leukocytes and reactive oxgygen species (ROS) in the seminal plasma. METHODS: Seventy-eight semen samples from leukocytospermia patients and 31 from healthy donors were divided into 3 layers, supernatant fluid, 30% sperm and 80% sperm, by Percoll gradient centrifugation, their sperm mtDNA content and mtDNA4977bp deletion quantitatively analyzed by real-time PCR, and the level of ROS determined by flow cytometry. RESULTS: The ROS in the seminal plasma and the sperm mtDNA contents of the three layers were all significantly higher in the leukocytospermia group than in the healthy control (P < 0.01). In the supernatant fluid and 80% layers, mtDNA4977bp deletion showed no obvious difference between the control and the leukocytospermia group, but was significantly higher in the 30% layer of the latter (P < 0.01). The ROS level was found positively correlated with the mtDNA content in the 30% (r = 0.347, P < 0.01) and the 80% layer (r = 0.456, P < 0.01), but not in the supernatant layer. CONCLUSION: The increase of leukocytes and ROS may be one of the causes of the enhanced sperm mtDNA content, but has no significant impact on the mtDNA4977bp deletion.

Identification and characterization of a novel spermatogenesis related gene LM23 in rat testis.

The aim of this study is to screen the novel gene related to the spermatogenesis. A novel rat testis-specific gene LM23 was identified and characterized by differential display PCR with total RNA from rat type A spermatogonia, pachytene spermatocytes, and round spermatids. LM23 cDNA consists of 1896 base pairs (bp) with a complete open reading frame of 936 bp, and encodes a putative protein including 312 amino acids, which shares no significant homology with any known gene. The sequence of LM23 was submitted to GenBank and the Accession No. was AF492385. Multitissue Northern blot and RT-PCR analysis showed LM23 was specific expression in testis, while its expression was not detected in other tissues. Real-time PCR analysis showed that the expression level of LM23 was highest in spermatocytes and very low in spermatogonia. In situ hybridization revealed strong cytoplasmic positive signal in spermatocytes and weak signal in spermatids and spermatogonia. These results indicated LM23 possessed the testis-specific and stage-specific expression characteristics, and possibly involved in rat spermatogenesis.

[Screening differentially expressed genes in human bone marrow stromal cells at defined stage of differentiation.].

To screen differentially expressed genes involved in osteogenic differentiation of human bone marrow stromal cells (BMSCs) at defined stages, subtractive cDNA library was established by means of suppression subtractive hybridization. The BMSCs cultured for 12 and 21 d were used as driver and tester, respectively. A subtract library was successfully constructed and five positive clones were selected from the library. Sequencing analysis and homology comparison showed that the five clones differentially expressed in BMSCs cultured for 21 d were at least 90% homologous with the known genes in human GenBank. It was interestingly found that the osteogenic BMSCs cultured for 21 d differentially expressed decorin and Bax inhibitor 1. RT-PCR was performed to confirm the differentially expressed genes. The results showed that the expression of Bax inhibitor 1 was significantly higher in the cells of 21-day than that of 12-day, while the expression of decorin was only detected in the cells of 21-day.

[Specific expression of beta-actin during spermatogenesis in rats].

OBJECTIVE: To screen the stage-specific expression proteins during rats spermatogenesis, and to investigate the beta-actin expression and localization in the tissues of rat testicular. METHODS: Highly enriched type A spermatogonia, pachytene spermatocytes and round spermatids were isolated by STAPUT method (sedimentation velocity at unit gravity, with 2% - 4% BSA gradient in DMEM/F12 medium) respectively to get the total proteins. The difference of protein expression between the three kinds of cells was analyzed by two-dimensional electrophoresis. Then the distribution of beta-actin in rat testicular tissues was investigated using specific anti-beta-actin antibodies by immunohistochemical method. RESULTS: beta-actin was identified as a stage-specific expression protein by two-dimensional electrophoresis. beta-actin protein was more strongly expressed in type A spermatogonia and pachytene spermatocytes, but not in round spermatids. The immunohistochemical results showed that beta-actin was mainly located in the cytoplasm of type A spermatogonia and pachytene spermatocytes and in the nuclei of nearly mature spermatids. CONCLUSION: beta-actin protein is a stage-specific expressed protein and may play an important role in spermatogenesis.