Overexpression of human SPATA17 protein induces germ cell apoptosis in transgenic male mice.

SPATA17 is a new testis-specific-expressed gene that is involved in Spermatogenesis process. Previous studies show that SPATA17 was involved in acceleration of cell apoptosis in GC-1 cell lines. To further investigate specific roles of SPATA17 in Spermatogenesis in vivo, we generated transgenic mice in which the human SPATA17 gene was expressed specifically in spermatocytes using the human phosphoglycerate kinase 2 (PGK2) promoter. The SPATA17 transgenic mice did not show any significant defect in gross testicular anatomy as well as in fertility. However, a significant increase was observed in defective spermatogenic cells, such as apoptotic cells in the SPATA17 transgenic mice. These results revealed that elevated production of the SPATA17 protein disturbed the normal development of male germ cells.

Cloning and primarily function study of two novel putative N5-glutamine methyltransferase (Hemk) splice variants from mouse stem cells.

Methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. Beginning with the ESTs which were highly expressed in undifferentiated human ES cells and using homology research in mouse dbEST database, we cloned two novel putative (N (5))-glutamine methyltransferase (Hemk) splice variants termed mHemk1 and mHemk2 (Genbank accession number AY456393 and AY583759). Sequence analysis revealed that mHemk1 and mHemk2 cDNAs are 1,792 bp and 1,696 bp in length respectively. The deduced proteins have 214 amino acid residues (mHemk1) and 138 residues (mHemk2) in length and both share significant homology with (N (5))-glutamine methyltransferase (Hemk proteins) in database. Northern blot and RT-PCR analysis showed that mHemk mRNAs were abundantly expressed in undifferentiated ES cells, testis and brain, weakly expressed in differentiated ES cells and kidney, and not expressed in muscle, heart, placenta, pancreas, lung and stomach. Immunohistochemical analysis further revealed that the protein was most abundant in undifferentiated ES cells. The green fluorescent protein produced by pEGFP-C3/mHemk1 was detected mainly in the nucleus of COS7 cell lines after 24 h post-transfection. RNA interference (RNAi)-mediated knock-down method was established. Cell cycle analysis suggests that the cell proliferation decreases after RNAi with mHemk1. In vitro bioactivity assay showed that no evidence for a DNA adenine-methyltransferase activity was detected. The accumulating functional information from Hemk homology proteins in bacteria and yeast suggests that it may be an uncharacterized new mammalian N(5)-glutamine methyltransferase.

Molecular cloning and preliminary function study of a novel human gene, TSARG7, related to spermatogenesis.

A novel human gene TSARG7 (GenBank accession No. AY513610) was identified from a human testis cDNA library by using the mTSARG7 gene (GenBank accession No. AY489184) as an electronic probe. The gene whose full cDNA length is 2,463 bp containing 12 exons and 11 introns is located in the human chromosome 8p11.21. The predicted protein encoded by this gene contains 456 amino acids with a theoretical molecular weight of 56,295 dalton and isoelectric point of 9.13. It is a new member of the acyltransferase family since its sequence possesses the highly conserved PlsC domain existing in all acyltransferase-like proteins. Two groups, the TSARG7 and mTSARG7, the TSARG7 and Au041707, share 97% identity in the 456 amino acids. Expression of the TSARG7 gene is restricted to the testis. Subcellular localization studies show that the EGFP-tagged TSARG7 protein was localized in the cytoplasm of GC-1 cells. The TSARG7 mRNA expression was initiated in the testis of a 13-year-old boy, and its level increased steadily along with spermatogenesis and sexual maturation of the human. The results of heat stress experiment demonstrate that TSARG7 expression has a relation with temperature. In conclusion, our study suggests that we have cloned a novel human gene and this gene may play an important role in human spermatogenesis and sexual maturation.

Molecular cloning of a novel rat gene Tsarg1, a member of the DnaJ/HSP40 protein family.

Beginning with a mouse gene mTSARG3, which was related to apoptosis of spermatogenic cells, bioinformatics was applied and a predicted novel rat gene full-length cDNA sequence was attained. Gene-specific primers were designed for PCR in rat testis cDNA library. A new gene Tsarg1 (GenBank Accession No. AY380804) was cloned, which is related to apoptosis in rat spermatogenic cells. The gene whose full cDNA length is 1176 bp containing 8 exons and 7 introns is located in rat chromosome 1q32-1q33, which encoded a protein containing 316 amino acid residues and being a new member of HSP40 protein family since the sequence contains the highly conserved J domain, which is present in all DnaJ-like proteins and is supported to have a critical role in DnaJ-DnaK protein-protein interactions. The results of RT-PCR and Northern blot analysis showed that Tsarg1 was specifically expressed in rat testis, which probably inhibits rat testis spermatogenic cell apoptosis.

Molecular cloning and primary functional analysis of a novel human testis-specific gene.

In this study, a new data mining tool called Digital Differential Display (DDD) from the NCBI was used to predict testis-specific expressed genes from the expressed sequence tag (EST) database. DDD (digital differential display) was performed between nine testis libraries and seventy libraries derived from other tissues. We identified a new contig of ESTs (HS. 326528) which was from testis libraries. To validate the use of bioinformatic approaches in gene discovery, the ESTs (HS. 326528), which were predicted to be testis-specific, were chosen for further study. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of matched sets of cDNAs from testis and other tissues indicated that the ESTs were specifically expressed in testis. This result was further validated by multi-tissue Northern blot. The full-length cDNA encompassing the entire open reading frame was cloned and, in view of its apparent specificity to testis, the gene was termed homo sapiens spermatogenesis-related gene 8---SRG8 (GenBank accession number: AY489187). The gene whose full cDNA length is 1 044 bp containing 3 exons and 2 introns is located in human chromosome 15q26.2, the cDNA encodes a novel protein of 105 amino acides with a theoretical molecular weight of 11.7 kD and isoelectric point of 10.09 which shares no significant homology with any known proteins in database. Real time PCR analysis of testis of different developmental periods revealed that SRG8 gene is significantly expressed in adult testis. The green fluorescent protein produced by pEGFP-C3/SRG8 was detected in the nucleus of HeLa cells after 24 h post-transfection. Cell cycle analysis showed that SRG8 can accelerate HeLa cells to traverse the S-phase and enter the G2-phase compared with the control without transfection of SRG8, which suggested that this gene plays an important role in the development of testis. The discovery of SRG8 showed that DDD combined with experiments is a feasible, time-saving strategy to identify new candidate genes for testis-specific development

[Effect of ditching for drain on control of Oncomelania hupensis snail in beaches of Dongting Lake].

OBJECTIVE: To evaluate the effect of ditching for drain on the control of the breed of Oncomelania hupensis snails in beaches of Dongting Lake. METHODS: From November, 2009 to November, 2012, an 0. hupensis snail infested beach of the Yueyang jail and an O. hupensis snail infested beach of Junshan District were selected as research fields in the eastern Dongting Lake area, and the former, as the intervention field, was performed with the ditching for drain by excavators and the latter, as the control field, was not. RESULTS: Before the project implemented, the average soil moisture contents on the beaches in dry seasons of the two fields were both about 35.56%. After the project implemented, in the intervention field, the average soil moisture content was 26.53% which was significantly lower than that (35.56%) in the control field (F = 6.53, P < 0.05). The underground water levels in different heights in the intervention field were lower than those in the control field (χ2 = 33.33, P < 0.05). Before the project implemented, the natural death rates of the snails were 0.98% and 0.89% in the two research fields respectively (P > 0.05), and after the project implemented (in 2012), no adult and young snails were found in the interventional field, but in the control field, the average densities of living snails and young snails were 29.37 snails/0.1 m2 and 213 ± 108.45 snails/0.1 m2 respectively. CONCLUSION: The intervention of ditching for drain can decrease the soil moisture contents quickly and change the ecological condition, therefore, can control the breed of O. hupensis snails in the beaches of Dongting Lake.

[The analysis of heterogeneity of HWTX-I expressed in Pichia pastoris].

To seek the reason of heterogeneity of recombinant HWTX-I (rHWTX-I) expressed in Pichia pastoris. We expressed HWTX-I gene of interest in Pichia pastoris GS115/HWTX-I. The heterogenous product expressed was separated, purified and identified by using Ion exchange HPLC, reverse HPLC, Tricine SDS-PAGE and MALDI-TOF Mass Spectrometry and then sequenced in both N-terminus and C-terminus. These results show that the heterogeneity of rHWTX-I results from the incomplete processing of signal peptide of N-terminus and the internal degradation of C-terminus. Biological activity assay shows that the activity of the heterogenous rHWTX-I only showed 30% activity compared with the native HWTX-I. The Solutions to how to avoid the heterogeneity are also discussed.

Identification of a novel testis-specific gene mtLR1, which is expressed at specific stages of mouse spermatogenesis.

A novel testis-specific gene termed mtLR1 was identified by digital differential display. Sequence analyses revealed that mtLR1 protein contains an amino terminus leucine-rich repeat domain and shows 33% similarities to PIDD which functions in p53-mediated apoptosis. Northern blot analysis showed that mtLR1 mRNA was specifically expressed in adult mouse testis, and RT-PCR results also showed that mtLR1 was exclusively expressed in adult testis and not in spermatogonial cells. The expression of mtLR1 mRNA was developmentally upregulated in the testes during sexual maturation and was, conversely, downregulated by experimental cryptorchidism in vivo. We also showed that the expression of mtLR1 mRNA was relatively highly sensitive to heat stress in vitro. The green fluorescent protein produced by pEGFP-C3/mtLR1 was only detected in the cytoplasm of spermatogonia cell line GC-1 after 24 h posttransfection. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of spermatocytes and round spermatids within seminiferous tubules of the adult testis. The time-dependent expression pattern of mtLR1 in postnatal mouse testes suggested that mtLR1 gene might be involved in the regulation of spermatogenesis and sperm maturation.

In vitro hematopoietic differentiation of human embryonic stem cells induced by co-culture with human bone marrow stromal cells and low dose cytokines.

Human embryonic stem (hES) cells randomly differentiate into multiple cell types during embryoid body (EB) development and limited studies have focused on directed hematopoietic differentiation. Here, we report that the treatment of hES cells during EBs development with a combination of low dose hematopoietic cytokines, including stem cell factor (SCF), Flt-3 ligand, vascular endothelial growth factor (VEGF) and human bone marrow stromal cells (hBMSCs), generated cell clusters that contained 8.81% KDR-positive hemangioblasts, 9.94% CD34-positive hematopoietic stem cells and 25.7% CD45-positive mature hematopoietic cells, and expressed hematopoietic genes such as KDR, stem cell leukemia (scl) and runt-related transcription factor 1 (Runx1). We provide the first evidence for the role of the cytokine-hBMSCs combination in promoting hematopoietic differentiation of hES cells, and thus provide the potential for generation of hematopoietic cells, as well as for understanding early developmental events that govern the initiation of hematopoiesis in humans.

Cloning, characterization and primary function study of a novel gene, Cymg1, related to family 2 cystatins.

Cystatins are cysteine proteinase inhibitors. We found two expression sequence tags (ESTs), CA463109 and AV042522, from a mouse testis library using Digital differential display (DDD). By electrical hybridization, a novel gene, Cymg1 (GenBank accession No. AY600990), which has a full length of 0.78 kb, and contains four exons and three introns, was cloned from a mouse testis cDNA library. The gene is located in the 2G3 area of chromosome 2. The full cDNA encompasses the entire open reading frame, encoding 141 amino acid residues. The protein has a cysteine protease inhibitor domain that is related to the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the CRES subfamily, which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract. CYMG1 has a 44% (48/108) identity with mouse CRES and 30% (42/140) identity with mouse cystatin C. Northern blot analysis showed that the Cymg1 is specifically expressed in adult mouse testes. Cell location studies showed that the GFP-tagged CYMG1 protein was localized in the cytoplasm of HeLa cells. Immunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium, spermatocytes, round spermatids, elongating spermatids and spermatozoa. RT-PCR results also showed that Cymg1 was expressed in mouse testes and spermatogonium. The Cymg1 expression level varied in different developmental stages: it was low 1 week postpartum, steadily increased 2 to 5 weeks postpartum, and was highest 7 weeks postpartum. The expression level at 5 weeks postpartum was maintained during 13 to 57 weeks postpartum. The Cymg1 expression level in the testes over different developmental stages correlates with the mouse spermatogenesis and sexual maturation process. All these indicate that Cymg1 might play an important role in mouse spermatogenesis and sexual maturation.

Molecular cloning of MSRG-11 gene related to apoptosis of mouse spermatogenic cells.

Beginning with a new contig of the expressed sequence tags (Mm.63892) obtained by comparing testis libraries with other tissue and cell line libraries using the digital differential display program, we cloned a new gene which is related to the apoptosis of mouse spermatogenic cells using the Genscan program and polymerase chain reaction (PCR) technology. The sequence data have been submitted to the GenBank database under accession number AY747687. The full cDNA length is 1074 bp, and the gene with 7 exons and 6 introns is located in mouse chromosome 1 H5. The protein is recognized as a new member of calmodulin (CaM) binding protein family because the sequence contains three short calmodulin-binding motifs containing conserved Ile and Gln residues (IQ motif) and is considered to play a critical role in interactions of IQ motif-containing proteins with CaM proteins. The putative protein encoded by this gene has 192 amino acid residues with a theoretical molecular mass of 23.7 kDa and a calculated isoelectric point of 9.71. The sequence shares no significant homology with any known protein in databases. RT-PCR and Northern blot analyses revealed that 1.3 kb MSRG-11 transcript was strongly expressed in adult mouse testis but weakly expressed in the spleen and thymus. The MSRG-11 gene was expressed at various levels, faintly at two weeks postpartum and strongly from three weeks postpartum in adult testes. The green fluorescence produced by pEGFP-C2/MSRG-11 was detected in the cytoplasm of COS7 cells 24 h post-transfection. The pcDNA3.1(?-)/MSRG-11 plasmid was constructed and introduced into COS7 cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA). MSRG-11 can accelerate COS7 cell apoptosis, which suggests that this gene may play an important role in the development of mouse testes and is a candidate gene of testis-specific apoptosis. Based on these observations, it was considered that we cloned a new gene which probably accelerates spermatogenetic cell apoptosis in mouse.

[Differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells].

Embryonic stem cells are pluripotent and their differentiation in vitro can serve as an experimental model to explore the molecular mechanisms of early embryonic development. To investigate the effect of stromal cell conditioned medium combined with cytokines (sccm + cys) on the differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells, the mouse fibroblast feeder cells to make human embryonic stem cells grown into embryonic bodies (EBs) were initially deleted. After culture for 3 days, EB cells were trypsinized into single cells and induced for 8 days by sccm + cys. Then, the differentiated cells were cultured in the semisolid medium containing 0.9% methylcellulose and cytokines to study the colony forming and self-renewal ability of cells. Immunocytochemical staining was used to check the surface markers of the colony cells. During the induction, mRNA expression of flk-1, BMI-1, scl, and Zeta-globin genes was tested by RT-PCR. Surface markers, such as flk-1, CD34 were tested by the flow cytometry. The results demonstrated that: (1) cell clusters containing 20-30 cells were formed after culture for 8 - 14 days in the semisolid medium, replanting these cells resulted in similar cell cluster forming. In addition, CD45 positive in big cell colonies were also found in the semisolid medium; (2) attached cell colonies appeared after culture for 8 days in the semisolid medium and VIII factor, UEA and KDR could be detected as negative by immunocytochemical staining; (3) on the 4(th) day of induction, mRNAs of flk-1, BMI-1, scl and Zeta-globin were all expressed. On the 8(th) day of induction, all of the above genes except Zeta-globin were expressed, while ES cell and EB cells which served as controls did not express scl and Zeta-globin genes; (4) on the 8(th) day of induction, the proportions of flk-1(+) cells and CD34(+) cells among all the inducing population were 9.8% and 16.8%, respectively, while the corresponding positive populations were 0.36% and 1.16% in spontaneously differentiated 11(th) day's EB, and 0.04% and 0.16%, respectively, in ES cells. If is concluded that embryonic stem cells can differentiate into hematopoietic cells and endothelial cells in combinant culture system of this study.

Molecular cloning of a novel mouse testis-specific spermatogenic cell apoptosis inhibitor gene mTSARG7 as a candidate oncogene.

A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and 11 introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFP-tagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC-1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.

[Expression and purification of recombinant huwentoxin-I in Pichia pastoris].

HWTX-I is a peptide neurotoxin purified from the crude venom of the Chinese bird Spider Selenocosmia Huwena, which has analyesic activity. rHWTX-I expressed by P. pastoris and secreted to culture supernatant was first precipitated by (NH4)2SO4, then it was isolated and desalted by ultrofiltration following by ion exchange chromatography of CM column, after reverse phase HPLC of C18 column and vacuum drying, the pure HWTX-I protein was obtained which was proved to be recombinant HWTX-I by Tricine SDS-PAGE, MALDI-TOF mass spectrometry, amino acid composition analysis, the N-terminal amino acid sequence and its biological activity. The final yield of the purified HWTX-I was about 80 mg/L accounting for 23.6% of its total secretory proteins.