Lrrc34 Is Highly Expressed in SSCs and Is Necessary for SSC Expansion In Vitro / 中国医学科学杂志(英文版)

Objective To discover critical genes contributing to the stemness and maintenance of spermatogonial stem cells (SSCs) and provide new insights into the function of the leucine-rich repeat (LRR) family member Lrrc34 (leucine-rich repeat-containing 34) in SSCs from mice. Methods Bioinformatic methods, including differentially expressed gene (DEG), gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, were used to uncover latent pluripotency-related genes. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence analyses were utilized to verify the mRNA and protein expression levels, respectively. RNA interference of Lrrc34 using siRNA was performed to detect its transient impact on SSCs. Results Eight DEGs between ID4-EGFP+ (G) and ID4-EGFP+/TSPAN8High (TH), eight DEGs between G and ID4-EGFP+/TSPAN8Low (TL) and eleven DEGs between TH and TL were discovered, and eleven protein-protein interaction (PPI) modules were found to be significant in the PPI network of DEGs. One of the DEGs, Lrrc34, was selected as a potential pluripotency-related gene due to its differential expression among ID4-EGFP+ spermatogonia subsets and its interaction with fibroblast growth factor 2 in the fifth module. Immunofluorescence experiments exhibited specific expression of Lrrc34 in a subpopulation of undifferentiated spermatogonia marked by LIN28A, and RT-PCR experiments confirmed the high expression of Lrrc34 in SSCs from P7 and adult mice. The transient knockdown of Lrrc34 in SSCs resulted in reduced colony sizes and significant changes in the transcriptome and apoptotic pathways. Conclusion Lrrc34 is highly expressed in mouse SSCs and is required for SSC proliferation in vitro through effects on transcriptome and signaling transduction pathways.

Construction and characterization of QKI knockout GC1-spg cell strain with CRISPR/CAS9 / 基础医学与临床

Objective To investigate whether QKI protein plays any important role in the process of spermatogene-sis.Constructing GC1-spg cell strain which knocked out QKI by the technology of CRISPR/Cas9,and detecting its effect on the proliferation and differentiation of QKI protein in vitro.Methods The plasmid PX330 was used to construct QKI knockout recombinant plasmid, then transfected it to GC1-spg wild-type cells and selected by puromycin.GC1-spg knock-QKI cell strain was identified by Western blot and gene sequencing; The wild-type and knockout cell strain was cultured normally,then detected the growth curve by cell counting kit(CCK8),and using quantitative PCR to get the changes of meiotic-related gene differentiation.Results QKI knockout GC1-spg cell strain was successfully constructed.Compared with the control group,the growth of QKI knockout cell strain was significantly decreased(P<0.05), and the expression of meiosis related molecular marker gene of c-kit, Mtl5 and Pspa2 was significantly decreased(P<0.05).Conclusions QKI proteins can affect reproductive sper-matogenesis by acting on proliferation and differentiation.

Expression Patterns of the Proteins Associated with Cell Junctions in Mouse Testes / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study on the expression patterns of proteins associated with cell junctions in the developing mouse testes.</p><p><b>METHOD</b>The expression levels of reproductive related cell lines spermatogonia cell line GC1 spg, spermatocyte cell line GC2 spg, leydig cell line TM3, and sertoli cell line TM4, primary sertoli cells, and 1-6-week mouse testes were analyzed using Western blot.</p><p><b>RESULTS</b>The sertoli cell junction-associated membrane proteins adhesion molecule A, Occludin and Claudin, and the sertoli-germ cell junction-associated membrane proteins junctional adhesion molecule C, Nectin-3, and E-cadherin were stage-specific in the seminiferous tubules in the mouse testes. The adaptor proteins associated with cell juctions zonula occludens-1, zonula occludens-2, Afadin, Β-catenin, and CD2-associated protein were not stage-specific in the seminiferous tubules in the mouse testes.</p><p><b>CONCLUSIONS</b>In the seminiferous tubules in the mouse testes, the membrane proteins associated with cell junctions are stage-specific. However, the expressions of adaptor proteins associated with cell junctions do not obviously change.</p>

Identification of proteins interacted with Bat3 using tandem affinity purification / 中国医学科学院学报

<p><b>OBJECTIVE</b>To identify the specific protein interactions involved in Bat3-mediated apoptosis.</p><p><b>METHODS</b>Tandem affinity purification (TAP) was utilized to investigate Bat3-protein interactions, during which full-length human Bat3 fused with Strep2 and FLAG tag as a bait was used to screen the specific protein-protein interactions. The isolated proteins were identified with mass spectrometry.</p><p><b>RESULTS</b>TAP studies showed that Ubl4A was identified as a Bat3-binding partner. Further investigation using co-immunoprecipitation confirmed that Bat3 was associated with Ubl4A.</p><p><b>CONCLUSION</b>TAP was successfully established and is suitable for isolating the binding partners of Bat3.</p>

Damage of seminiferous tubules in mouse testis caused by active immunization of actin-like protein 7a / 中国医学科学院学报

<p><b>OBJECTIVE</b>To obtain recombinant sperm-protein actin-like protein 7a (ACTL7a) and detect the damage seminiferous tubules in mouse testis caused by anti-sperm antibodies generated by purified ACTL7a active immunization.</p><p><b>METHODS</b>The recombinant expression plasmid pET30a-ACTL7a was constructed and then transformed into E. coli Rosseta (DE3). The protein expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the protein was purified by nickel ions chelating resin. Finally, the protein was separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and harvested by excising the gel containing target. ICR (Institute of Cancer Research) mice were then immunized using purified ACTL7a protein. The antibody titers were determined by ELISA and the development of seminiferous tubules after active immunization was stained by PAS staining.</p><p><b>RESULTS</b>Induced by IPTG, the target protein ACTL7a was expressed in E. coli. After purification, it was used to immunize the ICR mice. As shown by PAS staining, spermatid expulsion, pyknotic cells, absence of germ cells, and germ cells degenerated were seen in the seminiferous tubules in the immunized testes.</p><p><b>CONCLUSIONS</b>The ACTL7a prokaryotic expression vector was successfully constructed. High-purity target protein was obtained after induction and purification. After the active immunization with the target protein, the seminiferous tubules in the mouse testes will be severely damaged.</p>

Construction and identification of nemo-like kinase gene recombinant adenovirus vector / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct the nemo-like kinase (NLK) gene recombinant adenovirus vector.</p><p><b>METHODS</b>The AdEasy system was used to construct the recombinant adenovirus vector. Using reverse transcriptase polymerase chain reaction (RT-PCR), the full-length gene of NLK and its mutants (K155M, T286V, and C425Y) were amplified from HEK293 cells. The FLAG tag was appended at the C-terminal of NLK. After ligation and transformation, the NLK gene and its mutants were cloned into the pAdTrack-CMV vector. It was detected by PCR, sequencing, and Western blot analysis. Using DNA recombination and homogenous recombination, the normally expressed plasmids were linearized by the restriction enzyme-PmeI and PacI, then the enzyme-digested products were recycled by using ethanol precipitation. The purified product was transfected to HEK293A packaging cells with FuGENE HD transfection reagent. After amplification of the recombinant adenovirus, Western blot analysis was performed to detect the expression of NLK gene and its mutants.</p><p><b>RESULTS</b>The successful construction of pAdtrack-CMV-NLK (and mutants) was confirmed by PCR and sequencing. Western blot analysis showed that the target genes and the recombinant adenovirus were obtained. This recombinant virus was able to express NLK protein and its mutants correctly in HCT 116 cells.</p><p><b>CONCLUSION</b>The NLK gene recombinant adenovirus vector was successfully constructed and identified.</p>

Separation and morphological observation of mouse testis seminiferous tubule epithelium / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore a simple and feasible technique to locate proteins during spermatogenesis.</p><p><b>METHODS</b>Various germ cells and somatic cells were separated by collagenase I and DNase I after the albuginea was removed. The cells were then smeared, dyed, and observed directly under fluorescence and confocal microscopy.</p><p><b>RESULTS</b>Germ cells at different steps were successfully identified by specific dyestuffs for acrosome and nucleolus.</p><p><b>CONCLUSION</b>A simple method for locating proteins during spermatogenesis was successfully developed.</p>

Establishment of a targeting protein for Xenopus kinesin-like protein 2 C' terminal SBP-3 x Flag tagged HCT 116 colorectal cancer cell model / 中国医学科学院学报

<p><b>OBJECTIVE</b>To develop a targeting protein for Xenopus kinesin-like protein 2 (TPX2) C' terminal SBP-3 x Flag-tagged HCT 116 cell model.</p><p><b>METHODS</b>Homologous arms were amplified by polymerase chain reaction (PCR), and then the adeno-associated virus (AAV) -targeting vector of TPX2 was constructed. HCT 116 cells were targeted after the viruses were packaged. Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening. Finally, the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase, and the TPX2 C' terminal SBP and 3 x Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening.</p><p><b>RESULTS</b>Two positive cell clones with neomycin resistance gene were obtained by PCR screening. The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection, and the knock-in of SBP-3 x Flag gene was verified by Western blot analysis.</p><p><b>CONCLUSION</b>The TPX2 C' terminal SBP-3 x Flag tagged HCT 116 cell model was successfully established.</p>

Expression, purification, and crystallization of a novel galactose mutarotase from Thermoanaerobacter tengcongensis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction.</p><p><b>METHODS</b>The tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl beta-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose 4B affinity, ion chromatography (Resource Q 6 mL), and gel filtration chromatography (10/300 superdex 200).</p><p><b>RESULT</b>The purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9 A were obtained.</p><p><b>CONCLUSIONS</b>We successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.</p>

Characteristics of a novel human testis-specific gene, HSD-9, and its encoding protein / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the characteristics of a novel human testis-specific gene, HSD-9, and its encoding protein.</p><p><b>METHODS</b>HSD-9 was a novel gene from a human germ cells-specific ESTs library established in our laboratory. We used an electronic cloning method to obtain HSD-9 gene, and analyzed the characteristics of this novel gene and encoding product by bioinformatics methods, detected its expressing profile using a Northern blot assay, prepared specific rabbit polyclonal antibodies against HSD-9 protein, observed the localization of this protein in the germ cells and some somatic cells with confocal microscopy.</p><p><b>RESULTS</b>HSD-9 was expressed in human testes, and its rat homolog was found in the varying germ cells. HSD-9 protein could partly be colocalized with clathrin.</p><p><b>CONCLUSIONS</b>HSD-9 is specific in human testes, and the expression pattern of its encoding product is similar to those of some endocytosis proteins. It is speculated that HSD-9 protein may function in the endocytosis.</p>

Cloning and characterization of a novel rat gene RSD-7 differentially expressed in testis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To isolate and identify the differentially expressed genes in spermatogenesis for the understanding molecular mechanism of spermatogenesis.</p><p><b>METHODS</b>Screening of the cDNA library, Northern blot, expression and purification in E. coli with GST expression system, immunocytochemical staining of testis sections were used.</p><p><b>RESULTS</b>(1) A cDNA fragment designated as RSD-7 was isolated from rat testis cDNA library. It was 1,238 bp in length, coding a protein of 232 amino acids with the GenBank accession number AF315467. The encoding protein of RSD-7 cDNA had a Ubiquitin-like domain. (2) Northern blot indicated that RSD-7 was uniquely expressed in rat testis, and in the testis RSD-7 emerged on the 30th postnatal day and expressed until 120th postnatal day. (3) Expression and purification of RSD-7 protein in E. coli with GST expression system and were used to obtain anti-RSD-7 antibody. (4) Immunolocalization of RSD-7 in rat testis revealed that it is expressed only in Sertoli cells.</p><p><b>CONCLUSIONS</b>Transcription pattern of RSD-7 and localization of RSD-7 protein in testis have been made, which established the base for the functional study of RSD-7.</p>

Study on the function of HSD-3.8 gene encoding a testis-specific protein with yeast two-hybrid system / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the protein factors that could interact with the testis-specific protein encoded by HSD-3.8 gene (GenBank Accession Number AF311312) related with female fertilization.</p><p><b>METHODS</b>Yeast two-hybrid system was used to screen the human ovary MATCHMAKER cDNA library with constructed "bait plasmid" containing the 0.7 kb fragment (HSD-0.7) of HSD-3.8. The interaction with the positive fragments using a series of truncated bait plasmids was investigated.</p><p><b>RESULTS</b>One positive gene fragment was obtained, which coded for 144 amino acids of the C-terminus of human G protein beta subunit 1. Truncated bait plasmids couldn't interact with the fish protein fragment in yeast.</p><p><b>CONCLUSIONS</b>The protein encoded by HSD-3.8 gene may function through G protein signal transduction pathway and the interaction depends on the integration of the bait protein.</p>