The roles of intraflagellar transport (IFT) protein 25 in mammalian signaling transduction and flagellogenesis / 亚洲男科学杂志(英文版)

Cilium, an organelle with a unique proteome and organization, protruding from the cell surface, generally serves as a force generator and signaling compartment. During ciliogenesis, ciliary proteins are synthesized in cytoplasm and transported into cilia by intraflagellar transport (IFT) particles, where the inner counterparts undergo reverse trafficking. The homeostasis of IFT plays a key role in cilial structure assembly and signaling transduction. Much progress has been made on the mechanisms and functions of IFT; however, recent studies have revealed the involvement of IFT particle subunits in organogenesis and spermatogenesis. In this review, we discuss new concepts concerning the molecular functions of IFT protein IFT25 and how its interactions with other IFT particle subunits are involved in mammalian development and fertility.

Intraflagellar transport is essential for spermiogenesis / 中华男科学杂志

Intra flagellar transport (IFT) is an evolutionarily conserved mechanism thought to be essential for the assembly and maintenance of most eukaryotic cilia and flagella. Development of the sperm tail axoneme resembles the cilia formation, which is organized by intraflagellar transport (IFT). Of all mammalian cells, sperm have the longest motile cilia, but few studies are reported on the role of IFT in the formation of sperm flagella and the mechanisms of IFT in spermiogenesis. This article focuses on the role of IFT in spermatogenesis and the importance of IFT in male fertility.

The role of SPAG6/SPINK2 protein complex in the formation of sperm acrosome in mice / 中华男科学杂志

Objective@#To explore the expression and regulatory function of sperm-associated antigen 6 (SPAG6) in the formation of the sperm acrosome in mice.@*METHODS@#The expression of SPAG6 during the first wave of spermatogenesis on postnatal days (PN) 8, 12, 16, 20, 24, 28, 30 and 35 was examined by Western blot and the localization of SPAG6 in the testicular germ cells was determined by immunofluorescence. The expression plasmids of SPAG6 and serine protease inhibitor Kazal-type 2 (SPINK2) were constructed, the interaction between SPAG6 and SPINK2 in the AH109 and CHO cells examined by yeast two-hybrid and co-localization assays, and the expression and localization of SPINK2 in the testicular germ cells of the SPAG6-knockout (SPAG6 KO) mice detected by immunofluorescence.@*RESULTS@#SPAG6 was highly expressed between PN 16 and 28 and localized in the acrosome of the round spermatids. Yeast two-hybrid assay showed the growth of SPAG6 and SPINK2 in the selective culture medium SD/-Leu/-Trp/-His, and the transfection of the CHO cells revealed the co-localization of SPAG6 and SPINK2 around the nuclei. The expression and acrosomal localization of SPINK2 were not found in the testicular germ cells of the SPAG6-KO mice.@*CONCLUSIONS@#SPAG6 interacts with SPINK2 and probably participates in the formation of the sperm acrosome by stabilizing the expression of SPINK2 during spermatogenesis.

Construction of a GFP-fused mouse PACRG baculovirus recombinant vector and expression of the fusion protein in Sf9 inset cells / 中华男科学杂志

<p><b>Objective</b>To construct a GFP-fused mouse Parkin co-regulated gene (PACRG) baculovirus recombinant PACRG/GFP-pFastBac1 vector and express the fusion protein in Sf9 insect cells.</p><p><b>METHODS</b>Full-length mouse PACRG cDNA was amplified by PCR and cloned in frame to the vector pFastBac1 with eGFP (rpFBac-PACRG-GFP recombinant vector). The plasmid was transformed into DH10Bac cells to obtain the recombinant bacmid plasmid, the bacmid was transfected into Sf9 insect cells, and the expressed PACRG/GFP fusion protein was analyzed by Western blot and fluorescence microscopy.</p><p><b>RESULTS</b>The construction of the PACRG/GFP-pFastBac1 baculovirus plasmid was confirmed by sequencing and restriction enzyme digestion. Western blot showed the expression of the fusion protein carrying a green fluorescence in the Sf9 insect cells.</p><p><b>CONCLUSIONS</b>Conclusion: A PACRG/GFP-pFastBac1 recombinant baculovirus vector was successfully constructed and the fusion protein was highly expressed in the Sf9 insect cells. Our findings have provided a basis for further studies on the structure of the PACRG protein and regulation of spermatogenesis.</p>

Interaction between SPAG6 and TAC1 proteins / 中华男科学杂志

<p><b>Objective</b>To construct eukaryotic expression plasmids of the Tac1 gene and explore the interaction between TAC1 and sperm-associated antigen 6 (SPAG6).</p><p><b>METHODS</b>RNA was extracted from the heart, liver, spleen, lung, kidney, brain, muscle, and testis of 10 Kunming male mice and, after reverse transcription into cDNA, the expression of Tac1 in the above tissues was observed by RT-PCR. Tac1/pEGFP-N2 and Tac1/pGADT7 recombinant plasmids were constructed and Tac1/pEGFP-N2 was transfected into CHO and COS-1 cells, followed by localization and detection of the protein expression of TAC1 by immunofluorescence staining and Western blot. The interaction between TAC1 and SPAG6 was determined by yeast two-hybrid experiment and Western blot.</p><p><b>RESULTS</b>Tac1 was expressed mainly in the testis, brain and heart. The results of restriction enzyme digestion and sequencing indicated successful construction of the recombinant plasmids, with the restriction fragment length of 390 bp. TAC1 was localized in the whole body of the CHO cells when transfected alone, but expressed in the microtubule of the cells when cotransfected with SPAG6, with the molecular weight of 40 000. Yeast two-hybrid experiment showed the colonies of TAC1 and SPAG6 on the culture plate without Leu, Trp and His, both contained in the yeast fusion protein.</p><p><b>CONCLUSIONS</b>The Tac1 recombinant plasmid was constructed successfully and the interaction between TAC1 and SPAG6 was confirmed with the plasmid.</p>

Construction of a recombinant plasmid of BC022687 and identification of its expression and localization in CHO cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To construct a mammalian expression plasmid of the BC022687 gene and investigate the expression and localization of the fusion protein in Chinese hamster ovary (CHO) cells.</p><p><b>METHODS</b>The BC022687 coding sequence was amplified by polymerase chain reaction (PCR) and subcloned into the pEGFP-C1 vector carrying the gene of green fluorescence protein (GFP). After the target region was sequenced, the recombinant plasmid was transfected into CHO cells, and its expression in the CHO cells was determined by Western blot. The localization of GFP-tagged BC022687 in the CHO cells was observed by laser scanning confocal microscopy.</p><p><b>RESULTS</b>BC022687 was successfully cloned into the mammalian expression vector pEGFP-C1, with the restriction fragment length of 950 bp. The expression of the fusion protein was confirmed, with the relative molecular weight of 64 000. The GFP-tagged BC022687 protein was mainly localized in the cytoplasm, and also presented in the centrioles in the transfected CHO cells.</p><p><b>CONCLUSION</b>The successful construction of the plasmid expressing BC022687 in CHO cells has laid a foundation for further studies on the role of this protein in ciliogenesis.</p>