[Damage of seminiferous tubules in mouse testis caused by active immunization of actin-like protein 7a].

OBJECTIVE: 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. METHODS: The recombinant expression plasmid pET30a-ACTL7a was constructed and then transformed into E. coli Rosseta (DE3). The protein expression was induced by isopropyl ß-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. RESULTS: 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. CONCLUSIONS: 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.

Regulation of the G2/M phase of the cell cycle by sperm associated antigen 8 (SPAG8) protein.

Sperm associated antigen 8 (SPAG8), a testis-specific protein produced during male germ cell differentiation, was isolated from a human testis expression library using antibodies found in the serum obtained from an infertile woman. It was found to have a close functional relationship with microtubules. In this study, we generated a stably expressing SPAG8 CHO-K1 cell line. Immunofluorescence confocal microscopy showed that SPAG8 was concentrated at the microtubule-organizing center (MTOC) during prophase. As the cells progressed into metaphase, it co-localized with alpha-tubulin on the spindle. In anaphase, it was detected on both astral microtubules and mid-zone. Following cytokinesis, SPAG8 resumed its localization on the MTOC. Meanwhile, flow cytometry analysis found that SPAG8 prolonged the G2/M phase of CHO-K1 cells stably expressing SPAG8. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that SPAG8 inhibited the proliferation of the stable cells. SPAG8 might be involved in the regulation of cell cycle by changing the phosphorylation level of Tyr15 on cdc2. These results suggest that SPAG8 might play a role in cell division during spermatogenesis.

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique.

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.

Characterization of rtSH3p13 gene encoding a development protein involved in vesicular traffic in spermiogenesis.

A cDNA, designated as rtSH3p13, was isolated from a rat testis cDNA library. It consists of 1463 bp nuclear acids, which encodes a protein of 312 amino acids and was assigned the GenBank accession number AF227439. The deduced rtSH3p13 protein is a truncated isoform of SH3p13 as a result of mRNA alternative splicing. It is mainly expressed in the rat testis, detected in spermatids at the steps 8-19 of spermiogenesis, and found around the acrosome. During postnatal development, rtSH3p13 appears on day 18 and reaches maximum on day 60. Further experimental results suggested that rtSH3p13 forms a complex with activated epidermal growth factor receptor (EGFR) and interacts with synaptojanin I. Surprisingly, similar to SH3 domain, the V region of rtSH3p13 also inhibits endocytosis in CHO cells. Our results reveal a link between an rtSH3p13-synaptojanin-clathrin complex-mediated formation of pits and the process of spermiogenesis.

[Cloning and characterization of a novel rat gene RSD-7 differentially expressed in testis].

OBJECTIVE: To isolate and identify the differentially expressed genes in spermatogenesis for the understanding molecular mechanism of spermatogenesis. METHODS: Screening of the cDNA library, Northern blot, expression and purification in E. coli with GST expression system, immunocytochemical staining of testis sections were used. RESULTS: (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. CONCLUSIONS: 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.

Expression and localization of VCX/Y proteins and their possible involvement in regulation of ribosome assembly during spermatogenesis.

Variable Charge X/Y (VCX/Y) is a human testis-specific gene family that localized on X and Y chromosomes. In this study, VCY protein was expressed in E. coli in the form of glutathione-S-transferase (GST) fusion protein. With the purified fusion protein as antigen, the anti-GST-VCY antibody was generated and the localization of VCY protein in human testis was determined by immunohistochemistry. In the testis seminiferous epithelium, VCY proteins were highly expressed in nuclei of germ cells. Using propidium iodide staining and green fluorescent protein (GFP) tag technologies, VCY and VCX-8r proteins were mainly localized in the nucleoli of COS7 cells. In addition, the colocalization for VCY and VCX-8r in COS7 cells was also observed. With VCY cDNA as bait, a cDNA fragment of acidic ribosomal protein PO was obtained using yeast two-hybrid system. All the information above indicates that VCX/Y protein family might be involved in the regulation of ribosome assembly during spermatogenesis.