Dynamic alterations in the expression and localization of ACTL7a during capacitation in mouse spermatozoa.

OBJECTIVE: To demonstrate that capacitation in mouse spermatozoa involves alterations in the expression and localization of ACTL7a. DESIGN: Determine the alteration in the expression level and localization of ACTL7a in the induction of capacitation in mouse spermatozoa. SETTING: The Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, People's Republic of China. ANIMAL(S): ICR (Institute of Cancer Research) mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Western blot, indirect immunostaining. RESULT(S): The expression of ACTL7a is upregulated via the PKA pathway and undergoes remodeling during the early period of capacitation in mouse spermatozoa. CONCLUSION(S): ACTL7a is an essential component of capacitation in mouse spermatozoa. The alteration in the expression and localization of ACTL7a may be the primary biochemical event in the induction of capacitation in mouse spermatozoa.

Anti-ACTL7a antibodies: a cause of infertility.

OBJECTIVE: To show that antibodies against ACTL7a, a spermatozoon-specific protein, may be a cause of immunologic infertility. DESIGN: Determine the presence of anti-ACTL7a antibodies in infertile blood, raise antibodies against ACTL7a in rabbits, and demonstrate that the in vitro treatment of mouse spermatozoa with infertile sera markedly reduces their fertilizing capacity. Demonstrate that the active immunization of mice with ACTL7a protein reduces fertility. SETTING: National Research Institute for Family Planning, Beijing, World Health Organization Collaboration Center of Human Reproduction, China. ANIMAL(S): Rabbits, ICR mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Mass spectrometry, indirect immunostaining, spermatozoa agglutination test, and standard fertility assay. RESULT(S): The fertilizing potential of mouse spermatozoa was markedly reduced after in vitro treatment with ACTL7a antibody-containing serum from a vasectomized man. Active immunization with ACTL7a significantly reduced the fertility of mice. Anti-ACTL7a antibodies caused the agglutination of mouse and human spermatozoa in vitro. Furthermore, the antibodies were detected in the sera of additional vasectomized men. CONCLUSION(S): Anti-ACTL7a antibodies may cause infertility in mice because the in vitro treatment of mouse spermatozoa with ACTL7a antibody-containing serum markedly reduced the fertilizing potential of the spermatozoa. In addition, the active immunization of mice with ACTL7a resulted in significant reductions in fertility.

Sperm associated antigen 8 (SPAG8), a novel regulator of activator of CREM in testis during spermatogenesis.

cAMP response element modulator (CREM)-mediated gene expression is an essential regulatory mechanism for germ cell differentiation. CREM and its coactivator in testis, ACT, activate the transcription of many essential genes for spermatogenesis. Sperm associated antigen 8 (SPAG8) is a testis-specific component that is expressed during germ cell differentiation. In this study, we found the pattern of SPAG8 expression largely overlapped with that of ACT during spermatogenesis and verified the association of SPAG8 with ACT. Furthermore, we showed that SPAG8 enhanced the transcriptional activation of ACT-mediated CREMtau by strengthening the binding of ACT to CREMtau. These results indicate that SPAG8 acts as a regulator of ACT and plays an important role in CREM-ACT-mediated gene transcription during spermatogenesis.

Interaction of SH3P13 and DYDC1 protein: a germ cell component that regulates acrosome biogenesis during spermiogenesis.

The N-terminal BAR domain of endophilin has unique functions, such as affecting the curvature of the lipid membrane through its lysophosphatidic acid acyltransferase activity, binding of ATP and GTP and participating in tubulating activity. We recently demonstrated that SH3P13, a BAR domain-containing protein, assists in regulating clathrin-coated vesicle traffic that is crucial for acrosome biogenesis during spermatogenesis. DYDC1 was identified in a yeast two-hybrid screen from a human testis library by using the SH3P13 BAR domain as the bait. Consistent with the expression pattern of SH3P13, DYDC1 is exclusively expressed in the brain and testis and accumulates in the acrosome area during late stage of spermiogenesis. Here, we report that DYDC1 plays a crucial role during acrosome biogenesis. This relationship has been verified by a novel approach that involves germ cell transplantation and RNA interference. We found that knockdown of endogenous Dydc1 interfered with the formation of acrosomes, and thus spermatid differentiation during mouse spermiogenesis. These data provide important insight into the crucial process of acrosome biogenesis. In addition, our approach can also be applied to study functions of other genes related to spermatogenesis in vivo.

A novel testis protein, RSB-66, interacting with INCA1 (inhibitor of Cdk interacting with cyclin A1).

rsb-66 is a novel gene from a suppression subtracted hybridization (SSH) library of round spermatid-specific cDNAs against those of primary spermatocytes. It was found to be specifically expressed in round spermatids. To explore the function of RSB-66, a yeast two-hybrid system was used to screen for potential interacting partners in a human testis cDNA library. HSD45, also known as INCA1 (inhibitor of Cdk interacting with cyclin A1), was identified as one of the positive clones. The interaction between RSB-66 and INCA1 was demonstrated to occur by GST pull down and coimmunoprecipitation. Using immunofluorescence, RSB-66 was found to be specifically expressed in round spermatids, mainly in the cytoplasm. When being transfected into HeLa cells, RSB-66 and INCA1 were found to be co-localized principally in the cytoplasm. The alpha helix in the RSB-66 C terminal and two amino acid residues (tyr117 and his119) appear to be crucial for its function.

Delineation of the functional domains of the extracellular region of YWK-II Protein/APLP2 of sperm membrane.

The sperm membrane protein, designated as YWK-II protein/APLP2, is a member of the amyloid precursor protein (APP) superfamily and is a type I transmembrane protein involved in fertilization. Here, the structure-function of the domains of YWK-II protein was examined. Five segments with overlapping ends encompassing the entire extracellular region of mouse YWK-II gene were prepared, cloned and separately expressed in E. coli. The recombinant YWK-II segments were fused with glutathione S-transferase (GST), purified and evaluated for their antifertility activities by measuring their capacity to block in vitro mouse sperm-egg interaction. The structural domain(s) involved in the fertilization process was identified. The polypeptide segment corresponding to position 22-207 of YWK-II-763 inhibited the early stage of fertilization when the spermatozoa interacted with zona-free eggs; whereas the polypeptide segment 201-395 (lacking 309-364) of YWK-II-763 blocked sperm-egg membrane fusion. The remaining three segments, 201-395, 389-574 and 517-704 (lacking 613-624) of YWK-II-763, did not influence the in vitro fertilization process. The present results suggest that segment 22-308 of YWK-II-763 participates in the binding and fusion of sperm and egg plasma membranes thereby promoting fertilization.

Identification and characterization of rDJL, a novel member of the DnaJ protein family, in rat testis.

Applying the method of segmentation of seminiferous tubules combined with DDRT-PCR and cDNA library screening, a novel DnaJ homologue, rDJL was identified in rat testis. The reading frame encodes a protein of 223 amino acid residues containing J domain in the NH2 terminal region. rDJL gene is expressed mainly in testis and rDJL protein was immunolocalized notably in the acrosome region of spermatozoa. Immunoprecipitation experiments showed that rDJL interacted with Hsc70 and clathrin protein. When CHO cells were treated with EGF, rDJL and clathrin protein were found to be colocalized and be concentrated as endosome vesicles. The present findings suggest that rDJL functions as co-chaperone to Hsc70, participates in vesicular trafficking and may play an important role in acrosomogenesis.

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.

Characterization and potential function of a novel testis-specific nucleoporin BS-63.

A 1933 bp cDNA fragment, coding a truncated testis-specific novel nucleoporin, was isolated from a human testis lambdaZAPII cDNA library, designated as BS-63 and assigned GenBank accession number: U64675. By applying the methods of rapid amplification of cDNA ends (5' RACE) and PCR, a full-length BS-63 cDNA composed of 5475 bp was obtained. BS-63 cDNA contained an open reading frame consisting of 1765 codons and XFXFG or GLFG repetitive sequence motifs. These repetitive motifs are structural characteristic of nucleoporins. BS-63 cDNA has high homology with Nup358/Ran BP2. A 1599 bp fragment, corresponding to the C-terminus of BS-63 cDNA, was prepared and expressed in E. coli BL21(DE3). The recombinant product was purified by affinity chromatography and SDS-PAGE and polyclonal antibodies raised. In rat testis section, the BS-63 protein was localized at the sites of nuclear pores in spermatids by immuno-gold transmission electron microscopy and on the nuclear membrane of Triton X-treated sperm by colloidal silver immuno-gold scanning electron microscopy. The recombinant BS-63 protein can be phosphorylated in vitro with PKC and p34(cdc2). A yeast two-hybrid system was used to screen a mouse testis cDNA library to identify proteins capable of interacting with BS-63. Using the 1.6 kb cDNA fragment as bait, the following interacting proteins were identified: Ran, transportin (karyopherin beta2), two proteins related to the nucleocytoplasmic transporter and aF10 protein. The latter protein is a putative transcriptor containing a cysteine-rich N-terminus, a LAP/PHD finger, a leucine zipper domain and a glutamine-rich C-terminus. Also it is highly expressed in murine testis and is located in the cell nucleus and cytoplasm. The interaction of BS-63 with aF10 (696-1001aa) was validated by surface plasmon resonance and by affinity precipitation combined with Western blot. aF10 (696-1001aa) interacted in vitro with BS-63 extracted from rat testis germ cells. It is hypothesized that BS-63 is a testis-specific nucleoporin and possibly acts as a docking site and a cotransporter of Ran and transportin. The complex performs the task of a carrier system in transporting aF10 into the nucleus of germ cells during spermiogenesis.

INHIBITION OF GERMINAL VESICLE BREAKDOWN AND POLAR BODY FORMATION BY NICOTINAMIDE IN STARFISH AND SURF CLAM OOCYTES.

Nicotinamide inhibited both germinal vesicle breakdown (GVBD) and polar body formation (PBF) in surf clam and starfish oocytes. In the surf clam nicotinamide at 0.3 mM completely blocked PBF in the fertilized oocytes. For blockage of GVBD higher concentration was required. In the starfish, nicotinamide (30 mM) prevented PBF but not GVBD, when added 7 min after the commencement of 1-methyladenine (1-MeAde) administration. These results suggest that PBF is blocked by nicotinamide independent of its effect on GVBD. In the case of starfish, NAD+ was more effective than nicotinamide in inhibiting oocyte maturation. Nicotinamide also blocked GVBD induced by microinjection of the cytoplasm containing maturation-promoting factor (MPF) obtained from 1-MeAde-treatcd oocytes. These results suggest that nicotinamide prevents the action of MPF rather than inhibiting the interaction of 1-McAde with cell membrane or the induction of MPF.