The need of MMP-2 on the sperm surface for Xenopus fertilization: its role in a fast electrical block to polyspermy.

Monospermic fertilization in the frog, Xenopus laevis, is ensured by a fast-rising, positive fertilization potential to prevent polyspermy on the fertilized egg, followed by a slow block with the formation of a fertilization envelope over the egg surface. In this paper, we found that not only the enzymatic activity of sperm matrix metalloproteinase-2 (MMP-2) was necessary for a sperm to bind and/or pass through the extracellular coat of vitelline envelope, but also the hemopexin (HPX) domain of MMP-2 on the sperm surface was involved in binding and membrane fusion between the sperm and eggs. A peptide with a partial amino acid sequence of the HPX domain caused egg activation accompanied by an increase in [Ca(2+)]i in a voltage-dependent manner, similar to that in fertilization. The membrane microdomain (MD) of unfertilized eggs bound the HPX peptide, and this was inhibited by ganglioside GM1 distributed in the MD. The treatment of sperm with GM1 or anti-MMP-2 HPX antibody allows the sperm to fertilize an egg clamped at 0 mV, which untreated sperm cannot achieve. We propose a model accounting for the mechanism of voltage-dependent fertilization based on an interaction between the positively charged HPX domain in the sperm membrane and negatively-charged GM1 in the egg plasma membrane.

The egg membrane microdomain-associated uroplakin III-Src system becomes functional during oocyte maturation and is required for bidirectional gamete signaling at fertilization in Xenopus laevis.

In Xenopus laevis, sperm-egg interaction promotes partial proteolysis and/or tyrosine phosphorylation of uroplakin III (UPIII) and the tyrosine kinase Src, which both localize to the cholesterol-enriched egg membrane microdomains (MDs). Here we show that sperm promote proteolysis and/or tyrosine phosphorylation of UPIII and Src in MDs isolated from ovulated and unfertilized eggs (UF-MDs). An antibody against the extracellular domain of UPIII interferes with these events. Inhibition of fertilization by anti-UPIII antibody is rescued by co-incubation with UF-MDs. This suggests that, like MDs in intact eggs, the isolated UF-MDs are capable of interacting with sperm, an interaction that does not interfere with normal fertilization but rather augments the ability of sperm to fertilize eggs pretreated with anti-UPIII antibody. This unexpected effect of UF-MDs on sperm requires UPIII function in UF-MDs and protein kinase activity in sperm. MDs isolated from progesterone-treated mature oocytes, but not ovarian immature oocytes, are similarly functional as UF-MDs. The anti-UPIII extracellular domain antibody binds more effectively to the surface of mature than immature ovarian oocytes. We propose that the structural and functional competency of the UPIII-Src signaling system in MDs is strictly regulated during oocyte maturation and subsequently in sperm-mediated egg activation and fertilization. The fertilization-related signaling properties seen in UF-MDs can be partially reconstituted in MDs of human embryonic kidney 293 cells (293-MDs) expressing UPIII, Src and uroplakin Ib. However, 293-MDs expressing a proteolysis-resistant mutant of UPIII are less functional, suggesting that the availability of UPIII to protease action is important for MD function.

Male mice express spermatogenic cell-specific triosephosphate isomerase isozymes.

Triosephosphate isomerase 1 (TPI1) is a member of the glycolytic pathway, which is a critical source of energy for motility in mouse sperm. By immunoblotting, we detected two male, germ line-specific TPI1 bands (Mr 33,400 and 30,800) as well as the somatic-type band (Mr 27,700). Although all three bands were observed in spermatogenic cells, somatic-type TPI1 disappeared from sperm during epididymal maturation. In vitro dephosphorylation analysis suggested that the two male, germ line-specific TPI1 bands were not the result of phosphorylation of the 27,700 Mr TPI1 band. The Mr 33,400; 30,800; and 27,700 TPI1 bands corresponded to the respective sizes of the proteins predicted to use the first, second, and third possible initiation codons of the Tpi1 cDNA. We performed immunofluorescence on epididymal sperm and determined that TPI1 specifically localized in the principal piece. The antibody staining was stronger in cauda epididymal sperm than in caput epididymal sperm, a finding consistent with the identification of TPI1 as a cauda epididymal sperm-enriched protein. Immunofluorescence with sodium dodecyl sulfate (SDS)-insoluble flagellar accessory structures showed a strong TPI1 signal only in the principal piece, indicating that TPI1 is a component of the fibrous sheath. Northern blot hybridization detected longer Tpi1 transcripts (1.56 kb) in mouse testis, whereas somatic tissues had shorter transcripts (1.32 kb). As there is only one triosephosphate isomerase gene in the mouse genome, we conclude that the three variants we see in sperm result from the use of alternative translation start codons in spermatogenic cells.

Protein-tyrosine kinase signaling in the biological functions associated with sperm.

In sexual reproduction, two gamete cells (i.e., egg and sperm) fuse (fertilization) to create a newborn with a genetic identity distinct from those of the parents. In the course of these developmental processes, a variety of signal transduction events occur simultaneously in each of the two gametes, as well as in the fertilized egg/zygote/early embryo. In particular, a growing body of knowledge suggests that the tyrosine kinase Src and/or other protein-tyrosine kinases are important elements that facilitate successful implementation of the aforementioned processes in many animal species. In this paper, we summarize recent findings on the roles of protein-tyrosine phosphorylation in many sperm-related processes (from spermatogenesis to epididymal maturation, capacitation, acrosomal exocytosis, and fertilization).

Heads or tails? Structural events and molecular mechanisms that promote mammalian sperm acrosomal exocytosis and motility.

Sperm structure has evolved to be very compact and compartmentalized to enable the motor (the flagellum) to transport the nuclear cargo (the head) to the egg. Furthermore, sperm do not exhibit progressive motility and are not capable of undergoing acrosomal exocytosis immediately following their release into the lumen of the seminiferous tubules, the site of spermatogenesis in the testis. These cells require maturation in the epididymis and female reproductive tract before they become competent for fertilization. Here we review aspects of the structural and molecular mechanisms that promote forward motility, hyperactivated motility, and acrosomal exocytosis. As a result, we favor a model articulated by others that the flagellum senses external signals and communicates with the head by second messengers to affect sperm functions such as acrosomal exocytosis. We hope this conceptual framework will serve to stimulate thinking and experimental investigations concerning the various steps of activating a sperm from a quiescent state to a gamete that is fully competent and committed to fertilization. The three themes of compartmentalization, competence, and commitment are key to an understanding of the molecular mechanisms of sperm activation. Comprehending these processes will have a considerable impact on the management of fertility problems, the development of contraceptive methods, and, potentially, elucidation of analogous processes in other cell systems.

Identification and validation of mouse sperm proteins correlated with epididymal maturation.

Sperm need to mature in the epididymis to become capable of fertilization. To understand the molecular mechanisms of mouse sperm maturation, we conducted a proteomic analysis using saturation dye labeling to identify proteins of caput and cauda epididymal sperm that exhibited differences in amounts or positions on two-dimensional gels. Of eight caput epididymal sperm-differential proteins, three were molecular chaperones and three were structural proteins. Of nine cauda epididymal sperm-differential proteins, six were enzymes of energy metabolism. To validate these proteins as markers of epididymal maturation, immunoblotting and immunofluorescence analyses were performed. During epididymal transit, heat shock protein 2 was eliminated with the cytoplasmic droplet and smooth muscle γ-actin exhibited reduced fluorescence from the anterior acrosome while the signal intensity of aldolase A increased, especially in the principal piece. Besides these changes, we observed protein spots, such as glutathione S-transferase mu 5 and the E2 component of pyruvate dehydrogenase complex, shifting to more basic isoelectric points, suggesting post-translational changes such dephosphorylation occur during epididymal maturation. We conclude that most caput epididymal sperm-differential proteins contribute to the functional modification of sperm structures and that many cauda epididymal sperm-differential proteins are involved in ATP production that promotes sperm functions such as motility.

Characterization of a novel tektin member, TEKT5, in mouse sperm.

Tektins are important components of flagella. Alterations in the expression of or mutations in mouse tektins are correlated with defective sperm motility, a cause of male infertility. Our proteomic studies of flagellar accessory structures previously identified a novel tektin, TEKT5, whose function is unknown. To understand the role of TEKT5 in mouse sperm, we characterized the expression of the mouse Tekt5 gene and the presence of TEKT5 in spermatogenic cells and spermatozoa. A complete cDNA encoding the Tekt5 transcript was assembled following reverse transcription-polymerase chain reaction (RT-PCR) and 3'-rapid amplification of cDNA ends and predicted that TEKT5 is a 62 730-dalton protein with an unusual, long C-terminus. Tekt5 mRNA was highly expressed during late stages of spermiogenesis. Among examined tissues, Tekt5 mRNA was present only in testis and brain, and quantitative RT-PCR showed that the expression level of mRNA in testis was 6.8-fold higher than that in brain. At the protein level, TEKT5 was present in sperm and was enriched in the accessory structures of flagella. Immunofluorescence confirmed that TEKT5 was localized throughout the sperm tail in flagellar accessory structures. The expression pattern suggests that TEKT5 plays an important role in flagella formation during spermiogenesis as well as being implicated in sperm motility.

DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes.

Silencing of transposable elements occurs during fetal gametogenesis in males via de novo DNA methylation of their regulatory regions. The loss of MILI (miwi-like) and MIWI2 (mouse piwi 2), two mouse homologs of Drosophila Piwi, activates retrotransposon gene expression by impairing DNA methylation in the regulatory regions of the retrotransposons. However, as it is unclear whether the defective DNA methylation in the mutants is due to the impairment of de novo DNA methylation, we analyze DNA methylation and Piwi-interacting small RNA (piRNA) expression in wild-type, MILI-null, and MIWI2-null male fetal germ cells. We reveal that defective DNA methylation of the regulatory regions of the Line-1 (long interspersed nuclear elements) and IAP (intracisternal A particle) retrotransposons in the MILI-null and MIWI2-null male germ cells takes place at the level of de novo methylation. Comprehensive analysis shows that the piRNAs of fetal germ cells are distinct from those previously identified in neonatal and adult germ cells. The expression of piRNAs is reduced under MILI- and MIWI2-null conditions in fetal germ cells, although the extent of the reduction differs significantly between the two mutants. Our data strongly suggest that MILI and MIWI2 play essential roles in establishing de novo DNA methylation of retrotransposons in fetal male germ cells.

Isolation and characterization of novel testis-specific genes from mouse pachytene spermatocyte-enriched cDNA library.

Background and Aims: Isolation and analysis of spermatogenesis-specific genes provide important information for elucidating the mechanisms of human infertility. The aim of the present study was to suggest an effective strategy for the comprehensive isolation of novel genes associated with spermatogenesis in mice. Methods: To isolate novel testis-specific genes associated with meiosis in mice, we constructed a mouse pachytene spermatocyte-enriched cDNA library by the centrifugal elutriation method, and sequenced 120 cDNA clones isolated from the cDNA library. A basic local alignment search tool (BLAST) search was carried out on the cDNA clones to find novel genes and then a detailed expression analysis was carried out by Northern blot hybridization and in situ hybridization. Results: Of the 120 cDNA clones, 35 clones (29%) were novel and 18 clones (15%) were expressed only in the testis. The expression patterns of seven novel testis-specific clones were examined on the testis sections. Three clones were expressed in spermatocytes and other germ cells, and two clones were exclusively expressed in spermatocytes. Amino acid sequences of seven novel testis-specific clones were deduced from their nucleotide sequences, suggesting that two of them contain known functional repeat structures. Conclusions: This method provides a powerful strategy to isolate novel testis-specific genes efficiently. (Reprod Med Biol 2005; 4: 231-237).

Mili, a mammalian member of piwi family gene, is essential for spermatogenesis.

The piwi family genes, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. To reveal the function of the mammalian homolog of piwi, we produced and analyzed mice with targeted mutations in the Mili gene, which is one of three mouse homologs of piwi. Spermatogenesis in the MILI-null mice was blocked completely at the early prophase of the first meiosis, from the zygotene to early pachytene, and the mice were sterile. However, primordial germ cell development and female germ cell production were not disturbed. Furthermore, MILI bound to MVH, which is an essential factor during the early spermatocyte stage. The similarities in the phenotypes of the MILI- and MVH-deficient mice and in the physical binding properties of MILI and MVH indicate a functional association of these proteins in post-transcriptional regulation. These data indicate that MILI is essential for the differentiation of spermatocytes.