Mammalian spermatozoa become motile at ejaculation, but before they can fertilize the egg, they must acquire more thrust to penetrate the cumulus and zona pellucida. The forceful asymmetric motion of hyperactivated spermatozoa requires Ca 2؉ entry into the sperm tail by an alkalinization-activated voltage-sensitive Ca 2؉ -selective current (ICatSper). Hyperactivation requires CatSper1 and CatSper2 putative ion channel genes, but the function of two other related genes (CatSper3 and CatSper4) is not known. Here we show that targeted disruption of murine CatSper3 or CatSper4 also abrogated I CatSper , sperm cell hyperactivated motility and male fertility but did not affect spermatogenesis or initial motility. Direct protein interactions among CatSpers, the sperm specificity of these proteins, and loss of I CatSper in each of the four CatSper ؊/؊ mice indicate that CatSpers are highly specialized flagellar proteins.calcium ͉ contraception ͉ flagella S permatozoa first acquire the potential for motility in the epididymis. They are capacitated in the female reproductive tract (1), where they acquire hyperactivated motility and other attributes that enable fertilization (2). During hyperactivation, the sperm tail motion changes from symmetric, fast, and low amplitude (sinusoidal) to asymmetric, slow, and large amplitude (whip-like; refs. 3-5). Hyperactivation is required for fertilization, providing the force needed to free the sperm cell from the oviductal reservoir and to penetrate the cumulus and zona pellucida surrounding the egg (1, 6, 7).Sperm cells become motile and progress directionally once they enter the female reproductive tract. Ca 2ϩ -independent flagellar dynein and ATP orchestrate the low-amplitude sinusoidal-activated motility of the tail. As the sperm cells encounter a more alkaline environment in the higher female reproductive tract, they hyperactivate, a process that requires Ca 2ϩ entry (3,8,9). Studies with antibodies or nucleotide probes have labeled several Ca 2ϩ -permeant channels, including voltage-sensitive Ca 2ϩ -selective channels (CatSpers and CaVs), cyclic nucleotide-gated channels, and transient receptor potential channels, in spermatocytes or spermatozoa (10-18). However, recent patch-clamp recordings of mouse epididymal spermatozoa (19) show that the predominant Ca 2ϩ -carrying current requires the CatSper1 gene that encodes a six-transmembrane-spanning protein of the voltage-gated ligand ion channel superfamily (20). In both whole-cell and perforatedpatch configurations, the Ca 2ϩ -selective current (I CatSper ) originated from the principal piece of the sperm tail and was absent in spermatozoa from CatSper1 Ϫ/Ϫ mice. CatSper1 Ϫ/Ϫ and CatSper2 Ϫ/Ϫ male mice are infertile (11,21), and sperm cells from CatSper1 Ϫ/Ϫ and CatSper2 Ϫ/Ϫ mice are unable to hyperactivate (4, 21). I CatSper was dramatically potentiated by a rise in intracellular pH, suggesting that the alkalinization that occurs during sperm capacitation activates I CatSper to increase intracellular [Ca 2ϩ ] and induce hypera...

Many eukaryotic extracellular proteins share a sequence of unknown function, called the zona pellucida (ZP) domain. Among these proteins are the mammalian sperm receptors ZP2 and ZP3, non-mammalian egg coat proteins, Tamm-Horsfall protein (THP), glycoprotein-2 (GP-2), alpha- and beta-tectorins, transforming growth factor (TGF)-beta receptor III and endoglin, DMBT-1 (deleted in malignant brain tumour-1), NompA (no-mechanoreceptor-potential-A), Dumpy and cuticlin-1 (refs 1,2). Here, we report that the ZP domain of ZP2, ZP3 and THP is responsible for polymerization of these proteins into filaments of similar supramolecular structure. Most ZP domain proteins are synthesized as precursors with carboxy-terminal transmembrane domains or glycosyl phosphatidylinositol (GPI) anchors. Our results demonstrate that the C-terminal transmembrane domain and short cytoplasmic tail of ZP2 and ZP3 are not required for secretion, but are essential for assembly. Finally, we suggest a molecular basis for dominant human hearing disorders caused by point mutations within the ZP domain of alpha-tectorin.

Many secreted eukaryotic glycoproteins that play fundamental roles in development, hearing, immunity, and cancer polymerize into filaments and extracellular matrices through zona pellucida (ZP) domains. ZP domain proteins are synthesized as precursors containing C-terminal propeptides that are cleaved at conserved sites. However, the consequences of this processing and the mechanism by which nascent proteins assemble are unclear. By microinjection of mutated DNA constructs into growing oocytes and mammalian cell transfection, we have identified a conserved duplicated motif [EHP (external hydrophobic patch)͞IHP (internal hydrophobic patch)] regulating the assembly of mouse ZP proteins. Whereas the transmembrane domain (TMD) of ZP3 can be functionally replaced by an unrelated TMD, mutations in either EHP or IHP do not hinder secretion of full-length ZP3 but completely abolish its assembly. Because mutants truncated before the TMD are not processed, we conclude that the conserved TMD of mammalian ZP proteins does not engage them in specific interactions but is essential for C-terminal processing. Cleavage of ZP precursors results in loss of the EHP, thereby activating secreted polypeptides to assemble by using the IHP within the ZP domain. Taken together, these findings suggest a general mechanism for assembly of ZP domain proteins.T he zona pellucida (ZP) domain is a protein polymerization module of Ϸ260 aa (1). Since its identification in mouse ZP glycoproteins (2), this domain has been found in many extracellular eukaryotic proteins of diverse molecular architecture and biological function (2, 3). These include egg coat proteins, inner ear proteins, urinary and pancreatic proteins, transforming growth factor-␤ receptors, immune defense proteins, nematode cuticle components, and fly proteins involved in transmission of mechanical stimuli and in wing and tracheal morphogenesis (4).We study the ZP (3), an extracellular coat secreted by growing mouse oocytes, as a model for ZP domain protein maturation and assembly. The ZP consists of long filaments composed of ZP2 and ZP3, crosslinked by a third ZP domain protein, ZP1. Nascent ZP polypeptides have features in common with other ZP domain proteins (2-4) that include an N-terminal signal sequence (SP), a ZP domain, and a consensus furin cleavage site (CFCS). The latter is followed by a C-terminal propeptide containing a transmembrane domain (TMD) and short cytoplasmic tail (Fig. 1A) that are replaced by a glycosyl phosphatidylinositol anchor in some ZP domain proteins. During secretion, but before incorporation into the ZP, a furin-like enzyme excises the propeptide from ZP precursors by cleavage at the CFCS (5-8). C-terminal processing of precursors also occurs for ZP homologues from fish to birds (9-11), as well as for other . Recombinant ZP proteins synthesized from cDNAs mutated at the CFCS are not secreted and accumulate in the endoplasmic reticulum of transfected cells (15)(16)(17). Recently, assembly of ZP proteins was studied by microinjection of epitope-tagge...

The extracellular coat, or zona pellucida, of the mouse egg consists of three glycoproteins, called mZP1-3. The glycoproteins are synthesized and secreted concomitantly by growing oocytes during their 2-3-week growth phase. Each of the glycoproteins has a consensus furin cleavage site (-Arg-X-Lys/Arg-Arg-) near the C-terminus of their polypeptide. Here, several approaches were employed to determine whether nascent mZP2 and mZP3 are cleaved at the consensus sites, -Arg-Ser-Lys-Arg- and -Arg-Asn-Arg-Arg-, respectively, prior to secretion. Molecular mass determinations of deglycosylated mZP2 and mZP3 suggest that their polypeptides are approximately 9 and approximately 7 kDa smaller, respectively, than predicted from exon sequences. Two-dimensional thin-layer chromatographic analyses were also carried out to identify amino acids released from the C-terminus of mZP2 and mZP3 by carboxypeptidase B. On the basis of exon sequences, there are no Arg residues at the predicted C-terminus of the mature glycoproteins. However, for both mZP2 and mZP3, Arg residues were released by carboxypeptidase B, consistent with processing at the consensus furin cleavage site. Furthermore, an antiserum raised against an mZP3 peptide, located downstream of the consensus furin cleavage site, failed to label purified mZP3 on Western immunoblots. The antiserum also failed to label the zona pellucida of oocytes examined by laser scanning confocal microscopy. Collectively, these results strongly suggest that mZP2 and mZP3 are processed at their consensus furin cleavage site prior to secretion and incorporation into the zona pellucida.