Egg Zona Pellucida Research Articles

Maturation conditions and boar affect timing of cortical reaction in porcine oocytes

The cortical reaction induces changes at the egg's Zona pellucida (ZP), perivitelline space and/or oolemma level, blocking polyspermic fertilization. We studied the timing of sperm penetration and cortical reaction in pig oocytes matured under different conditions and inseminated with different boars. Immature (germinal vesicle stage) and in vitro matured (IVM) (metaphase II stage) oocytes were inseminated and results assessed at different hours post insemination. Penetrability and polyspermy rates increased with gamete coincubation time and were higher in IVM oocytes. A strong boar effect was observed in IVF results. Cortical reaction (assessed as area occupied by cortical granules) and galactose-β(1-3)-Nacetylgalactosamine residues on ZP (area labeled by peanut agglutinin lectin, PNA) were assessed in IVM and in vivo matured (IVV) oocytes at different hours post insemination. After maturation, IVM and IVV oocytes displayed similar area occupied by cortical granules and it decreased in fertilized oocytes compared to unfertilized ones. Cortical reaction was influenced by boar and was faster in polyspermic than in monospermic oocytes, and in IVM than in IVV oocytes. The outer ZP of inseminated oocytes appeared stained by PNA and the labeled area increased along with gamete coculture time. This labeling was also observed after insemination of isolated ZP, indicating that this modification in ZP carbohydrates is not induced by cortical reaction. The steady and maintained cortical reaction observed at 4 to 5 h post insemination in IVV monospermic oocytes might reflect the physiological time course of this important event in pigs. Both maturation conditions and boar affect cortical granules release.

Sperm arylsulfatase A binds to mZP2 and mZP3 glycoproteins in a nonenzymatic manner

We have shown previously that sperm surface arylsulfatase A (ASA) of mouse, pig, and human is involved in sperm-egg zona pellucida (ZP) binding. By treating capacitated mouse sperm with A23187 to induce the acrosome reaction, we demonstrated by immunoblotting that ASA also existed in the acrosomal content and on the inner acrosomal membrane. Since mZP2 and mZP3 are known as sperm receptors, whereas mZP1 as a cross-linker of mZP2/mZP3, we determined whether purified ASA bound to mZP2 and mZP3 selectively. The three mZP glycoproteins were purified from solubilized ovarian ZP by size exclusion column chromatography. Immuno-dot blot analyses revealed that purified sperm ASA bound to mZP2 at the highest level followed by mZP3, whereas the binding of ASA to mZP1 was minimal. The results confirmed the physiological significance of sperm ASA in the ZP binding process. The binding of ASA to mZP2 and mZP3 was, however, not dependent on the active site pocket amino acids, Cys69, Lys123, and Lys302, which are pertinent to the capturing of an arylsulfate substrate, since ASA mutant with Ala substitution at these three residues still bound to mZP2 and mZP3. The availability of the active site pocket of ASA bound to the ZP suggested that ASA would still retain enzymatic activity, which might be important for subsequent sperm penetration through the ZP.

Sperm Epidermal Growth Factor Receptor (EGFR) Mediates α7 Acetylcholine Receptor (AChR) Activation to Promote Fertilization

To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca(2+) influx, and the acrosome reaction.

Influence of the zona pellucida of the mouse egg on folliculogenesis and fertility

All mammalian eggs are surrounded by a relatively thick extracellular coat, called the zona pellucida (ZP), that plays vital roles during oogenesis, fertilization, and preimplantation development. The mouse egg ZP consists of three glycoproteins, called mZP1-3, that are synthesized solely by oocytes during their 2-to-3 week growth phase. The ZP is seen initially as isolated extracellular deposits of nascent ZP fibrils that coalesce to form a thickening matrix. Elimination of ZP glycoprotein synthesis by targeted mutagenesis yields mice that are heterozygous or homozygous for the null mutations. Homozygous null males are unaffected by the mutations and heterozygous females are as fertile as wild-type females. On the other hand, eggs from mZP2-/- and mZP3-/- females lack a ZP and the mice are completely infertile due to a severely reduced number of ovulated eggs in their oviducts. Development of ovarian follicles is retarded in homozygous null females and manifested as reduced ovarian weight, number of Graafian follicles, and number of ovulated eggs. Eggs from mZP1-/- females have a distorted ZP and, although the mice are fertile, they exhibit reduced fertility due to early embryonic loss. Potential relationships between ZP biogenesis, folliculogenesis, and fertility are addressed.

Loss of Cysteine-Rich Secretory Protein 4 (Crisp4) Leads to Deficiency in Sperm-Zona Pellucida Interaction in Mice1

Mammalian sperm gain their ability to fertilize the egg during transit through the epididymis and by interacting with proteins secreted by the epididymal epithelial cells. Certain members of the CRISP (cysteine-rich secretory protein) family form the major protein constituent of the luminal fluid in the mammalian epididymis. CRISP4 is the newest member of the CRISP family expressed predominantly in the epididymis. Its structure and expression pattern suggest a role in sperm maturation and/or sperm-egg interaction. To study the relevance of CRISP4 in reproduction, we have generated a Crisp4 iCre knock-in mouse model through insertion of the iCre recombinase coding cDNA into the Crisp4 locus. This allows using the mouse line both as a Crisp4 deficient model and as an epididymis-specific iCre-expressing mouse line applicable for the generation of conditional, epididymis-specific knockout mice. We show that the loss of CRISP4 leads to a deficiency of the spermatozoa to undergo progesterone-induced acrosome reaction and to a decreased fertilizing ability of the sperm in the in vitro fertilization conditions, although the mice remain fully fertile in normal mating. However, removal of the egg zona pellucida returned the fertilization potential of the CRISP4-deficient spermatozoa, and accordingly we detected a reduced number of Crisp4-deficient spermatozoa bound to oocytes as compared with the wild-type spermatozoa. We also demonstrate that iCre recombinase is expressed in a pattern similar to endogenous Crisp4 and is able to initiate the recombination event with its target sequences in vivo.

Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs

Many investigators maintain that spermatozoa that have initiated the acrosome reaction (AR) before reaching the surface of the egg's zona pellucida (ZP) are unable to bind and penetrate the ZP. A recent study has revealed that most fertilizing mouse spermatozoa initiate the AR before contacting the ZP. We found that acrosome-reacted spermatozoa collected from the perivitelline space of Cd9-null mice (whose egg plasma membranes are incapable of fusing with spermatozoa) were able to pass through both the cumulus and ZP of WT mouse eggs and produced live offspring. This means that the spermatozoa we used had the ability to pass through the ZP at least twice. Apparently, some spermatozoa that had undergone the AR long before contact with the ZP remained capable of crossing the ZP and fertilizing eggs. Thus, the concept that acrosome-reacted spermatozoa are unable to bind to the ZP and have lost their fertilizing capacity must be reconsidered.

Purification of N-acetyllactosamine-binding Activity from the Porcine Sperm Membrane: Possible Involvement of an ADAM Complex in the Carbohydrate-binding Activity of Sperm

Although the importance of carbohydrate recognition by sperm during egg zona pellucida binding has been widely reported, the sperm molecular species that recognize the carbohydrates are poorly characterized. Our previous cytochemical study indicated that two kinds of carbohydrate-binding proteins are expressed on porcine sperm heads-one recognizes N-acetyllactosamine (Galβ1-4GlcNAc-), and the other recognizes the Lewis X structure (Galβ1-4(Fucα1-3)GlcNAc-). For this report, we used proteomic techniques to characterize the sperm proteins that bind N-acetyllactosamine. Porcine sperm plasma membrane was solubilized with a detergent solution and subjected to sequential chromatography with dextran sulfate agarose, affinity, and hydroxyapatite, and the binding activities in the eluates were monitored by a solid-phase binding assay. The tryptic peptides of two proteins most likely associated with the binding activities were subjected to tandem mass spectrometry sequencing. A subsequent database search identified one of the two proteins as predicted disintegrin and metalloprotease domain-containing protein 20-like (XP_003128672). The other protein was identified as disintegrin and metalloprotease domain-containing protein 5 (AB613817) by database searches for homologous amino acid sequences, cDNA cloning, nucleotide sequencing and nucleotide database searches. Furthermore, two-dimensional blue native/SDS-PAGE demonstrated that they formed a variety of non-covalent complexes. Therefore, these ADAM complexes probably are responsible for the N-acetyllactosamine-binding activity. An affinity-purified fraction containing these ADAM complexes showed zona pellucida-binding activity, though the activity was relatively weak, and the presence of another zona pellucida-binding protein that probably works in concert with these ADAM complexes was suggested. Immunofluorescence testing suggested that ADAM20-like was localized on the anterior part of the sperm plasma membrane.

Transitional states of acrosomal exocytosis and proteolytic processing of the acrosomal matrix in guinea pig sperm

In this study, we adapted a FluoSphere bead-binding assay to study the exposure and release of guinea pig sperm acrosomal components during the course of capacitation and acrosomal exocytosis. Prior to capacitation or the initiation of exocytosis, acrosomal proteins were not accessible to FluoSpheres coated with antibodies against two acrosomal matrix (AM) proteins, AM67 and AM50; during the course of capacitation and ionophore-induced acrosomal exocytosis, however, we detected the transient exposure of the solid-phase AM proteins on the surface of guinea pig sperm using the antibody-coated fluorescent beads. Several different transitional stages leading to complete acrosomal exocytosis were classified, and we propose these represent true, functional intermediates since some of the AM proteins are orthologues of mouse proteins that bind the zona pellucida (ZP) of unfertilized eggs. In addition, we present evidence that implicates acrosin in the proteolytic processing of AM50 during AM disassembly. Thus, we propose that the transitional states of acrosomal exocytosis involve early binding of AM proteins to the ZP (by what visually appear to be "acrosome-intact" sperm), maintenance of ZP binding that coincides with the progressive exposure of AM proteins, and gradual proteolytic disassembly of the AM to allow sperm movement through the ZP. We feel this "transitional states" model provides a more refined view of acrosomal function that supports a move away from the widely held, overly simplistic, and binary "acrosome-reaction" model, and embraces a more dynamic view of acrosomal exocytosis that involves intermediate stages of the secretory process in ZP binding and penetration.

Mouse sperm undergo GPI-anchored protein release associated with lipid raft reorganization and acrosome reaction to acquire fertility

Mammalian sperm undergo several maturation steps after leaving the testis to become competent for fertilization. Important changes occur in sperm within the female reproductive tract, although the molecular mechanisms underlying these processes remain unclear. To investigate sperm membrane remodeling upon sperm maturation, we developed transgenic mouse lines carrying glycosylphosphatidylinositol (GPI)-anchored enhanced green fluorescent protein (EGFP-GPI) and traced the fate of this fluorescent protein during the fertility-acquiring process in sperm in vitro and in vivo. When the GFP-labeled sperm were treated with compounds for promoting the acrosome reaction, EGFP-GPI was released from the sperm surface crosslinked with characteristic relocation of a lipid raft marker ganglioside GM1. Sperm ejaculated into the uterus strongly expressed EGFP-GPI in the head region, whereas a part of the oviductal sperm lost fluorescence in a manner that was dependent on the presence of angiotensin-converting enzyme (ACE). Moreover, sperm on the zona pellucida of eggs in the oviduct were all found to have low levels of GFP. These results suggest that sperm undergoing GPI-anchored protein release associated with reorganization of lipid rafts and the acrosome reaction acquire fertilization potential.

Sperm Proteasomes Degrade Sperm Receptor on the Egg Zona Pellucida during Mammalian Fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

The Molecular Basis of Sex: Linking Yeast to Human

Species-specific recognition between egg and sperm, a crucial event that marks the beginning of fertilization in multicellular organisms, mirrors the binding between haploid cells of opposite mating type in unicellular eukaryotes such as yeast. However, as implied by the lack of sequence similarity between sperm-binding regions of invertebrate and vertebrate egg coat proteins, these interactions are thought to rely on completely different molecular entities. Here, we argue that these recognition systems are, in fact, related: despite being separated by 0.6-1 billion years of evolution, functionally essential domains of a mollusc sperm receptor and a yeast mating protein adopt the same 3D fold as egg zona pellucida proteins mediating the binding between gametes in humans.

Evolutionary patterns of two major reproduction candidate genes (Zp2 and Zp3) reveal no contribution to reproductive isolation between bovine species

BackgroundIt has been established that mammalian egg zona pellucida (ZP) glycoproteins are responsible for species-restricted binding of sperm to unfertilized eggs, inducing the sperm acrosome reaction, and preventing polyspermy. In mammals, ZP apparently represents a barrier to heterospecific fertilization and thus probably contributes to reproductive isolation between species. The evolutionary relationships between some members of the tribe Bovini are complex and highly debatable, particularly, those involving Bos and Bison species for which interspecific hybridization is extensively documented. Because reproductive isolation is known to be a major precursor of species divergence, testing evolutionary patterns of ZP glycoproteins may shed some light into the speciation process of these species. To this end, we have examined intraspecific and interspecific genetic variation of two ZP genes (Zp2 and Zp3) for seven representative species (111 individuals) from the Bovini tribe, including five species from Bos and Bison, and two species each from genera Bubalus and Syncerus.ResultsA pattern of low levels of intraspecific polymorphism and interspecific divergence was detected for the two sequenced fragments each for Zp2 and Zp3. At intraspecific level, none of neutrality tests detected deviations from neutral equilibrium expectations for the two genes. Several haplotypes in both genes were shared by multiple species from Bos and Bison.ConclusionsHere we argue that neither ancestral polymorphism nor introgressive hybridization alone can fully account for haplotype sharing among species from Bos and Bison, and that both scenarios have contributed to such a pattern of haplotype sharing observed here. Additionally, codon-based tests revealed strong evidence for purifying selection in the Zp3 coding haplotype sequences and weak evidence for purifying selection in the Zp2 coding haplotype sequences. Contrary to a general genetic pattern that genes or genomic regions contributing to reproductive isolation between species often evolve rapidly and show little or no gene flow between species, these results demonstrate that, particularly, those sequenced exons of the Zp2 and the Zp3 did not show any contribution to reproductive isolation between the bovine species studied here.

Molecular models for mouse sperm-oocyte binding

Binding of sperm to egg in the mouse has been proposed to depend primarily on interactions between lectin-like egg-binding proteins on the sperm plasma membrane and complementary carbohydrates on the specialized extracellular matrix of the egg known as the zona pellucida. An alternative model posits that initial sperm-egg binding depends on the interaction of a sperm surface protein with a supramolecular complex of the three mouse zona pellucida glycoproteins (mZP1, mZP2, mZP3); the role of carbohydrate recognition in this paradigm is thought to be minimal (Gahlay G, Gauthier L, Baibakov B, Epifano O,Dean J. 2010. Gamete recognition in mice depends on the cleavage status of an egg's zona pellucida protein. Science.329:216-219). This perspective reviews these recent findings,and considers them in light of evidence favoring a major role for lectin-like interactions. An alternative model, the domain specific model for mammalian gamete binding, could reconcile some of the conflicting observations.

Role and Regulation of Sperm Gelsolin Prior to Fertilization

To acquire fertilization competence, spermatozoa should undergo several biochemical changes in the female reproductive tract, known as capacitation. The capacitated spermatozoon can interact with the egg zona pellucida resulting in the occurrence of the acrosome reaction, a process that allowed its penetration into the egg and fertilization. Sperm capacitation requires actin polymerization, whereas F-actin must disperse prior to the acrosome reaction. Here, we suggest that the actin-severing protein, gelsolin, is inactive during capacitation and is activated prior to the acrosome reaction. The release of bound gelsolin from phosphatidylinositol 4,5-bisphosphate (PIP(2)) by PBP10, a peptide containing the PIP(2)-binding domain of gelsolin, or by activation of phospholipase C, which hydrolyzes PIP(2), caused rapid Ca(2+)-dependent F-actin depolymerization as well as enhanced acrosome reaction. Using immunoprecipitation assays, we showed that the tyrosine kinase SRC and gelsolin coimmunoprecipitate, and activating SRC by adding 8-bromo-cAMP (8-Br-cAMP) enhanced the amount of gelsolin in this precipitate. Moreover, 8-Br-cAMP enhanced tyrosine phosphorylation of gelsolin and its binding to PIP(2(4,5)), both of which inactivated gelsolin, allowing actin polymerization during capacitation. This actin polymerization was blocked by inhibiting the Src family kinases, suggesting that gelsolin is activated under these conditions. These results are further supported by our finding that PBP10 was unable to cause complete F-actin breakdown in the presence of 8-Br-cAMP or vanadate. In conclusion, inactivation of gelsolin during capacitation occurs by its binding to PIP(2) and tyrosine phosphorylation by SRC. The release of gelsolin from PIP(2) together with its dephosphorylation enables gelsolin activation, resulting in the acrosome reaction.

Role and regulation of EGFR in actin remodeling in sperm capacitation and the acrosome reaction

To bind and fertilize the egg, the spermatozoon should undergo few biochemical and motility changes in the female reproductive tract collectively called capacitation. The capacitated spermatozoon binds to the egg zona pellucida, and then undergoes the acrosome reaction (AR), which allows its penetration into the egg. The mechanisms regulating sperm capacitation and the AR are not completely understood. In the present review, we summarize some data regarding the role and regulation of the epidermal growth factor receptor (EGFR) in these processes. In the capacitation process, the EGFR is partially activated by protein kinase A (PKA), resulting in phospholipase D (PLD) activation and actin polymerization. Protein kinase C alpha (PKCα), which is already activated at the beginning of the capacitation, also participates in PLD activation. Further activation of the EGFR at the end of the capacitation enhances intracellular Ca(2+) concentration leading to F-actin breakdown and allows the AR to take place. Under in vivo conditions, the EGFR can be directly activated by its known ligand epidermal growth factor (EGF), and indirectly by activating PKA or by transactivation mediated by G protein-coupled receptors (GPCRs) activation or by ouabain. Under physiological conditions, sperm PKA is activated mainly by bicarbonate, which activates the soluble adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP), the activator of PKA. The GPCR activators angiotensin II or lysophosphatidic acid, as well as ouabain and EGF are physiological components present in the female reproductive tract.

Zonadhesin Is Essential for Species Specificity of Sperm Adhesion to the Egg Zona Pellucida

Interaction of rapidly evolving molecules imparts species specificity to sperm-egg recognition in marine invertebrates, but it is unclear whether comparable interactions occur during fertilization in any vertebrate species. In mammals, the sperm acrosomal protein zonadhesin is a rapidly evolving molecule with species-specific binding activity for the egg zona pellucida (ZP). Here we show using null mice produced by targeted disruption of Zan that zonadhesin confers species specificity to sperm-ZP adhesion. Sperm capacitation selectively exposed a partial von Willebrand D domain of mouse zonadhesin on the surface of living, motile cells. Antibodies to the exposed domain inhibited adhesion of wild-type spermatozoa to the mouse ZP but did not inhibit adhesion of spermatozoa lacking zonadhesin. Zan(-/-) males were fertile, and their spermatozoa readily fertilized mouse eggs in vitro. Remarkably, however, loss of zonadhesin increased adhesion of mouse spermatozoa to pig, cow, and rabbit ZP but not mouse ZP. We conclude that zonadhesin mediates species-specific ZP adhesion, and Zan(-/-) males are fertile because their spermatozoa retain adhesion capability that is not species-specific. Mammalian sperm-ZP adhesion is therefore molecularly robust, and species-specific egg recognition by a protein in the sperm acrosome is conserved between invertebrates and vertebrates, even though the adhesion molecules themselves are unrelated.

Regulation of the sperm EGF receptor by ouabain leads to initiation of the acrosome reaction

The sperm acrosome reaction occurs after the binding of the capacitated sperm to the egg zona pellucida. This study describes a novel mode of regulation of the sperm epidermal growth factor receptor (EGFR) under physiological conditions and its relevance to the acrosome reaction. Ouabain, a known Na/K ATPase blocker is present in the blood and in the female reproductive tract. We show here that physiological concentrations (nM) of ouabain enhance phosphorylation of EGFR on tyr-845, stimulate Ca 2+ influx and induce the acrosome reaction in sperm. These effects could be seen only in the presence of very low concentrations of EGF (0.1 ng/ml or 0.016 nM) added together with nano-molar ouabain. Phosphorylation, Ca 2+ influx, and the acrosome reaction are inhibited by an EGFR blocker, suggesting that trans-activation of the EGFR is involved. Moreover, our data revealed that protein kinase A and the family of tyrosine kinase, SRC, shown before to be involved in EGFR activation in sperm, mediate the acrosome reaction induced by ouabain. Ouabain alone (without EGF) at relatively high concentration (10 µM) could enhance EGFR phosphorylation, Ca 2+ influx and acrosome reaction, and these processes were inhibited by EGFR blockers. Moreover, we show here that PKA and SRC family are involved in the activation of EGFR by 10 µM ouabain, further demonstrating that ouabain induces the acrosome reaction by a mechanism mediated by the trans-activation of EGFR. In conclusion, this study describes an interesting regulatory path of EGFR by physiological concentrations of ouabain and EGF found in the female reproductive tract. Neither of these compounds can activate the EGFR alone at such low physiological levels; however, when both are present, the interaction of ouabain with the Na/K ATPase leads to the priming of the EGFR, which undergoes its full activation by EGF.

Infertility with Impaired Zona Pellucida Adhesion of Spermatozoa from Mice Lacking TauCstF-641

Fertilization is a multistep process requiring spermatozoa with unique cellular structures and numerous germ cell-specific molecules that function in the various steps. In the highly coordinated process of male germ cell development, RNA splicing and polyadenylation help regulate gene expression to assure formation of functional spermatozoa. Male germ cells express tauCstF-64 (Cstf2t gene product), a paralog of the X-linked CstF-64 protein that supports polyadenylation in most somatic cells. We previously showed that loss of tauCstF-64 causes male infertility because of major defects in mouse spermatogenesis. Surprisingly, although Cstf2t(-/-) males produce very few recognizable spermatozoa, some of the spermatozoa produced are motile. This led us to ask whether these Cstf2t(-/-) sperm were fertile. A motile cell-enriched population of spermatozoa from Cstf2t-null males dispersed cumulus cells of cumulus-oocyte complexes normally. However, motile spermatozoa from Cstf2t-null males failed to fertilize cumulus-intact mouse eggs in vitro. In addition, sperm adhesion to the zona pellucida (ZP) of cumulus-free eggs was significantly decreased, indicating tauCstF-64 is required for production of spermatozoa capable of ZP interaction. Acrosomal proteins involved in sperm-ZP recognition, including zonadhesin, proacrosin, SPAM1/PH-20, and ZP3R/sp56, were normally distributed in the apical head of Cstf2t(-/-) spermatozoa. We conclude that tauCstF-64 is required not only for expression of genes involved in morphological differentiation of spermatids but also for genes having products that function during interaction of motile spermatozoa with eggs. To our knowledge, this is the first demonstration that a gene involved in polyadenylation has a negative consequence on sperm-ZP adhesion.

Impaired sperm aggregation in Adam2 and Adam3 null mice

Previous studies have shown that sperm from Adam2 and Adam3 knockout mice have defective migration in the female reproductive tracts and cannot bind to the egg's zona pellucida (ZP), which leads to infertility. Here, we report that Adam2 and Adam3 knockout sperm have severely impaired sperm aggregation and that this defect is not restored over time during in vitro cultivation, suggesting the requirement of ADAM2 and ADAM3 in normal sperm association.

Loss of SED1/MFG‐E8 results in altered luminal physiology in the epididymis

SED1/MFG-E8, herein referred to as SED1, is a bimotif adhesive protein with ascribed functions in a range of cell-cell interactions, including sperm-egg binding. In the male reproductive tract, SED1 is secreted by the initial segment of the epididymis, where it coats sperm and subsequently facilitates binding to the egg zona pellucida. We have recently reported that SED1-null epididymides show an unexpected incidence of spermatic granulomas, reflecting breakdown of the epithelium and a consequent autoimmune response against sperm antigens. However, spermatic granulomas are most often manifest in the distal segments of the epididymis, whereas the bulk of SED1 is expressed in the proximal epididymis. In some models, the presence of granulomas in the distal epididymis is associated with an underlying defect in the maintenance of luminal fluid homeostasis. Herein, we report that SED1-null epididymal fluid is both hypo-osmotic and alkaline, relative to wildtype epididymal fluid. Furthermore, the SED1-null epididymal epithelium exhibits various hallmarks of disrupted fluid reabsorption and pH regulation, including altered morphology of clear cells, increased intracellular vesicles, and apical distribution of VATPase. Results indicate that the SED1-null epididymal pathologies are not the secondary consequences of defective testes or efferent ducts or of improper epididymal differentiation, unlike that seen in other epididymal models. The expression and distribution of various ion exchangers, channels, and enzymes that mediate fluid transport and pH regulation are examined in wildtype and SED1-null epididymides, and models to account for how SED1 functions in luminal fluid dynamics are discussed.