Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.     

Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization

The 26S proteasome, which is a multi-subunit protease with specificity for substrate proteins that are postranslationally modified by ubiquitination, has been implicated in acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs increases the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). Ubiquitin aldehyde (UA), which is a specific nonpermeating UCH inhibitor, significantly (P < 0.05) increased polyspermy during porcine IVF and reduced (P < 0.05) UCH enzymatic activity measured in motile boar spermatozoa using a specific fluorometric UCH substrate, ubiquitin-AMC. Antibodies against two closely related UCHs, UCHL1 and UCHL3, detected these UCHs in the oocyte cortex and on the sperm acrosome, respectively, and increased the rate of polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with the meiotic spindle. Sperm-borne UCHL3 was localized to the acrosomal surface and coimmunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T reduced polyspermy when added to the fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and was partially degraded 6-8 h after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-ZP interactions and antipolyspermy defense. Modulation of UCH activity could facilitate the management of polyspermy during IVF and provide insights into male infertility.     

An intermediate state of fertilization involved in internalization of sperm components

The pathway of sperm entry during sea urchin fertilization was analyzed by using sperm covalently labeled with fluorescent and radioactive tracers. Sperm that have been covalently labeled on their surfaces with fluorescein isothiocyanate (FITC) or a radioactive congener, diiodofluorescein isothiocyanate (¹²⁵IFC), transfer labeled components to the egg that persist throughout early development. In order to study the transfer of sperm components and their fate after fertilization, cytochalasin B-dependent inhibition of fertilization, previously shown to permit the cortical reaction of sea urchin eggs but block sperm pronuclear incorporation, was investigated. Under certain conditions cytochalasin B or D (CB or CD) results in about half of the activated eggs having both the sperm nucleus and the fluorescently labeled sperm components arrested apparently at the level of the egg plasma membrane. This arrest of internalization was reversed by removal of CB or CD, and the sperm derivatives entered the egg. When sperm were labeled noncovalently with ethidium bromide or rhodamine 123, fluorescence was transferred to the egg in the cytochalasin-inhibited state in a fashion similar to that found in normal fertilization; in both cases the sperm fluorescence disappeared within a few minutes of fertilization, due to the repartitioning of the noncovalent dyes into the egg cytoplasm. It is concluded that cytochalasin arrests fertilization at an intermediate step in which the sperm has fused with the egg to achieve cytoplasmic continuity, but in which the subsequent internalization of sperm components is inhibited. After removal of cytochalasins the fluorescent sperm components move from the egg surface to an internal site, a process that can be monitored by time-lapse video microscopy with an image intensifier to permit extended observations of sperm fluorescence. The cytoplasmic location of labeled sperm components was substantiated by autoradiography of early embryos fertilized with ¹²⁵IFC-labeled sperm; transfer of sperm components to an internal site was seen after fertilization of either sea urchin or mouse eggs. Taken together, the data suggest that the fate of the labeled sperm surface components, as well as that of the sperm nucleus, is to be transferred to the egg cytoplasm, and that this transfer is mediated by the actin-dependent cytoskeleton of the egg.     

Studies on the specificity of sperm binding in echinoderm fertilization

Using gametes from the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus, we have evaluated the role of the acrosome reaction and the sperm-egg binding process in the block to interspecific fertilization among echinoids. The results indicate that sperm preinduced to undergo the acrosomal reaction by two different methods still bind to homologous eggs in a species specific manner. These results, taken in conjunction with an earlier study on species specificity of jelly coat induction of the acrosomal reaction (SeGall and Lennarz 1978), indicate that both the acrosome reaction and the sperm binding process contribute to the species specificity of fertilization in S. purpuratus and A. punctulata.     

Oocyte fertilization triggers acid phosphatase activity during Rhodnius prolixus embryogenesis

Acid phosphatase activity, previously identified in Rhodnius prolixus oocytes, was studied during egg development. Fertilized eggs exhibited a five fold increase of total acid phosphatase activity during the first days of development. In contrast non-fertilized oviposited eggs showed no activation of this enzyme. An optimum pH of 4.0 for pNPP hydrolysis in a saturable linear reaction and a strong inhibition by lysosomal acid phosphatase inhibitors such as NaF (10 mM) and Na(+)/K(+) tartrate (0.5 mM) are the major biochemical properties of this enzyme. Fractionation of egg homogenates through gel filtration chromatography revealed a single peak of activity with a molecular mass of 94 kDa. The role of this enzyme in VT dephosphorylation was next evaluated. Western blots probed with anti-phosphoserine polyclonal antibody demonstrated that VT phosphoaminoacid content decreases during egg development. In vivo dephosphorylation during egg development was confirmed by following the removal of (32)P from (32)P-VT in metabolically labeled eggs. Vitellin was the only phosphorylated molecule able to inhibit pNPPase activity of partially purified acid phosphatase. These data indicate that acid phosphatase activation follows oocyte fertilization and this enzyme seems to be involved in VT dephosphorylation.     

Egg and sperm recognition systems during fertilization

Fertilization is a programmed process that has many molecules and sequential events amenable to study. The biochemistry of fertilization has identified cellular and acellular components fundamental to the interactions between sperm and egg. Recent studies highlight the molecular details of the species-specificity of fertilization that involve protein–protein and protein–carbohydrate interactions. Although the diversity of structure and mechanism may imply rapid evolution of fertilization proteins, understanding the structure–function relationships has become important. Here, we introduce the molecules controlling the sperm AR, sperm attachment to, and penetration through, the egg investments.     

Effect of recombinant boar beta-acrosin on sperm binding to intact zona pellucida during in vitro fertilization

In a previous paper we demonstrated that boar beta-acrosin recombinant proteins were able to bind non-enzymatically to solubilized pig zona pellucida (ZP) glycoproteins. Here we report the participation of boar beta-acrosin in the secondary binding of sperm to intact pig ZP. This was achieved by using two boar recombinant proteins: beta-acrosin and a mutant of the catalytic site, beta-acrosin Ser/Ala(222). Assays of binding between the iodinated recombinant beta-acrosin and whole ZP showed that this binding could be saturated, was specific, and was stable over time. Using autoradiography, we determined that recombinant beta-acrosin bound on the entire surface of the ZP but initially was distributed heterogeneously. This suggests that the ligands for beta-acrosin may not be homogeneously distributed on the ZP. To study the contribution of acrosin in sperm secondary binding to the ZP, we preincubated in vitro-matured oocytes with these recombinant proteins and then performed in vitro fertilization assays. Under the experimental conditions used, binding of beta-acrosin recombinant proteins did not block sperm penetration. These results suggest that there may be other proteins that participate in the secondary binding, and that these proteins may recognize ligands that are different from those blocked by beta-acrosin recombinant proteins.     

Monoclonal antibodies to intra-acrosomal proteins inhibit gamete binding in vitro

The sperm-zona pellucida-binding assay in vitro was used as a functional test for zona pellucida-binding ability of boar spermatozoa after co-incubation with monoclonal antibodies against intra-acrosomal proteins. The effect of monoclonal antibodies ACR.2 against boar acrosin (55, 53, 45 and 38 kDa), and Hs-8 against boar intra-acrosomal protein (230, 110, 88, 60, 48 kDa) on boar spermatozoa-porcine oocyte binding was examined. The sperm-zona pellucida-binding was reduced when medium was supplemented with monoclonal antibodies during sperm-oocyte co-incubation, but not when capacitated spermatozoa were pretreated with monoclonal antibodies before incubation with oocytes. Our results show that the monoclonal antibodies (ACR.2, Hs-8) against intra-acrosomal proteins reduce the secondary sperm-zona pellucida-binding with statistically significant difference. This suggests the role of these proteins in the early phases of fertilization.     

Calcium-mediated mitochondrial movement in ascidian sperm during fertilization.

Sperm of the solitary ascidian Ascidia ceratodes shed their mitochondria on contact with the chorion. The mitochondrion forms a sphere which slides down the sperm tail ultimately to be released in about 2 min. During this process the sperm emits acid into the surrounding seawater, lowering the pH. Raising the external pH to 9.5 stimulates the shedding process in the absence of eggs. This requires Ca²⁺ but not Na⁺. Removal of Na⁺ from the medium also induces the reaction in the presence of Ca²⁺. Sperm freshly diluted in pH 9.5 seawater require 2.5 min for 50% fertilization, while the same sperm 13 min later require only 1 min, suggesting that mitochondrial shedding is rate limiting in fertilization. Acid release can be induced by diluting the sperm in Na⁺-free seawater containing Ca²⁺ or by the ionophores A23187 or Nigericin. The necessity of Ca²⁺ for both acid release and the morphological changes suggests that an exchange of Ca²⁺ for H⁺ may be occurring which triggers the shedding process.     

Understanding fertilization through intracytoplasmic sperm injection (ICSI)

Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.     

Autophagy is not involved in the degradation of sperm mitochondria after fertilization in mice

In almost all animals, mitochondrial DNA (mtDNA) is transmitted only from the female, while the paternal mitochondria and mtDNA are thought to be eliminated during early embryogenesis. Autophagy is involved in the elimination of sperm mitochondria and mtDNA in early embryos in Caenorhabditis elegans; however, solid evidence is still lacking in mammals. Recently, we found that despite the fact that some autophagy-related proteins, such as SQSTM1 and LC3 could localize nearby sperm mitochondria before the 2-cell stage, autophagy did not participate in the elimination of sperm mitochondria and mtDNA. Instead, the pre-elimination of sperm mtDNA before fertilization and the restriction of sperm mitochondria in one blastomere before 4-cell stage embryos are the most important mechanisms of maternal mitochondrial inheritance in mice.     

Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida

Glioma pathogenesis‐related 1‐like protein1 (GliPr1L1) was identified by liquid chromatography‐tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine‐rich secretory proteins, Antigen 5 and pathogenesis‐related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N‐glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N‐glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti‐GliPr1L1 showed that this protein is involved in sperm–zona pellucida interaction. J. Cell. Physiol. 227: 3876–3886, 2012. © 2012 Wiley Periodicals, Inc.     

Cloning and sequencing of cDNA encoding for a human sperm antigen involved in fertilization

A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-λgt11 expression library by using antibodies to human sperm surface antigens belonging to 14–18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5′ and 139-bp 3′ noncoding regions. The translated protein has a calculated molecular weight of ∼19 kD, and has two casein kinase II (CK-2) sites at aa 94–97 and 149–152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935–76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of ∼20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The ∼20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans. Mol. Reprod. Dev. 51:176–183, 1998. © 1998 Wiley-Liss, Inc.     

Receptor function of mouse sperm surface galactosyltransferase during fertilization

Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GlcNAc) residues in the zona pellucida (Shur, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GlcNAc-agarose and alpha-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn++-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away. The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GlcNAc substrate in the zona pellucida. Under identical conditions, UDP-glucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time- and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites. Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.     

Molecular identification of the sperm selection involved porcine sperm binding glycoprotein (SBG) as deleted in malignant brain tumors 1 (DMBT1)

Porcine sperm binding glycoprotein (SBG) is involved in sperm-oviduct interaction. Here we use mass spectrometry to identify SBG, finding peptides corresponding to deleted in malignant brain tumors 1 (DMBT1), at scavenger receptor cysteine-rich (SRCR) and CUB domains. RT-PCR allowed the cloning of unique sequences, belonging to porcine DMBT1. Western blot and immunofluorescence of oviductal tissues using anti-SBG and anti-hDMBT1 antibodies showed identical results. The biochemical characteristics of both proteins are coincident. We conclude that porcine SBG is an oviductal form of DMBT1, and thus assign this protein a novel location and function.     

Identification of human sperm peptide sequence involved in egg binding for immunocontraception

Development of a vaccine based on sperm antigens represents a promising approach to contraception. The sperm-zona pellucida (ZP) interaction constitutes the most important event in the fertilization process, and the molecular sequences involved at this site may provide the most attractive candidates for immunocontraception. In the present study, using the phase peptide display technique, a novel dodecamer sequence, designated as YLP(12), was identified that is involved in sperm-ZP recognition/binding. The synthetic 12-mer peptide based on this sequence and its monovalent Fab' antibodies specifically and significantly (P < 0.05) inhibited human sperm-ZP binding. In Western blot and immunoprecipitation procedures, the YLP(12) peptide recognized the ZP3 component of solubilized human ZP proteins. In the Western blot procedure involving 10 different human tissue extracts, the anti-YLP(12) Fab' antibodies recognized a protein band of approximately 72 +/- 2 kDa only in the testis lane. The peptide sequence was localized on the acrosomal region of the human sperm cell. These findings indicate that the novel testis-specific 12-mer YLP(12) that is present in the acrosomal region and is involved in human sperm-ZP interaction may find applications in contraceptive vaccine development, as well as in diagnosis and treatment of male infertility mediated through sperm dysfunction.