Vitelline coat of Unio elongatulus: II. Biochemical properties of the 220- and 180-kD components

In a previous study we found that two glycoproteins with apparent molecular weights of 220 kD and 180 kD account for 80–90% of the material dissolved from the vitelline coat of the egg of the bivalve mollusk, Unio elongatulus (Focarelli and Rosati, 1993: Mol Reprod Dev 35:44–51). In this study we isolated and purified these glycoproteins by electroelution. The two proteins differ in many respects: the 180-kD molecule is acidic in nature and highly heterogeneous, whereas the 220-kD protein is neutral and less heterogeneous. Both seem to have O- and N-linked oligosaccharide chains. The 180-kD protein contains 13–16% carbohydrate, whereas the 220-kD molecular contains only 7–8%. Amino acid analysis and peptide mapping also show that each protein represents a unique polypeptide chain. © 1995 Wiley-Liss, Inc.     

A 140‐kDa glycopeptide from the sperm ligand of the vitelline coat of the freshwater bivalve Unio elongatulus,only contains O‐linked oligosaccharide chains and mediates sperm‐egg interaction

In previous studies we found that sperm binding activity in the vitelline coat of the freshwater bivalve Unio elongatulus is located on the O‐linked oligosaccharide chains of gp273, one of the two major components of the extracellular coat, and that fucose plays a key role in this interaction. In this paper we report the partial characterization of a large glycopeptide (about 140 kDa) obtained by cyanogen bromide fragmentation of gp273, that maintains sperm binding activity. Lectin blotting revealed that the glycopeptide reacted with lectins from Arachis hypogaea (PNA) and Lotus tetragonolobus (LTA) but not Canavalia ensiformis (ConA). No other PNA‐positive fragments could be detected in the electrophoretic pattern of fragmented gp273 but several ConA‐positive fragments of lower molecular weight were present indicating that all the O‐linked chains are clustered together in this fragment. Two‐dimensional gel electrophoresis of the fragment revealed it to be acidic in nature in contrast with the neutral character of the whole gp273 molecule. Competition binding assay showed that this fragment is a strong inhibitor of the interaction, whereas no effect was detected using the ConA‐positive peptides. This confirms that the sperm receptor activity of gp273 is related to its O‐linked chains. The immunodominance of this fragment is also discussed. Mol. Reprod. Dev. 54:203–207, 1999. © 1999 Wiley‐Liss, Inc.     

Direct evidence for the secretion of lactoferrin and its binding to sperm in the porcine epididymis

Lactoferrin has been for the first time purified from the porcine cauda epididymal fluid as a 70 kDa protein. Both Western and Northern blot analyses show that lactoferrin is synthesized in the regions from the distal caput to the cauda epididymis and secreted into the luminal fluid. Lactoferrin is first secreted as a 75 kDa glycoprotein and its carbohydrate moieties are gradually digested to form 70 kDa protein in the cauda epididymis. Lactoferrin has already bound to the surface of the epididymal sperm because the anti-lactoferrin antiserum induces the mature sperm tail-to-tail agglutination. These results strongly suggest new physiological functions of lactoferrin on the sperm maturation in the epididymis. Mol. Reprod. Dev. 47:490–496, 1997. © 1997 Wiley-Liss, Inc.     

Following passage through the oviduct, the coelomic envelope of Discoglossus pictus (amphibia) acquires fertilizability upon reorganization, conversion of gp 42 to gp 40, extensive glycosylation, and formation of a specific layer

This paper describes the morphological and biochemical changes in Discoglossus pictus coelomic oocyte envelope (CE) following passage through the oviduct. As in other anurans, in this species, the transformation of the envelope into vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein. In addition, several features, typical of Discoglossus pictus, were observed. A new layer, VE-D, forms underneath the VE region facing the site of sperm entrance, the dimple. In the VE, arrowhead-like bundles of fibrils are perpendicularly oriented toward the dimple. Ultrastructural observations and staining with UEA-I suggested that VE-D might have a role in supporting sperm penetration into the dimple by orienting VE bundles and exposing sugar residues such as fucose. In 'in vitro' tests, VE binding of sperm occurs only if sperm are exposed to A23187, in agreement with previous data (Campanella et al., 1997: Mol Reprod Dev 47:323-333). Sperm binding occurs all over the VE. Accordingly, extracts of the VE covering the animal or the vegetal hemisphere have the same affinity to lectins (DBA, DSA, GNA, MAA, SBA, SNA, UEA-I, WGA). The CE contains six main glycoproteins. Peptide mapping indicated that during CE transformation into VE, gp 42 shifts to an apparent M(r) of 40 and gp 61 is converted to an apparent M(r) of 63 kDa. Lectin blot analyses showed extensive changes in cross-reactivity of most glycoproteins during the CE-->VE transition. The fact that DBA and UEA-I stain gp 63 rather than gp 61 and that this change is related only to gp 63, suggested that O-glycosylation and terminal fucose might be acquired by gp 63 in preparation of fertilization. Gp 63 has recently been cloned (Vaccaro et al., submitted) and shown to exhibit high homology to Xenopus gp 69/64, a VE sperm ligand (Tian et al., 1997a: J. Cell Biol. 136: 1099-1108; Tian et al., 1997b: Dev Biol 187:143-153), and to ZP2 of mammals.     

Giant,accordioned sperm acrosomes of the greater bulldog bat,Noctilio leporinus

Sperm of the greater bulldog bat Noctilio leporinus display an architecture that is totally unique among mammalian spermatozoa. The sperm head of Noctilio is extraordinarily large and flat and lies eccentrically with respect to the sperm tail. The major portion of the atypically large acrosome lies anterior to the nucleus and is shaped into a dozen accordionlike folds that run parallel to the long axis of the sperm. The ridge of each fold is shaped into ∼60 minute, evenly spaced rises that extend along the entire length of the fold. We speculate that acrosome ridges may serve to strengthen the sperm head during transport. Mol. Reprod. Dev. 48:90–94, 1997 © 1997 Wiley-Liss, Inc.     

Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane,the other involving the follicle cells

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm–egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 μM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 μM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N-acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm–egg fusion. Mol. Reprod. Dev. 48:137-143, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of the guanidinobenzoatase in the epididymal fluids of the mouse

Guanidinobenzoatase (GB), a proteolytic enzyme found in the epididymal fluids of mice, was purified to apparent homogeneity by molecular sieving and affinity chromatography. It has a molecular mass of 71 kDa and its enzymatic activity is heat labile and sensitive to EGTA. Its kinetic parameters (Km of 6.66 μM and a Vmax of 4.38 nmol/min/mg) were determined using 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) as the substrate. GB activity is concentrated in the cauda epididymal region of the genital tract. Heat-solubilized whole zonae, biologically active ZP3, and several serine proteinase inhibitors, including a proteinase inhibitor endogenous to the male genital tract, effectively block the ability of GB to hydrolyze MUGB. Pretreating cumulus-free, zonae intact oocytes with purified GB reduces, in a concentration-dependent manner, the number of sperm able to bind to the zonae. The function of the soluble enzyme is not known. Its ability to bind both trypsin inhibitors and ZP3 suggests a possible role in gamete recognition. Mol. Reprod. Dev. 47:204–209, 1997. © 1997 Wiley-Liss, Inc.     

Rat MDC family of proteins: Sequence analysis,tissue distribution,and expression in prepubertal and adult rat testis

An increasing number of sequence-related, cysteine-rich membrane proteins containing metalloproteinase-like and disintegrin-like domains (the MDC protein family) have been identified in mammalian tissues. Here, we report the cloning and sequence analysis of cDNAs encoding several rat orthologues of this protein family, some of which are found to be expressed exclusively in the male reproductive tract, others exhibiting a broader tissue distribution. We also examine their expression in prepubertal and adult rat testis, which, in conjunction with the data on tissue distribution, form a necessary prelude to further studies aimed at establishing their individual functions. Mol. Reprod. Dev. 48:159–167, 1997. © 1997 Wiley-Liss, Inc.     

Glycosidases are present on the surface of Drosophila melanogaster spermatozoa

We investigated the presence of enzymes on the surface of Drosophila melanogaster spermatozoa that might bind to the carbohydrate residues of the egg shell. Spectrophotometric and fluorimetric studies were used on whole spermatozoa to assay galactosyltransferase and glycosidase activities. No galactosyltransferase is present on the sperm surface, whereas two glycosidases, β-N-acetylglucosaminidase (GlcNAc′ase) and α-mannosidase (Man′ase), have been evidenced. They have an optimal pH of 6–6.5 and 4, respectively. The same glycosidases were detected as soluble forms probably secreted by the seminal vesicle epithelium. We suggest that these enzymes might be involved in the recognition of α-mannose and β-N-acetylglucosamine residues present on the egg shell at the site of sperm entry. Mol. Reprod. Dev. 48:276–281, 1997. © 1997 Wiley-Liss, Inc.     

New member of the Snf1/AMPK kinase family,Melk, is expressed in the mouse egg and preimplantation embryo

The initial phase of mammalian preimplantation development is directed by stored maternal mRNAs and their encoded proteins, yet most of the molecules controlling this process have not been described. We have used differential display analysis of cDNA libraries prepared from unfertilized eggs and preimplantation embryos to isolate three maternal cDNAs that represent novel genes exhibiting different patterns of expression during this developmental period. One of these, Melk, encodes a protein with a kinase catalytic domain and a leucine zipper motif, a new member of the Snf1/AMPK family of kinases. This gene product may play a role in the signal transduction events in the egg and early embryo. Mol. Reprod. Dev. 47:148–156, 1997. © 1997 Wiley-Liss, Inc.     

Triangle Consortium for Reproductive Biology 22nd Annual Meeting

Mol. Reprod. Dev. 80: 504–507, 2013. © 2013 Wiley Periodicals, Inc. This article is a US government work and, as such, is in the public domain in the United States of America.     

Binding of rat and ovine epididymis-specific prealbumins (PES) to rat spermatozoa without effect of heterologous immunization on rat fertility

The epididymis, under control of testosterone, secretes proteins which bind to the membrane of the spermatozoa during their passage through the lumen. One such class is termed PES (prealbumin epididymal specific). Injection of heterologous oPES (ovine PES) into male rats caused antibody production but failed to induce sterility, unlike results previously obtained when rat PES was injected into male rats. This suggests that only very restricted species-specific epitopes of PES might be useful for causing immunocontraception. Despite this, the sperm binding properties of PES purified from the rat (rat PES) and from the ram (oPES) were shown to be similar. When either rat PES or oPES, conjugated with a fluorescent probe (dimethylamino-fluorescein), was incubated with washed rat spermatozoa originating from the caput, corpus or cauda epididymis, results of flow cytometric analysis showed: (1) the number of spermatozoa bound to isologous or heterologous fluorescent PES, and (2) the binding-affinity of spermatozoa for PES was greater for sperm collected from more distal sites in the epididymis. Mol. Reprod. Dev. 47:483–489, 1997. © 1997 Wiley-Liss, Inc.     

Isolation and characterization of a protein with homology to angiotensin converting enzyme from the periacrosomal plasma membrane of equine spermatozoa

The periacrosomal plasma membrane of spermatozoa is involved in sperm binding to oviductal epithelial cells and to the zona pellucida. A protein of 68–70 kD molecular mass was purified biochemically from the isolated periacrosomal plasma membrane of equine spermatozoa as a possible receptor for adhesion of spermatozoa to oviductal epithelial cells. A polyclonal antibody raised in rabbits against the purified equine sperm membrane protein recognized the 70 kD and an antigenically related 32 kD protein in preparations of isolated periacrosomal sperm plasma membrane and in detergent extracted ejaculated and epididymal spermatozoa. A larger protein (∼110 kD) was detected in equine testis. Two antigenically related proteins (64 and 45 kD) were recognized on the plasma membrane of cynomolgus macaque spermatozoa. In vitro sperm-binding assays were performed in the presence of antigen-binding fragments or IgG purified from the polyclonal antiserum to investigate a possible function of the isolated protein in binding of equine spermatozoa to homologous oviductal epithelial cells or zona pellucida. Incubation with antigen-binding fragments or IgG purified from the antiserum did not inhibit binding of equine spermatozoa either to oviductal epithelial cells or to the zona pellucida. On ultrastructural examination, the antibody bound exclusively to the cytoplasmic side of the periacrosomal plasma membrane of equine and macaque spermatozoa. Microsequence analysis of 13 residues of sequence showed strong homology with a number of angiotensin converting enzymes: An 84% identity was identified with testis specific and somatic forms of human and mouse angiotensin-converting enzyme. Immunocytochemistry and immunoblot analysis established that the protein is specific for the periacrosomal membrane of ejaculated, epididymal, and testicular stallion spermatozoa. Mol. Reprod. Dev. 48:251–260, 1997. © 1997 Wiley-Liss, Inc.     

cDNA sequence and deduced amino acid sequence of bovine oviductal fluid catalase

A bovine oviductal fluid catalase (OFC) which preferentially binds to the acrosome surface of some mammalian spermatozoa has recently been purified. The objectives of this study were to clone the OFC, obtain the full-length cDNA and protein sequence and determine which characteristics of the proteins are associated with the binding of the enzyme to sperm surface. Northern blot analysis revealed low levels of catalase mRNA in bovine oviducts and uterus compared to the liver and kidney. Screening of a cDNA library from the cow oviduct permit to obtain a full-length cDNA of 2282 bp, with an open reading frame of 1581 bp coding for a deduced protein of 526 amino acids (59 789 Da). The deduced protein contained four potential N-glycosylation sites and many potential O-glycosylation sites. The OFC protein exhibited high identity with catalase from other bovine tissues, likewise with catalases from human fibroblast and kidney, and with rat liver catalase. The homology of amino acid sequence of OFC with bovine liver catalase was about 99%. However the OFC posses an extended carboxyl terminus of 20 amino acids not present on the liver catalase. This result is supported by a lower mobility of the OFC compared to the liver catalase when both proteins are submitted on SDS-PAGE. Mol. Reprod. Dev. 51:265–273, 1998. © 1998 Wiley-Liss, Inc.     

Cryopreservation of mouse embryos affects later embryonic development possibly through reduced expression of the glucose transporter GLUT1

In order to study the effects of cryopreservation on later embryonic development, two-cell mouse embryos were frozen, thawed, and then allowed to develop into blastocysts. The percentage of cryopreserved embryos which developed into blastocysts was significantly lower than that of fresh two-cell embryos. The amount of glucose incorporation in terms of ³H-2-deoxyglucose uptake in blastocysts developed in vivo, and in vitro from fresh or frozen-thawed two-cell embryos, was 473 ± 108, 105 ± 75, and 43.0 ± 28.3 fmol per embryo per hour, respectively. Quantification of glucose transporter GLUT1 in these embryos by Western blotting was reflective of the degree of glucose incorporation. The implantation rate of blastocysts developed in vitro from frozen-thawed two-cell embryos (22.0%) was significantly lower than that developed in vivo (41.1%). These data suggest that cryopreservation may have later consequences on embryonic development through a mechanism that involves altered GLUT1 expression. Mol. Reprod. Dev. 48:496–500, 1997.© 1997 Wiley-Liss, Inc.     

Cytokeratin cytoskeleton in the differentiating ovarian follicle of the lizard Podarcis sicula Raf

By immunoblotting and immunocytochemical techniques, we characterized the cytokeratins previously localized by us in the previtellogenic ovarian follicle of Podarcis sicula. Our results show that these cytokeratins correspond to those expressed in the monolayered epithelia. In fact, the immunoblotting analysis showed that the NCL-5D3 antibody, specific for human low molecular weight cytokeratins expressed in monolayered epithelia, reacted with the cytokeratins extracted both from the ovary and from the monolayered intestinal mucosa of Podarcis sicula. Furthermore, this antibody, in this reptile as in humans, clearly immunolabeled sections of corresponding tissues. The organization of the cytokeratin cytoskeleton in the main steps of the ovarian follicle differentiation was also clarified. The reported observations suggest that in Podarcis sicula, the cytokeratin cytoskeleton is absent in the early oocytes. It first appears in the growing oocytes as a thin cortical layer in concomitance with its becoming visible also in the enlarging follicle cells. In the larger follicles, this cytoskeleton appears well organized in intermediate cells and in particular in fully differentiated pyriform cells. In both these cells a cytokeratin network connects the cytoplasm to the oocyte cortex through intercellular bridges. At the end of the previtellogenic oocyte growth, the intense immunolabeling of the apex in the regressing pyriform cells suggests that the cytokeratin, as other cytoplasmic components, may be transferred from these follicle cells to the oocyte. At the end of the oocyte growth, in the larger vitellogenic oocytes surrounded by a monolayer of follicle cells, the cytokeratin constitutes a heavily immunolabeled cortical layer thicker than in the previous stages. Mol. Reprod. Dev. 48:536–542, 1997. © 1997 Wiley-Liss, Inc.