Proteins from human oviductal tissue-conditioned medium modulate sperm capacitation.

BACKGROUND: Spermatozoa acquire the ability to fertilize an oocyte when they become capacitated. Capacitation takes place when sperm pass through the female reproductive tract, interacting with female fluids. Both tyrosine phosphorylation of sperm proteins and the ability to respond to acrosome reaction (AR) inducers have been associated with sperm capacitation. Recent data indicate that conditioned media (CM) from human oviductal tissue culture decrease sperm affinity for the zona pellucida in vitro. Since capacitation enables the sperm-oocyte interaction, the aim of the present study was to investigate the effect of CM on events related to sperm capacitation and to assess whether these effects were permanent. METHODS: Oviductal tissue was obtained from premenopausal patients (scheduled for hysterectomies because of uterine fibromyoma). The tissues were cultured as explants and CM were collected. Explant viability was assessed as tissue DNA integrity. Normozoospermic semen samples were obtained from healthy donors. Motile spermatozoa were incubated under capacitating conditions with or without increasing protein concentrations of CM for 6 or 22 h. Human follicular fluid-induced AR was detected by the Pisum sativum technique. Tyrosine phosphorylated proteins were detected with a monoclonal anti-phosphotyrosine antibody. RESULTS: The incubation of spermatozoa in the presence of increasing concentrations of conditioned medium (CM) proteins caused a dose-dependent decrease in both tyrosine phosphorylation of sperm proteins and in the level of AR induction. When CM was removed from the sperm incubation media, the effects were reversed. Heat-inactivated CM did not affect either tyrosine phosphorylation or the induction of AR. CONCLUSIONS: The present data suggest that proteins secreted from human oviductal tissue are able to inhibit events associated with sperm capacitation in vitro.

ACROSOMIC REACTION AND PROTEOLYTIC ACTIVITY IN THE SPERMATOZOA OF AN ANURAN AMPHIBIAN, LEPTODACTYLUS CHAQUENSIS (CEI).

Acrosomal reaction and acrosomal protease release in Leptodactylus chaquensis are correlated. When spermatozoa are included in gelatin films, the characteristic digestion halos around the sperm head can be observed only after the acrosomic reaction is accomplished. The addition of 10 mM EDTA to the gelatin films arrests the acrosomic reaction and no digestion halo is formed. Crude enzymatic preparations containing acrosomal proteases are equally active on gelatin and BAPNA substrates, irrespective of the presence of EDTA. Trypsin inhibitors (ovomucoid, lima bean and soybean) produce both diminution of the digestion halo and lowering of the activity of crude enzymatic preparations on gelatin or BAPNA substrates.

EFFECT OF TRYPSIN INHIBITORS AND CONCANAVALIN A ON BUFO ARENARUM FERTILIZATION.

The presence of some trypsin inhibitors (soybean trypsin inhibitor and lima bean trypsin inhibitor) in the inseminating media, is effective in blocking Bufo arenarum fertilization. Ovomucoid does not significantly affect fertilization, even at concentrations as high as 10 mg/ml. Similarly the presence of Concanavalin A in the inseminating media results in fertility impairment. The possible mechanisms of action of these substances are discussed.

Interaction of ovary lectin with homologous sperm from Bufo arenarum.

A soluble beta-galactoside lectin purified from Bufo arenarum ovary agglutinated homologous neuraminidase-treated spermatozoa. Microscopic observations of sperm clusters showed that spermatozoa agglutinated in a random way, but the head-to-head type of sperm agglutination was the most common (94-98%). The lectin activity was specifically inhibited by D-galactose and its derivatives, thio-digalactoside being the most active saccharide inhibitor.

Vitelline envelope formation during oogenesis in Bufo arenarum.

The formation of vitelline envelope (VE) during the oogenesis of Bufo arenarum (Amphibia Anura) is described. At the stage of early vitellogenesis, the first structures appear: the number of oocyte microvilli increases, and many cross sections of them are observed between the follicle cells and the oocyte. A filamentous material is observed inside the follicle cells and between the follicle cells and the oocyte. Multivesicular bodies are also found in the follicle cells, and in the perivitelline space. The micrographs also suggest the participation of the oocyte in the process of VE formation: large vesicles are present in the cortex of the oocyte, filled with an amorphous material of low and uniform electron density. Some of them are in the process of releasing their content to the perivitelline space. Many vesicles (probably resulting from microvilli fragmentation) are also observed in the perivitelline space. During late vitellogenesis the VE is a continuous structure between the layer of follicle cells and the oocyte. The filamentous material is aggregated in bundles, forming a net, and the spherical components are now either included in the orifices of the net, or free near the oocyte's surface. At the end of oogenesis, when the VE is completely formed, it is difficult to distinguish its components independently. Immunolocalization with antibodies against VE, show a positive reaction in follicle cells and oocytes in previtellogenic and full grown ovarian follicles. This analysis suggests that both the oocyte and the follicle cells are directly involved in the synthesis and secretion of the components of the vitelline envelope in Bufo arenarum.

Sperm concentration and fertilization rate in Bufo arenarum (Amphibia: Anura).

The influence of sperm concentration upon the fertilization rate of Bufo arenarum oocytes was determinated. The experimental results were analysed according to the theories of Rothschild and Swann, and Hultin and Hagström for Psammechinus miliaris. The experimental results agreed with the predictions of the latter theory. Since Bufo arenarum and Psammechinus miliaris oocytes differ in both size and disposition of jelly envelopes, the postulations of Hultin and Hagström's theory appear to have a general validity.

Diffusible highly glycosylated protein from Bufo arenarum egg-jelly coat: biological activity.

L-HGP is a highly glycosylated protein from Bufo arenarum egg-jelly coat that diffuses into the surrounding medium when the strings of oocytes are incubated in saline solutions. L-HGP was purified from egg water and the estimated percentage of L-HGP/total protein in egg water was estimated in 30%. In the present study we examine, by indirect immunofluorescence, the effect of L-HGP on acrosome status of homologous spermatozoa. A high percentage (77%) of sperm lost the acrosome when incubated in 10% Ringer solution buffered with 10 mM Tris-HCl, pH 7.6, during 60 min, a condition that resembles egg-jelly osmolarity. The addition of purified L-HGP to the incubation medium prevents acrosome breakdown. The acrosome integrity is maintained for at least 1 hr. This effect is specific for L-HGP at concentration ranging from 0.01 to 0.1 mg/ml since neither BSA nor fetuin seems to have similar activity at similar concentrations. The same effect was observed when spermatozoa were incubated in egg water. Preliminary results suggest that L-HGP binds to B. arenarum spermatozoan membranes.

Amphibian cross-fertilization and polyspermy.

A string of oocytes from Bufo arenarum (Ba) was inseminated with spermatozoa from Ba, Leptodactylus chaquensis (Lch), or Bufo paracnemis (Bp). Homologous insemination resulted in monospermic eggs and normal development, while in the case of heterologous fertilization both polyspermy and abnormal development were the rule. Oocytes from Ba inseminated with homologous spermatozoa, when reinseminated 30 sec after the first insemination with heterologous spermatozoa, exhibit a high rate of polyspermy and abnormal development. A lower rate of abnormalities was observed when reinsemination was carried out 60 sec after the first insemination. Normal development and an absence of polyspermy were observed in the case of eggs reinseminated 300 sec after initial insemination. This result indicated that the electrophysiological blockage of fertilization either was not triggered or was ineffective. The establishment of the permanent blockage of polyspermy, either by artificial activation of oocytes or as a result of their incubation with the products of the cortical granules, inhibited penetration of Ba eggs by heterologous sperm.

Some biophysicochemical properties of Bufo arenarum oocytes jelly coats.

The state of water in the oocyte jelly coat in Bufo arenarum has been studied by different methods. The water exchange and adsorption isotherm show a high interaction between water and the matrix of jelly coat. Some biological implications are discussed.

Assessment of the acrosome reaction in Bufo arenarum spermatozoa by immunostaining: comparison with other methods.

The acrosome reaction in Bufo arenarum spermatozoa has been visualised only by electron microscopy. Different staining procedures for spermatozoa are described in the present study. By light microscopy the acrosomal status cannot be determined. In some reacted sperm, stained with Coomassie blue, a filament that could be the "perforatorium' was observed, as seen by electron microscopy. Several fluorescent lectins were used, but only FITC-PNA binds to acrosomal proteins. However, the fluorescence observed was weak. Indirect immunofluorescence, with antibodies raised against acrosomal matrix, showed different staining patterns between acrosome-reacted and intact spermatozoa. The technique is specific for evaluating acrosomal status in a large population of spermatozoa. Moreover, immunostaining, in contrast with lectin staining, can be carried out in the presence of glycoconjugates such as oocyte extracellular matrix without interference.

Purification and biological characterization of N-acetyl beta-D glucosaminidase from Bufo arenarum spermatozoa.

Fertilization in Bufo arenarum requires the sperm to penetrate the egg envelopes. The incubation of isolated vitelline envelopes with sperm induces the acrosome reaction, releasing proteases and glycosidases to the media. In the present work N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, and alpha-D-glucosidase activities are measured in spermatozoa. N-acetyl-beta-D-glucosaminidase is the major sperm glycosidase activity assayed. However, N-acetyl-beta-D-galactosamine show competitive inhibitory effect. The glycosidase pH optimum is 3.5 being inhibited at pHs higher than 7.5. In our study, N-acetyl-beta-D-glucosaminidase is the only glycosidase that in vitro binds to vitelline envelopes in conditions that resemble natural fertilization media. The isolation of the active enzyme will allow studies of its role in fertilization. The enzyme has been purified in a two-step procedure. After native gel electrophoresis, the activity-stained band was cut out and the eluted enzyme was finally subjected to ConA-sepharose chromatography. In SDS-PAGE, the denatured enzyme migrates as a single band with a molecular mass of 45 kDa. Furthermore, analysis by size-exclusion on HPLC showed a peak of activity at around 45 kDa. Preliminary localization studies showed higher relative activity in the acrosomal content. In addition, 10% of the N-acetyl-beta-D-glucosaminidase activity was associated with the reacted sperm. By in vitro fertilization assay, it was observed that the inhibition of the enzyme results in the inhibition of fertilization. This last study shows that N-acetyl-beta-D-glucosaminidase plays an important role in toad fertilization.

Primary structure and developmental expression of Bufo arenarum cellular nucleic acid-binding protein: changes in subcellular localization during early embryogenesis.

A Bufo arenarum cellular nucleic acid-binding protein (bCNBP) full-length cDNA was cloned. bCNBP is a 19.4 kDa protein containing seven CCHC zinc finger motifs, an RGG box and a Ser-rich region. Amino acid comparisons showed high values of homology in vertebrates and smaller values in insects or inferior eukaryotes. Northern blot analysis during oogenesis and early development revealed two transcripts with different expressions of pattern behavior. One of them is present in all stages analyzed, whereas the other is only detected from the beginning of zygotic transcription. Immunocytochemistry assays carried out on sections of ovary and early embryos showed that there was no specific staining of previtellogenic oocytes. In early vitellogenic oocytes, in oocytes at stages V/VI and in embryos at early blastula stage, reaction was observed inside the cytoplasm. At mid-blastula stage, CNBP was mainly detected in the epiblast. At the late gastrula stage, two layers of cells were stained in the archenteron roof, in which the internal one presented as strong staining. Nuclei in this layer were stained even stronger than the cytoplasm. Changes in mRNA expression patterns, accompanied by changes in subcellular localization, suggest that CNBP might interact with both nuclear and cytoplasmic nucleic acids.

Spermatolysins in Bufo arenarum: their activity on oocyte surface.

The activity of spermatolysins from Bufo arenarum spermatozoa on spawned dejellied oocytes is studied at structural and ultrastructural levels. After adding spermatolysins to spawned dejellied oocytes, a wrinkling of the animal hemisphere is first observed under a stereomicroscope. Two or three minutes later, the vitelline envelope in the animal hemisphere is completely digested, which produces oocyte flattening. The vitelline envelope covering the vegetal hemisphere is not modified with the treatment. Ultrastructural observations indicate that while the vitelline envelope of the vegetal hemisphere remains unaltered, the animal counterpart gradually loses its components and finally all structures disappear. Scanning microscope observations reveal that the microvilii of the plasma membrane in the animal hemisphere decreases in number and length, while the vegetal region is not altered by the enzyme.

Effect of cholesterol and phosphatidylcholine of different chain length on acrosome breakdown and fertilizing capacity of amphibian spermatozoa.

The effect of different lipids on the fertilizing capacity of Bufo arenarum spermatozoa and on acrosome breakdown of Leptodactylus chaquensis spermatozoa was studied. Sonicated vesicles of egg yolk phosphatidylcholine (1 mM) were as effective as vesicles of egg yolk phosphatidylcholine:cholesterol (molar ratio 1:0.9) in inhibiting the fertilizing capacity of Bufo arenarum spermatozoa. This suggests that cholesterol depletion from the spermatozoa was not the cause of the fertility loss. Bufo arenarum spermatozoa were incubated with phosphatidylcholines with even chain length from 6 to 18 carbons. At a concentration of 0.01 mM, didecanoyl-phosphatidylcholine reduced fertilizing capacity to 10% in a few minutes and to 0% within 60 minutes. Didodecanoyl-phosphatidylcholine required 2 hours to reduce fertility to 10% and 4 hours to cause a 100% loss of fertilizing capacity. A concentration of didecanoyl-phosphatidylcholine as low as 5 x 10(-4) mM caused a more than 95% fertility loss in less than five minutes. At a concentration of 0.1 mM, didecanoyl-phosphatidylcholine induced complete acrosome breakdown in Leptodactylus chaquensis spermatozoa in 15 minutes, whereas didodecyl-phospatidylcholine required 2 hours. At a concentration 100-fold lower didecanoyl-phosphatidylcholine induced complete acrosome breakdown in 2 hours. Electron microscopic observations in both species showed loss of acrosome caused by the action of the didecanoyl-phosphatidylcholine. Longer chain phosphatidylcholines exerted an inhibitory effect on Bufo arenarum spermatozoa fertilizing capacity at a higher concentration when in a vesicular form.

Lectin binding sites in the vitelline envelope of Bufo arenarum oocytes: role in fertilization.

Incubation of dejellied spawned oocytes from Bufo arenarum with different lectins results in a decrease of oocyte fertility. Concanavalin A was the most effective lectin; phytohemagglutinin P and wheat germ lectin were less effective. Agglutinin from soybean was scarcely active. These lectin effects could be ascribed to a hindering of specific sites for some proteases, since the same treatment renders the oocyte vitelline envelope insensitive to spermatolysin (an essential requisite for fertilization) and to trypsin. Also in this case concanavalin A was the most effective lectin. Univalent concanavalin A was also effective in blocking the fertility of dejellied oocytes. These results indicate that the residues of alpha-D-glucose and alpha-D-mannose present in the vitelline envelope are involved in gamete interactions in Bufo arenarum. This idea is also supported by the finding that dejellied oocytes (fertilizables) have a number of binding sites for concanavalin A that is three or four orders of magnitude higher than coelomic or fertilized oocytes (both not penetrable by spermatozoa).

Bufo arenarum egg jelly coat: purification and characterization of two highly glycosylated proteins.

Egg jelly coats from Bufo arenarum are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they transit along the different oviductal portions. In this study, we have isolated two highly glycosylated proteins of the jelly coat, which are secreted almost all the way along the oviduct. Both glycoproteins [designated as highly glycosylated protein (HGP) and low-molecular-mass highly glycosylated protein (L-HGP)] were purified to homogeneity, from the secretion of the caudal oviduct portion, by CsCl density gradient ultracentrifugation. HGP is a high-molecular-mass protein with mucin-like characteristics: high viscosity, a high content of serine and threonine, about 70% carbohydrate by weight, and a protease-resistant domain. Cleavage of disulphide bridges with reducing agents resulted in the release of a single subunit (300000 Da). L-HGP is also a disulphide-cross-linked protein with lower apparent monomeric molecular mass, in the range 100-120 kDa and containing 50% carbohydrate by weight. HGP contains galactose, fucose, N-acetylgalactosamine and sialic acid, but no mannose, suggesting the presence of O-linked oligosaccharides exclusively. The secretion ratio of HGP increases from cephalic (16% of total protein in pars preconvoluta) to caudal (40% of total protein in pars convoluta) oviductal portions. It appears to be the major structural component of the jelly coat. Our purification data suggest that HGP is non-covalently linked to the other egg jelly proteins. Polyclonal antiserum to each purified glycoprotein from secretion was raised in rabbits and used to localize both glycoproteins in the different oviductal portions, total egg jelly and the aqueous medium where oocyte strings were incubated. HGP forms a stable fibre matrix around the oocyte. L-HGP is present in the jelly coat and is released into the incubation medium.

Newly synthesized extracellular ribonucleic acids in the amphibian gastrula.

The possible synthesis of RNA located in the extracellular compartment of Bufo arenarum gastrula was studied using a biochemical method. [3H]adenosine was microinjected into the blastocoel of late blastulae or early gastrulae, which were then dissociated at advanced gastrula stage. RNA was extracted from both, the cellular supernatant and the disaggregated cells, by the Kirby-phenol procedure. Most of the ethanol-precipitable radioactivity was sensitive to RNase and alkaline treatment. The partial characterization of these molecules indicate that the radioactive pattern of total RNA, found in sucrose gradients, the ratio Poly(A)+RNA/Poly(A)-RNA as well as the radioactive pattern of Poly(A) fraction in acrylamide gels were different in samples from cellular and from extracellular origin. Although not conclusive, these results are proposed as a new argument for the existence of an extracellular RNA in the amphibian embryo.

Effect of trypsin inhibitors and concanavalin A on the fertilization of Bufo arenarum coelomic oocytes.

The effect of trypsin inhibitors (obtained from soybean, lima bean and ovomucoid) and Concanavalin A on fertilization in Bufo arenarum was tested. In order to study the effect of these substances at the level of the vitelline envelope of the oocytes, a new bioassay was designed. This bioassay employs coelomic oocytes to which some oviducal factors necessary for their fertility was added. Trypsin inhibitors block both the lytic effect of the acrosomal proteases on the vitelline envelope and fertilization. This indicates that the blockade of fertilization is a consequence of the inhibition of the lytic effect of the acrosomal proteases. Concanavalin A is effective as well in blocking the lytic effect of acrosomal proteases and fertilization. These effects are reversed by some sugar antagonists of the lectin, thus indicating that the effect of Concanavalin A is through its saccharide-binding capacity. These results suggested the involvement of glucosidic residues of the vitelline envelope in amphibian fertilization (the saccharide residues might be involved in the attack of the vitelline envelope by the acrosomal proteases). The possible mechanism of action of these substances is discussed.

Exocytosis of cortical granules from activated oocytes of the toad, Bufo arenarum.

Observation of the cortical region of oocytes of Bufo arenarum by transmission electron microscopy reveals modifications on their surface and in the contents of the cortical granules (CG) during activation. In non-activated oocytes only amorphous cortical granules (ACG) can be observed. Activated oocytes display ACG, intermediate cortical granules containing both amorphous and membranous material (ICG), and a third type containing only membranous material (MCG). During exocytosis, CG release their contents into the perivitelline space, where the amorphous and membranous materials are found. The three types of CG found during oocyte activation suggest transformation of ACG to MCG and indicate that the different components of the cortical granules, when released into the perivitelline space, might play different roles in prevention of polyspermy.

Structural analysis of oligosaccharide-alditols released by reductive beta-elimination from the jelly coats of the anuran Bufo arenarum.

Amphibian egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they are transported toward the cloaca. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O-linked carbohydrate chains of the jelly coat surrounding the eggs of Bufo arenarum were released by alkaline borohydride treatment. Fractionation of the mixture of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography, ion-exchange chromatography and high-performance liquid chromatography using an amino-bonded silica column, afforded 11 fractions. The primary structures of these O-glycans were determined by one-dimensional and two-dimensional 1H-NMR spectroscopy in conjunction with matrix-assisted laser-desorption-ionization--time-of-flight mass spectrometry. 11 oligosaccharide structures, possessing a core consisting of Galbeta1-->3GalNAc-ol with or without branching through a GlcNAc residue linked beta1-->6 to the GalNAc residue (core type 2 or core type 1, respectively) are described. These oligosaccharide-alditols with these types of cores have been identified previously in mammalian mucins or in oviducal amphibian jellies. These glycans contain blood group determinants such as H, A or Cad antigens.