Organization of the Hamster Cumulus Extracellular Matrix: A Hyaluronate-Glycoprotein Gel which Modulates Sperm Access to the Oocyte

The mammalian oocyte-cumulus complex contains an extracellular matrix rich in hyaluronate. Recently, the microstructure of the hamster cumulus extracellular matrix was described (52). In the present work, we investigated the organization of this matrix. We employed freeze-substitution methodologies to investigate ultrastructural effects of various treatments, including sperm enzymes, on the matrix. Protease treatment resulted in disruption with a loss of the fibrillar structures and some expansion; in contrast, hyaluronidase treatment completely solubilized the matrix. EDTA extraction revealed that the fibrils are composed of fine filaments. A discrete region of the matrix immediately surrounding the oocyte, the corona radiata, was resistant to EDTA disruption. We found that hyaluronate is an ubiquitous constituent of the microstructural elements of this extracellular matrix. The matrix exhibits a carbohydrate:protein ratio of approximately 2:1. SDS-PAGE revealed that glycosylated polypeptides are bound to the matrix. The lectins LCA and WGA had differing affinities for these polypeptides, and bound ubiquitously to the intact matrix. The present data suggest that glycoprotein-hyaluronate interaction is critical for maintenance of the cumulus extracellular matrix microstructure and for its physical properties.     

Comparison of the Effects of Septoplasty and Sinus Lifting Simulation in Rats on Changes in Heart Rate Variability

Doklady Biochemistry and Biophysics - The effects of septoplasty and sinus lifting simulation in rats on changes in the frequency domain of heart rate variability were compared. In the early...     

Shared Signatures of Selection Related to Adaptation and Acclimation in Local Cattle and Sheep Breeds from Russia

Russian Journal of Genetics - The aim of this study was to identify common candidate genes related to environmental adaptation including the cold climates in Russian native cattle and sheep breeds....     

Stochastic properties of one-parameter discrete model of genetic diversity in the unique random parameter case

Stochastic properties of previously introduced one parameter discrete model of genetic diversity (M. Yu. Shchelkanov et al, J. Biomol. Struct. Dyn. 15, 887-894 (1998)) are investigated. Two approaches are compared: (A) when the on-step substitution number and/or the number of substitution steps are random variables; (B) referred parameters are replaced by mathematical expectations of the respective variables. It has been demonstrated, that estimations of sequence measure based on the number of replication steps are more under the assumption of case (A) as compared with (B). Thus, real biological situation relating to the case (A) could additionally promote the increasing of distinctions between different taxons (e.g. HIV, etc.). Peculiarities of one-parameter discrete model of genetic diversity during calculation of the distinctions between symbol (e.g. nucleotide) sequence sets are also discussed.     

Real-time observations of individual macaque sperm undergoing tight binding and the acrosome reaction on the zona pellucida

Changes in binding affinity, acrosomal status, and motility of living sperm on the zona pellucida were for the first time in any mammalian species directly observed and analyzed with video microscopy. A single zona was air-dried and rehydrated on a microscope slide, and a coverslip supported by glass beads was added. Capacitated sperm were added together with Alexa-SBTI, a probe for acrosin that can detect the acrosome reaction. The heads of loosely attached sperm oscillated on the zona and the flagella beat symmetrically with a sigmoid-shaped waveform. Tight binding was observed after 16 sec as the sperm head became fixed in place on the zona. The shape of the flagellar beat simultaneously shifted to a more rigid, C-shaped waveform. The first signs of the acrosome reaction were detected within 11 sec of tight binding. Rapid flushing removed approximately 65% of sperm that were loosely attached but only 2% of those that were tightly bound. In the 2 min following the onset of tight binding, the lateral displacement of the flagellum increased by approximately 30% and the beat frequency decreased by 25%. Lignosulfonic acid (LSA) inhibited loose sperm attachment and the development of tight binding. LSA had no effect on the time of the acrosome reaction following tight binding or on changes in motility that followed tight binding. These data suggest that LSA affects the initial attachment or docking of sperm to the zona, a step that may align or recruit one or more specific zona receptors to be responsible for mediating the acrosome reaction.     

ESP13.2, a member of the beta-defensin family,is a macaque sperm surface-coating protein involved in the capacitation process

Female macaques produced isoantibodies to a limited number of sperm surface proteins following immunization with sperm components released by phosphatidylinositol-specific phospholipase C (PI-PLC). Washed, acrosome-intact, fixed sperm injected into rabbits elicited a major immune response to one of the same PI-PLC-released proteins, which was shown to be a sperm surface-coating protein. After purification and digestion of the glycoprotein, four peptides were analyzed for amino acid sequence, and all had 100% homology with an epididymal secretory protein, ESP13.2, reported previously to be a small, cationic-rich peptide and a member of the beta-defensin family. Antibodies to purified ESP13.2 recognized a number of protein bands on Western blots of nonreduced PI-PLC-released sperm components and nonreduced whole-sperm extracts. After chemical disulfide reduction, only a single, broad band from 31 to 35 kDa was recognized by anti-ESP13.2 antibodies. Indirect immunofluorescence showed ESP13.2 over the entire surface of ejaculated macaque sperm. Fluorescence was only slightly reduced after sperm were washed through 80% Percoll. A 24-h incubation in capacitating medium significantly reduced the amount of ESP13.2 over the head and midpiece, whereas exposure of the incubated sperm to dbcAMP and caffeine (capacitation activators) resulted in almost complete loss of ESP13.2 from the sperm surface. After activation, ESP13.2 was the primary component released into the medium as judged electrophoretically. Lignosulfonic acid, a potent inhibitor of macaque fertilization in vitro, completely blocked release of ESP13.2 from the sperm surface, even following treatment with activators. These findings suggest that the beta-defensin, ESP13.2, has a function in the capacitation of macaque spermatozoa and may modulate sperm surface-receptor presentation at the time of fertilization.     

Acrosomal status and motility of guinea pig spermatozoa during in vitro penetration of the cumulus oophorus

Previous studies have suggested that both acrosome-intact and acrosome-reacted guinea pig sperm are capable of binding to the zona pellucida of cumulus-free oocytes, but the acrosomal status of guinea pig sperm during penetration of the cumulus has not been reported. We made video recordings of the interaction between capacitated guinea pig sperm and cumulus-invested guinea pig oocytes. The videotapes were analysed to identify sperm with hyperactivated motility and to classify the acrosomal status of sperm during penetration of the cumulus and after binding to the zona pellucida. The resolution of the video recordings was not sufficient to recognise sperm with swollen acrosomes. However, sperm that had completed the acrosome reaction were easily identified. Acrosome-reacted sperm were found adherent to the outer boundary of the cumulus, but were never observed to penetrate the cumulus. The percentage of acrosome-intact, hyperactivated sperm was higher in the cumulus oophorus than in culture medium, suggesting that changes in motility were elicited in response to contact with the cumulus. Fully acrosome-reacted sperm were found adherent to the zona pellucida, and solubilised guinea pig zona pellucida was capable of inducing acrosome reactions in capacitated guinea pig sperm. Acrosome-intact sperm were also observed on the zona, but they were not tightly bound and did not have hyperactivated motility, suggesting that these sperm were not functionally capacitated. Our observations demonstrate that guinea pig sperm penetrate the cumulus matrix in an acrosome-intact state. Although we did not observe sperm undergoing the acrosome reaction, our observations and experimental data suggest that the acrosome reaction of guinea pig sperm is completed on or near the surface of the zona pellucida.     

Dihydro-resveratrol—A potent dietary polyphenol

Dihydro-resveratrol (dihydro-R), a prominent polyphenol component of red wine, has a profound proliferative effect on hormone-sensitive tumor cell lines such as breast cancer cell line MCF7. We found a significant increase in MCF7 tumor cells growth rates in the presence of picomolar concentrations of this compound. The proliferative effect of dihydro-R was not observed in cell lines that do not express hormone receptors (MDA-MB-231, BT-474, and К-562).     

Molecular mechanisms of action of natural amino acids and serotonin on infusorian adenylyl cyclase and guanylyl cyclase

Earlier, it has been shown that some amino acids and their derivatives are able to regulate activities of adenylyl cyclase (AC) and guanylyl cyclase (GC) in free-living infusoria Dileptus anser and Tetrahymena pyriformis. The goal of this work consisted in studying the molecular mechanisms of action of methionine, tyrosine, alanine, and neurohormone serotonin on the activity of enzyme-cyclases and in identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both infusoria; the effect of serotonin on AC in T. pyriformis took place with participation of the Ca²⁺-dependent form of AC and of the heterotrimeric G-proteins. The AC-stimulating effect of tyrosine and alanine was expressed weakly and was revealed only in D. anser. Serotonin in both infusoria and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine did not affect GC. Methionine and serotonin were bound with a high affinity to the surface receptors of infusoria. The K_D for [methyl-³H]methionine binding to D. anser and T. pyriformis were equal to 7.5 and 35.6 nM, and for [³H]serotonin binding, they were 2.7 and 4.7 nM, respectively. Alanine and tyrosine were bound to infusoria with low affinity. Thus, in the infusoria D. anser and T. pyriformis, there are chemosignal systems regulated by amino acids and their derivatives, including enzymes with cyclase activity. These systems are suggested to be similar to the hormonal signal systems of the higher eukaryotes and to be their predecessors.