In the egg of the anuran Discoglossus pictus, the site of fertilization is restricted to the central portion of an animal hemisphere indentation (the dimple). Previous studies showed that the acrosome reaction of D. pictus sperm is triggered in the jelly, and yet sperm arrive at the dimple surface with the plasma membrane at an early stage of vesiculation. Reactivity of the dimple surface with specific lectins suggests that fucose might be utilized as a marker of glycoproteins located at the dimple surface. In this paper, proteins of the egg surface were labeled with the membrane impermeable sulfo-NHS-biotin. Four main bands of 200, 230, 260, and 270 kDa labeled only at the dimple surface, although they were detected in the cortex of the whole egg. The 270-kDa band reacted with Galanthus nivalis agglutinin only in the cortex of the dimple, suggesting that this band is differently glycosylated according to its localization. The alpha-l-fucose-specific lectin Ulex europaeus agglutinin I was utilized both in lectin blotting and in affinity chromatography and cross-reacted with the 200- and 270/260-kDa bands. Furthermore, two polypeptides were obtained by exposure of intact eggs to lysylendoproteinase C. They were also reactive to Ulex europaeus agglutinin I. The 200- and 270/260-kDa bands were eluted from the acrylamide gels and adsorbed to polystyrene beads. An assay for sperm binding to 200-kDa glycoprotein-bound beads was developed. Sperm stuck to the beads before but not after Ca-ionophore treatment. When the beads were coated with the 270/260-kDa glycoproteins, binding occurred after ionophore treatment. In these assays, the 200- and 270/260-kDa glycoproteins competitively inhibited sperm binding to the beads coated with the corresponding glycoprotein. These results indicate that the assayed glycoproteins, located either in the glycocalyx or in the plasma membrane of the fertilization site, are involved in sperm binding.
We have used ratiometric confocal microscopy and three fluorescence techniques to study the distribution and activity of mitochondria in frog oocytes during the early stages of oogenesis. Mitochondria in frog oocytes during oogenesis were characterised by a high ratio in the ‘mitochondrial cloud’ and peri-nuclear region and a low ratio in mitochondria freely dispersed within the cytoplasm. We tested whether the high ratio visualised by the three techniques represented mitochondrial membrane potential by perturbing the mitochondrial membrane potential. Carbonyl cyanide p-(trifluoromethyl)phenylhydrazone (FCCP) caused the immediate destruction of the membrane potential, and consequent loss of fluorescence from the membrane-potential-sensitive confocal channel. In contrast, nigericin caused an increase in membrane potential represented by a steady increase in fluorescence ratio. These data demonstrate that mitochondrial activity can be measured during oogenesis in frog oocytes, and suggest that the mitochondrial cloud and perinuclear regions are characterised by highly active mitochondria.
Introduction: D-Aspartate is an endogenous amino acid involved in LH and testosterone release in humans. In this study we investigate the impact of nutritional supplementation of sodium D-aspartate on the improvement of sperm quality in sub-fertile patients and the rate of pregnancies that occurred with their partners. Materials and Methods: A group of 30 patients affected by oligo-asthenozoospermia and a group of 30 patients affected by asthenozoospermia were treated with a daily dose of sodium D-aspartate for 90 days. After which, the change in spermatozoa concentration and their motility and the pregnancies that occurred with their partners were recorded. Results: We found that the supplementation of D-aspartate significantly increased the concentration and the motility of spermatozoa. In oligoasthenozoospermic patients the increase of sperm concentration was found to be 2.0-fold, P < 0.001 (from a mean of 8.2 ± 4.5 million spermatozoa/ml of seminal plasma before treatment to a mean of 16.5 ± 5.5 million after treatment). In asthenozoospermic patients, the increase of spermatozoa was 1.6-fold, P < 0.001 (from a mean of 29.9 ± 5.7 million spermatozoa/ml before treatment to a mean of 48.7 ± 12.8 after treatment). The same positive effects also occurred for sperm motility. Oligo-asthenozoospermic patients showed an increase of rapid progressive spermatozoa motility from a mean of 15.5% ± 4.4% before treatment to a mean of 23.1% ± 4.7% after D-aspartate treatment (1.49-fold increased, P < 0.001). The same effects occurred in oligo-asthenozoospermic patients. In these subjects the increase of rapid progressive spermatozoa motility was 1.86-fold (from 11.6% ± 3.9% before treatment to 21.6 ± 7.5 after treatment, P < 0.001). In addition, the treatment of D-aspartate in these patients consequently led to a significantly increased number of pregnancies occurring in the partners of the treated patients. Conclusions: Treatment of sub-fertile patients with sodium D-aspartate improved the number and the motility of the spermatozoa and consequently improved the rate of pregnancies of their partners.
Carbohydrate additives to modern embryo culture media are based on three basic energy sources, glucose, pyruvate and lactate. Although the use of these substrates is almost universal, debate continues as to the roles of the individual components in the human. This is mainly due to the lack of human embryos for research and the reliance on animal model systems. In the present work, the human embryo was used to study the role of the above simple substrates in the maintenance of the mitochondrial membrane potential and cell division. The mitochondrial membrane potential was measured with fluorescence techniques. Cell division was scored as the number of blastomeres on day 3. Both the mitochondrial membrane potential and cell division were dramatically lost in the absence of energy sources. The mitochondrial membrane potential and cell division were normal in media containing all three energy sources, or in pyruvate-containing media. Both glucose and lactate individually proved poor energy sources for the maintenance of the mitochondrial membrane potential. However, cell division continued in the presence of glucose, suggesting that some energy production can continue. These data suggest that pyruvate is an absolute requirement for mitochondrial respiration and cell cleavage during human preimplantation development. The role of lactate is as yet unclear.
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