Franca Porcelli

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 °C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.

Quantitative cytochemical study of some enzymatic activities in preovulatory bovine oocytes after in vitro maturation

In the present study quantitative cytochemical assays were used to measure some enzymatic activities in situ in bovine meiotically immature oocytes and oocytes matured in vitro, since the special metabolic activity of the growing oocytes may be a pivotal factor in stabilizing the meiotically arrested oocytes. Modifications of this particular metabolism might destabilize the arrested meiosis. Preovulatory oocytes, mostly at the germinal vesicle stage, were obtained by puncturing follicles ranging from 2 to 6 mm in diameter with a hypodermic needle. A group of collected oocytes was incubated in maturation medium CRML 1066 to obtain metaphase II oocytes. Succinate, lactate and glucose-6-phosphate dehydrogenase activities in just collected meiotically immature and in vitro matured oocytes were assayed cytochemically. Microdensitometric measurements were made with a Vickers M85a scanning microdensitometer. Our findings show that: 1) succinate dehydrogenase activity was significantly increased in matured oocytes; 2) lactate dehydrogenase activity was present and very strong in immature oocytes but was detectable in only about 50% of matured oocytes, with significantly lower integrated optical density values; 3) glucose-6-phosphate dehydrogenase activity was very high in immature oocytes but significantly decreased after in vitro maturation; 4) there was no linear correlation between the integrated optical densities of the three enzymatic activities and the diameters of the oocytes. We suggest that the ability to utilize glucose may appear earlier in bovine oocytes than in other species and takes place at the time of maturation.

Effects of lipid peroxidation on chromatin in rabbit and mouse spermatozoa: A cytochemical approach

Abstract To extend present knowledge on the possible interactions between lipid peroxidation and chromatin, we studied rabbit and mouse spermatozoa during spontaneous lipid peroxidation. Epididymal spermatozoa were aerobically incubated in high sodium or high potassium media to obtain spontaneous lipid peroxidation in the absence of added promoters. Quantitative cytochemical assays were performed in situ, in individual spermatozoa processed by Feulgen reaction and Gallocyanin-chrome alum (GCA) staining. Our findings show that in both species examined, the spermatozoa chromatin undergoes destabilization associated with marked alterations of the physico-chemical state of the DNA-protein complex. The decrease of Feulgen and GCA-positive material may be explained by DNA damage and implies the release from the nucleus of DNA fragments. Moreover, our data support the idea that during spontaneous lipid peroxidation, the spermatozoa chromatin undergoes destabilization sometimes correlated with increased acid lability of the DNA-protein complex. There are differences in the two species, probably correlated with differences in major protective enzymatic systems against cell damage by autoxidation and, especially, against different kinds of oxygen radicals responsible for initiation of lipid peroxidation.

Cytophotometric assay of cytochrome oxidase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities in human peroxidized spermatozoa

Human spermatozoa contain appreciable amounts of intracellular glutathione, which has a protective function against peroxidative degradation of spermatozoal polyunsaturated fatty acids by the NADPH-dependent glutathione peroxidase/reductase enzymatic system. The glutathione system provides a basic defense against peroxidative damage, without which the superoxide dismutase system would dominate. Since oxidative damage is said to include enzyme leakage and changes in metabolism, cytochrome oxidase and lactate dehydrogenase activities were used as indicators of the energy metabolism in unwashed and washed human spermatozoa during lipid peroxidation. Lipid peroxidation was induced by aerobic incubation of sperms in the presence of sodium ascorbate and ferrous sulphate. In addition, since NADPH concentrations influence the concentration of reduced glutathione, we studied glucose-6-phosphate dehydrogenase activity as an indicator of pentose phosphate shunt activity, the main source of NADPH. Microdensitometric measurements of the three enzymes were made by a Vickers M85a scanning microdensitometer. We found that the lipid peroxidation process greatly affects the 3 enzymatic activities examined and that seminal plasma protects against the extensive deleterious effects of lipid peroxidation.

ILA 147 immunoreactivity of the bull spermatozoa membrane during epididymal maturation

A cytochemical quantitative study was carried out to detect immunostaining of bull spermatozoa during epididymal maturation using an ILA 147 monoclonal antibody and a standard immunoperoxidase method. This antibody recognizes a bovine panleukocyte determinant. Microdensitometric measurements were made on spermatozoa collected from different sites of the male genital tract (caput, corpus and cauda epididymidis and ductus deferens). On the basis of ILA 147 staining, at least 5 subpopulations of sperm cells from each site of the genital tract were found. These subpopulations showed: 1) immunostaining in the acrosome domain and in the cytoplasmic droplet in a proximal position; 2) immunostaining in the acrosome domain and in the distal cytoplasmic droplet; 3) immunostaining in the acrosomal region and lack of a cytoplasmic droplet; 4) immunostaining only in the cytoplasmic droplet; 5) lack of a cytoplasmic droplet and absence of staining of the head. The possible relationship between the presence of ILA 147 on the sperm head and the maturation process was evaluated, and we suggest that the most significant changes in ILA 147 expression occur in the corpus epididymidis. The absence of immunocytochemical staining in some spermatozoa may be related to plasma membrane damage due to spontaneous peroxidative damage of lipids.

Cytochrome oxidase and lactate dehydrogenase activities in peroxidized rabbit epididymal spermatozoa.

Summary: We report a quantitative cytochemical study on cytochrome oxidase and lactate dehydrogenase activities on rabbit epididymal spermatozoa during spontaneous lipid peroxidation.

Microdensitometric assay of enzymatic activities in parthenogenetically activated and in vitro fertilized bovine oocytes

To examine the paternal genomes role in reprogramming metabolic activity in one-cell embryos, we investigated metabolic aspects of bovine oocytes after in vitro maturation and in vitro fertilization and after in vitro parthenogenetic activation with a Ca2+ ionophore and 6-dimethylaminopurine. We assayed succinate dehydrogenase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities by microspectrophotometry in immature oocytes and oocytes after maturation, in vitro fertilization and parthenogenetic activation. Succinate dehydrogenase activity significantly increased after in vitro maturation, significantly decreased after Ca2+ ionophore activation and further decreased after 6-dimethylaminopurine treatment. Lactate dehydrogenase activity showed a significant decrease in bovine oocytes after in vitro maturation, remained unchanged in Ca2+ ionophore-treated oocytes and rose significantly after 6-dimethylaminopurine treatment. This activity was dramatically reduced after in vitro fertilization, reaching absorbance levels that were not different from those in mature and Ca2+ ionophore-treated oocytes. Glucose-6-phosphate dehydrogenase activity was significantly lower in matured oocytes as compared to immature oocytes, was significantly higher after artificial activation with Ca2+ ionophore and remained constant after 6-dimethylaminopurine treatment or after in vitro fertilization. We suggest that metabolic changes involved in parthenogenetic activation are similar to those occurring after fertilization.

Microphotometric study of glucose-6-phosphate dehydrogenase activity in epididymal spermatozoa during spontaneous lipid peroxidation

Mammalian spermatozoa are highly sensitive to lipid peroxidation and the glutathione peroxidase/reductase system provides an effective defense against oxidative damage to different degree in different species. Rabbit spermatozoa rely on superoxide dismutase as the primary enzymatic defense against lipid peroxidation and contain only low detectable endogenous glutathione reductase activity while in mouse spermatozoa the glutathione system is the major protective enzyme against cell damage by autoxidation. We describe a cytochemical quantitative assay for glucose-6-phosphate dehydrogenase activity in rabbit and mouse spermatozoa undergoing spontaneous lipid peroxidation during in vitro incubation. Microdensitometric measurements were made by a Vickers M85 a scanning microdensitometer at lambda 585 nm wavelength. Our findings suggest that in mouse spermatozoa, the enhanced glutathione reductase and peroxidase activities induced by the spontaneous lipid peroxidation increases NADPH production from the pentose phosphate shunt, while in rabbit spermatozoa, NADPH production is much lower.

Cytochemical study on human spermatozoa metabolism during in vitro capacitation.

Summary: In eutherian mammalian spermatozoa the capacitation is coupled to a specific type of metabolism, that is glycolysis or oxidative respiration. A cytochemical study was carried out on cytochrome oxidase and lactate dehydrogenase in human spermatozoa collected at different times during in vitro capacitation.

Effect of lipid peroxidation on the immunocytochemical detection of a leukocyte antigenic determinant in fresh and cryopreserved bovine spermatozoa

Abstract Studies on different species, including rats, monkeys and humans, have shown the presence of leukocyte differentiation antigens in the spermatozoa. In some case the expression of these molecules is related to a specific functional state of the sperm cell, as was found for the CD 46 antigen, that in humans can be used as a marker of the acrosome reaction. The aim of the present study was to assess wether promoted lipid peroxidation of the spermatozoa induces any variations in their immunoreactivity with ILA 147 antibody that, in bull spermatozoa, recognizes bovine leukocyte antigens. Freshly ejaculated bovine spermatozoa and cryopreserved semen were tested for ILA 147 reactivity by standard immunoperoxidase staining, before and after promoted lipid peroxidation. Staining intensity was assessed in the individual cells using the microdensitometric method to measure integrated optical density (IOD), overcoming the disadvantage of an operator’s subjective interpretation of the results. After the lipid peroxidation there was significantly decreased staining intensity in the fresh spermatozoa, but not in the cryopreserved cells. Furthermore, in the preincubation conditions, the cryopreserved spermatozoa had a lower mean I.O.D. value than the fresh sperm, showing that the freezing and thawing processes induced an alteration in the antigen exposure. However the mean immunoreactivity of the cryopreserved cells was not significantly influenced by lipid peroxidation. The absorbance value maps, made following immunoperoxidase staining by the examined antibody, showed that the reaction sites in the fresh and cryopreserved spermatozoa fell mainly within the periacrosomal region. Moreover, after induced lipid peroxidation there were fewer reaction sites in this domain. The present research has confirmed the presence of the examined leukocyte antigenic determinant in the bull spermatozoa, and suggests that promoted lipid peroxidation and the freezing and thawing of spermatozoa can produce membrane damage, leading to reduced ILA 147 antigenic site exposure.