Claudio Barros

Immunolocalization of proacrosin/acrosin in bovine sperm and sperm penetration through the zona pellucida

The aim of the present work was to immunolocalize acrosin in bull spermatozoa incubated for up to 6 h in capacitating culture medium (TALP-heparin), in order to study the kinetics of its release during the acrosome reaction and in vitro sperm penetration. Six replicates from semen of one bull were used. Acrosin was localized by the silver-enhanced immunogold technique using anti-bovine acrosin monoclonal antibody ACRO-C2E5. Spermatozoa thus showed the presence of acrosin only at the acrosomal region. Four different patterns were seen: (1) no labeling: (2) intense labeling on the rim of the portion of the acrosome; (3) diffuse label over the entire acrosomal region; and (4) intense label over the entire acrosomal region. Spermatozoa incubated in capacitating medium for 4 h showed that unlabeled (pattern 1) spermatozoa decreased from 72% to 28% difference that was found to be significant (p<0.05). Patterns 3 and 4 increased from about 10% to 20-29%, (p<0.05). With further incubation (4-6 h), pattern 1 increased while patterns 3 and 4 decreased differences were not significant (p0.05). The incidence of pattern 2 did not change through the whole incubation period. Sperm penetration through the zona pellucida of in vitro matured bovine oocytes (57%) or empty zonae pellucida (70.5%) increased (p<0.05) as a function of sperm incubation time in capacitating medium. The presence of acrosin, as determined by the silver-enhanced immunogold technique, was highly correlated with sperm penetration of in vitro mature bovine oocyte (r=0.98) and cryopreserved zonae pellucidae (r=0.93) (p<0.01).

In vitro sperm penetration through the zona pellucida of immature and in vitro matured oocytes using fresh, chilled and frozen canine semen

The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1-10 h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10 h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p<0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p<0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P<0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. There was a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization.

Evaluation of glucose as a cryoprotectant for boar semen

Fertility parameters of boar spermatozoa were evaluated in vitro, after freeze-thawing the semen in three different extenders containing permeable and non-permeable cryoprotectants: A (111-OmM Tris, 31.4mM citric acid, 185.0mM glucose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G); B (200mM Tris; 70.8mM citric acid, 55.5mM glucose, 20 per cent egg yolk, three per cent glycerol and 100 iu/ml penicillin G); C (200mM Tris, 70.8mM citric acid, 55.5mM fructose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G). The freeze-thawing techniques were the same for each extender. Eight ejaculates from four boars were obtained; the sperm-rich fraction of each ejaculate was extended in each of the three media at a final concentration of 400 x 106 sperm/ml, loaded into 0.5 ml straws and frozen at a rate of 30°C/minute to - 196°C. The straws were thawed at 60°C for eight seconds. Sperm motility, acrosomal integrity and in vitro sperm penetration through the zona pellucida of gilt oocytes matured in vitro were evaluated. The motility of unfrozen spermatozoa was 93.1 per cent compared with 60.7 per cent, 48.2 per cent and 35 per cent for sperm frozen in extenders A, B and C respectively; these values were all significantly different (P<0.05). There was no significant decline in sperm motility after incubation for 30 minutes in extender A, but there were significant decreases in sperm motility after 30 minutes of incubation in B and C. The percentage acrosomal integrities were 97.2 per cent for the control and 45.5 per cent, 30.3 per cent and 16.8 per cent for the frozen-thawed spermatozoa in extenders A, B and C respectively. The results of the in vitro penetration assay were 80.7 per cent when using control spermatozoa, and 42.2 per cent, 18.4 per cent and 3.3 per cent when using frozen-thawed spermatozoa in extenders A, B and C respectively

Peroxisomal proteins in rat gametes.

Peroxisomes are essential subcellular organelles, which appear to be derived from pre-existing organelles. This biogenetic mechanism assumes the presence of peroxisomes in either or both mammalian gametes (sperms and/or oocytes). In order to test the presence and subcellular localization of peroxisomal proteins in rat sperms and oocytes, the authors carried out fractionation and immunofluorescence experiments. The results showed that rat oocytes contain peroxisomelike structures, which were detected by indirect immunofluorescence, using an antisera against total peroxisomal proteins. In contrast, such structures were not detected in rat sperms, which appear to contain catalase localized in the cell cytosol. The results reported herein show evidence for the first time of the presence of peroxisome-like structures in mammalian oocytes, and provide evidence for the peroxisome biogenesis hypothesis, by division of pre-existing organelles.

Evaluation of cortical granules and viability of canine oocytes during long-term in vitro maturation

THE morphological appearance of the canine cumulusoocyte complex (COC) is known to influence the rate of in vitro maturation (IVM) (Farstad 2000). The low rate of IVM in dogs might also be related to the unsuitability of the culture medium to support oocyte viability and development. Canine ovulation of oocytes occurs at the germinal vesicle stage; completion of the maturation process continues in the oviduct, requiring an extended period of two to five days (Renton and others 1991). In order to mimic this process in vitro, it is necessary to use prolonged culture periods, and thus the viability of the gametes becomes an issue. Correct culture conditions have been determined by the preservation of oocyte morphology, and thus sustained oocyte viability (Figueiredo and others 1994). In addition to nuclear maturation, proper cytoplasmic and membrane maturation are critical for continued viability of oocytes. Most studies on canine oocytes have evaluated nuclear maturation in culture, but there has been little attention given to cytoplasmic maturation. An oocyte that has not completed cytoplasmic maturation is of poor quality, and thus unable to successfully complete the normal developmental processes (Krisher 2004). Cytoplasmic maturation mainly involves the redistribution of cortical granules around the periphery of the oocyte (Sun 2003), which then contribute to the inhibition polyspermy (Hoodbhoy and Talbot 1994). Considering the long culture period required for IVM in dogs, the aims of this study were to evaluate the viability of the plasma membrane of canine oocytes recovered from the ovaries before and during culture, and to identify and observe the distribution of cortical granules before and after culture. In each experimental replicate, oocytes were obtained from normal bitch ovaries following ovariohysterectomy. In the laboratory, COCs were released by slicing the ovarian cortex. Oocytes with uniform ooplasm and a compact cumulus cell mass were selected. After two washes in phosphate buffered saline (PBS) the COCs were placed in maturation medium (TCM 199; Earle’s salt, buffered with 25mM Hepes; Invitrogen), supplemented with 10 per cent fetal calf serum and 2·5 μl/ml pyruvic solution (11·2 mg/ml pyruvic acid), 10 iu/ml of human chorionic gonadotrophin and 5 μl/ml antibiotic solution (12·2 mg/ml penicillin and 20 mg/ml streptomycin) (Sigma Chemical). The cultures were performed at 38·5°C in a humidified atmosphere of 5 per cent carbon dioxide for up to 96 hours (De los Reyes and others 2005). To verify the viability of oocytes matured in vitro, several oocytes (approximately 15 from each replicate) were submitted for IVM in parallel to the experiment carried out for the cortical granule evaluation. Oocyte viability was assessed before culture and then every 24 hours in culture up to 96 hours. After each culture time the COCs were incubated with 5 μg/ml fluorescein diacetate (FDA; Sigma) in PBS for 5 minutes, and evaluated under an epifluorescence microscope, with green fluorescence indicating cell viability (Barros and others 1982). Evaluation of cortical granules was performed before incubation and after 96 hours of culture. Oocytes were fixed in 2 per cent paraformaldehyde/PBS for 30 minutes, and permeabilised for 5 minutes in PBS containing 0·1 per cent Triton X-100 with 0·3 per cent BSA to block non-specific binding sites. The oocytes were incubated with 20 μg/ml lectin Lens culinaris agglutinin (LCA) coupled to fluorescein isothiocyanate, in PBS for 30 minutes. The oocytes were washed and subsequently mounted with Vectashield (Vector Laboratories) under coverslips supported with vaseline/paraffin dots. The samples were evaluated with an epifluorescence microscope (UV emission 480 nm) (Nikon Optiphot II; Nikon).

SUBCELLULAR LOCALIZATION OF CATALASE IN SEA URCHIN (TETRAPIGUS NIGER) GAMETES : IMPLICATIONS FOR PEROXISOME BIOGENESIS

Abstract Peroxisomes are essential subcellular organelles that appear to be derived from pre-existing organelles. To test the presence of peroxisomes in sea urchin (Tetrapigus niger) sperm and eggs, we performed biochemical and morphological experiments to evaluate the subcellular distribution of catalase as the typical peroxisomal marker. In sea urchin sperm, we found that catalase is localized in the cell cytosol. In contrast, sea urchin eggs contain sedimentable catalase, presumably contained in peroxisome-like structures detected by immunomicroscopy and by cytochemistry. Our results show, for the first time, evidence for the presence of peroxisome-like structures in sea urchin eggs and provide evidence for the peroxisome biogenesis hypothesis by division of pre-existing organelles.