M. De los Reyes

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).

Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.

A scanning electron microscopy study of frozen/thawed dog sperm during in vitro gamete interaction.

The aim of this work was to study the effects of cryopreservation on the binding and penetration of dog spermatozoa to the zona pellucida (ZP) by scanning electron microscopy (SEM). The sperm-rich fraction of six ejaculates from five dogs was divided into two aliquots and washed by centrifugation. One aliquot was processed as fresh control sample and the other aliquot frozen in Tris-fructose extender. Gamete interaction was assessed using in vitro matured bitch oocytes, which were co-incubated for up to 3 h. At hourly intervals after the start of co-incubation, in vitro fertilized (IVF) oocytes were processed by SEM. The results were analysed statistically using the anova test. Differences in binding and penetration of the spermatozoa to the ZP occurred; a lower proportion of oocytes with spermatozoa bound to ZP was observed using frozen sperm (p < 0.05) than with fresh sperm (61%, 57% and 53% vs 42%, 40% and 44% at 1, 2 and 3 h, respectively). The percentage of ZP penetration by fresh sperm was directly proportional to the time of co-incubation (9%, 25% and 34%; p < 0.05); in contrast, no differences were observed in the penetration rate with frozen-thawed sperm (21%, 17% and 21%). More acrosome reacted sperm were observed in frozen sperm than in fresh sperm on the surface of the ZP. The differences in the percentage of binding and penetration between fresh and frozen sperm during the co-culture could indicate that the time course of penetration is faster in frozen-thawed dog spermatozoa than in fresh sperm, but that fresh spermatozoa can penetrate more oocytes over a given period of time, which may be related to their reacted or non-reacted initial status.

Ultrastructural Study of the Canine Zona Pellucida Surface During In Vitro Maturation

The aim of the present study was to compare the ultrastructure of the surface of the zona pellucida (ZP) of immature and in vitro matured dog oocytes using scanning electron microscopy (SEM). Bitch oocytes were collected after ovariohysterectomy; the ovaries were sliced and the released cumulus-oocyte complexes (COCs) were washed with phosphate buffered saline (PBS). The selected COCs were randomly allocated into three groups, two groups were processed after in vitro maturation at both 72 and 96 h and a third group was processed immediately at immature state in PBS medium. After that, oocytes were fixed, critical point dried and viewed by using SEM. The diameters of the outer holes of the ZP were measured on a total of 93 oocytes; the results were analyzed with anova. The mean diameters of holes were different between groups (p < 0.05): 0.69 +/- 0.12, 1.56 +/- 0.19 and 1.42 +/- 0.27 mum, for immature and in vitro matured oocytes for 72 and 96 h, respectively. The difference in the hole sizes between immature and in vitro matured canine oocytes indicates that the ZP surface is related to oocyte maturity in canines.

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

Histological Characteristics and Steroid Concentration of Ovarian Follicles at Different Stages of Development in Pregnant and Non-pregnant Dairy Cows

The aim of the study was to investigate the histological characteristics and steroid concentrations in follicular fluid of different populations of follicles at different stages of development, during pregnancy and the oestrous cycle in cows. Follicles from ovaries collected at a slaughterhouse were allocated into three size categories (small, 2–5.9 mm; medium, 6–13.9 mm; and large, 14–20 mm) in pregnant and non-pregnant cows. Slices were stained with HE and PAS for histological analysis. Follicular fluid was pooled according to size and pregnancy status and estradiol, testosterone and progesterone concentrations in follicular fluid were determined by RIA. Characteristics of healthy follicles did not differ, regardless of follicle size or pregnancy status. Total histological atresia was significantly higher in pregnant cows than in non-pregnant cows (p < 0.05). Estradiol increased and testosterone decreased significantly, while follicles increased in size, in both non-pregnant and pregnant cows (p < 0.05). Nonpregnant cows had the highest estradiol values in follicles of all sizes. Medium and large follicles from pregnant cows showed the lowest testosterone concentration (p < 0.05). Progesterone levels increased with follicle size only in non-pregnant animals. In large follicles, progesterone concentration was significantly higher in non-pregnant cows than in pregnant cows (p < 0.05). Considering steroid concentration and histological findings, most large follicles might be atretic during pregnancy in cattle.

Western Blot Analysis of Proacrosin/Acrosin in Frozen Dog Sperm During In Vitro Capacitation

Acrosin is an acrosomal protease synthesized as an inactive precursor, proacrosin, which is processed via autoproteolysis into active forms alpha- and beta-acrosin. In this paper, a comparative study on the immunoreactivity of acrosin during in vitro capacitation of frozen and fresh (control) canine sperm using Western blot analysis is reported. Semen samples were obtained by digital stimulation and ejaculates processed as fresh and frozen samples and then capacitated for 0, 30, 60 and 90 min. At each time period, samples were analyzed with monoclonal antibody C5F10 by Western blot. The antibody specifically recognized, in fresh and frozen/thawed spermatozoa, a 40-, 32- and 27-kDa bands corresponding to proacrosin, alpha- and beta-acrosin, respectively, during capacitation. Western immunoblots showed that the beta-acrosin reactivity in fresh sperm was directly proportional to the time of capacitation, whereas a decreased reactivity of active form of acrosin was observed with frozen-thawed sperm (p < 0.05). These results suggest that proacrosin is activated to beta-acrosin earlier in frozen/thawed dog spermatozoa than in fresh dog spermatozoa.

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).

Golgi apparatus and endoplasmic reticulum dynamic during meiotic development in canine oocytes.

The Golgi apparatus (GA) and endoplasmic reticulum (ER) play a central role in the events related to intracellular trafficking distribution. This work evaluated the dynamics and localization of the GA and ER in canine oocytes during meiotic development in vitro. Cumulus-oocytes complexes (COCs) from ovaries of adult bitches were incubated for IVM for 0, 48, 72 and 96 h. At each time, the nuclear status was determined using DAPI staining, and the GA was evaluated by immunofluorescence using two antibodies against Golgi proteins: GM130 and Giantin. ER was analysed with fluorescent lipid probes (ER-Tracker), for living cells. Golgi structures were homogeneous in the cytoplasm in non-matured oocytes, mainly in those GV-arrested oocytes. In contrast, at 48 h and from GVBD stage, the immunolocalization began to be subcortical, increasing at 72 h and 96 h. Meiotic development increased with time and the majority of oocytes at MI-MII stages showed cortical distribution of Golgi structure. Living ZP intact non-matured oocytes showed a reticular pattern of ER that covered oocyte cortex. Confocal microscopy showed that, in all levels cuts the fluorescence marks were located in the cortical region, irrespective of culture time. The changes and localization in these organelles during IVM might be related to meiotic development, but in a non-synchronous manner.

Pannexin channels increase propidium iodide permeability in frozen–thawed dog spermatozoa

Pannexins (Panx) are proteins that form functional single membrane channels, but they have not yet been described in dogs. The aim of the present study was to detect Panx1, Panx2 and Panx3 in frozen-thawed dog spermatozoa using flow cytometry and immunofluorescence analyses, evaluating the relationship of these proteins with propidium iodide (PI) in frozen-thawed spermatozoa. Fresh and frozen-thawed dog spermatozoa from eight dogs were preincubated with 3μM PI with or without 15μM carbenoxolone (CBX) or 1mM probenecid (PBD), two Panx channel inhibitors, and then incubated with rabbit anti-Panx1, anti-Panx2 and anti-Panx3 antibodies (1:200). Panx immunolocalisation was assessed by fluorescence microscopy. Flow cytometry data were evaluated by analysis of variance. All three Panx proteins were found in dog spermatozoa: Panx1 was mostly localised to the acrosomal and equatorial segment, Panx2 was found in the posterior region of the head and tail and Panx3 was localised to the equatorial and posterior head segment. The percentage of PI-positive cells determined by flow cytometry was reduced (P<0.05) in the presence of Panx inhibitors. These results show that Panx proteins are present in dog spermatozoa and increase PI permeability in frozen-thawed dog sperm, suggesting that the percentage of PI-positive spermatozoa used as an indicator of non-viable cells may lead to overestimation of non-viable cells.