RESUMEN
Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self-renew and give rise to differentiated progeny. Previous studies have reported that MSC may be induced in vitro to develop into different types of specialized cells including male gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals and conservation of endangered species. This study aimed at investigating the in vitro effect of BMP4, TGFß1 and RA on the potential for germ cell (GC) differentiation of bovine foetal MSC (bfMSC) derived from bone marrow (BM). The effect of BMP4, TGFß1 and RA was analysed on the expression of pluripotent, GC and male GC markers on bfMSC during a 21-day culture period. bfMSC cultured under in vitro conditions expressed OCT4, NANOG and DAZL, but lacked expression of mRNA of VASA, STELLA, FRAGILIS, STRA8 and PIWIL2. Treatment with exogenous BMP4 and TGFß1 induced a transient increase (p < .05) in DAZL and NANOG mRNA levels, respectively. However, exposure to RA was more effective in increasing (p < .05) expression of DAZL and regulating expression of OCT4 and mRNA levels of NANOG. These data suggest that bfMSC may possess potential for early GC differentiation, where OCT4, NANOG and specially DAZL may play significant roles in controlling progression along the GC lineage.
Asunto(s)
Células de la Médula Ósea/citología , Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Espermatozoides/citología , Animales , Proteína Morfogenética Ósea 4/farmacología , Bovinos , Técnicas de Cultivo de Célula , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Masculino , Factor de Crecimiento Transformador beta1/farmacología , Tretinoina/farmacologíaRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Conexinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Propidio/farmacología , Espermatozoides/metabolismo , Acrosoma/efectos de los fármacos , Acrosoma/metabolismo , Animales , Carbenoxolona/farmacología , Membrana Celular/efectos de los fármacos , Criopreservación/veterinaria , Perros , Masculino , Permeabilidad , Probenecid/farmacología , Preservación de Semen/veterinaria , Espermatozoides/efectos de los fármacosRESUMEN
At high altitude, hypoxia and/or oxidative stress may compromise fertility. This study tested the relative effect of short- or long-term exposure to high-altitude hypobaric hypoxia and oxidative stress in sheep on preovulatory follicle dynamics and gonadotrophin secretion. Thus, growth dynamics, stereidogenic function and competence to ovulate of preovulatory follicles, as well as FSH and LH availability throughout the entire oestrous cycle, were compared among sheep native from low and high altitude, and sheep newcomers to high altitude. The results indicates that short-term exposure in sheep newcomers to high altitude has a deleterious effect on both the ovarian function (affecting preovulatory follicular development) and the pituitary function (diminishing plasma LH availability). On the other hand, there were no detected differences in the preovulatory follicular development in sheep adapted to high altitude for generations and, conversely, LH secretion was increased, which suggests an adaptive mechanism. The treatment with antioxidant agents during a relative short period for the time of folliculogenesis (approximately 1 month and a half) changed substantially the development of preovulatory follicles in short-term exposed sheep to similar patterns than in sheep native and living to both high and low altitude. These results highlight the role of oxidative stress in the detriment of the reproductive function in individuals recently exposed to high-altitude hypoxic environment.
Asunto(s)
Altitud , Hormona Luteinizante/sangre , Folículo Ovárico/fisiología , Ovulación/fisiología , Ovinos/fisiología , Alimentación Animal/análisis , Animales , Antioxidantes/farmacología , Ácido Ascórbico/administración & dosificación , Ácido Ascórbico/farmacología , Dieta/veterinaria , Suplementos Dietéticos , Estradiol/sangre , Femenino , Hormona Folículo Estimulante/sangre , Oxígeno/sangre , Vitamina E/administración & dosificación , Vitamina E/farmacologíaRESUMEN
The objectives of this study were to evaluate the sperm nuclear decondensation capacity of ovulated and in vitro-matured (IVM) canine oocytes during different culture times and correlate this decondensation ability with the state of oocyte nuclear maturation in vitro and in vivo. Fresh ejaculates from three dogs were used for in vitro fertilization (IVF). Dog spermatozoa were cocultured with ovulated or IVM oocytes after each culture period (0, 48, 72 and 96 h) for 24 h. The nuclear stage of the oocytes and the appearance of the sperm nucleus were determined, and data were analysed with a chi-square test. The rates of decondensation and meiotic development in IVM oocytes increased up to 72 h of culture. In contrast, almost all in vivo-matured oocytes showed MII nuclear stage and sperm chromatin decondensation. The percentages of oocytes at MII stage were much lower (p < 0.05) in all IVM groups compared with ovulated oocytes; the rate of sperm chromatin decondensation was higher in ovulated oocytes than in those matured in vitro. Thus, IVM canine oocytes are able to decondense the sperm chromatin during IVF, and this ability increases with time. Nevertheless, sperm chromatin decondensation is less efficient than in ovulated oocytes and may not be completely synchronized with nuclear development as it occurs in vivo.
Asunto(s)
Perros/fisiología , Fertilización In Vitro/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/citología , Animales , Femenino , Masculino , Espermatozoides/fisiologíaRESUMEN
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.
Asunto(s)
Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Meiosis/fisiología , Oocitos/citología , Oocitos/fisiología , Animales , Perros , Femenino , Técnica del Anticuerpo Fluorescente , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Sensibilidad y Especificidad , Coloración y EtiquetadoRESUMEN
Cyclooxygenase 2 (COX-2) encoded by the Cox-2 gene within the periovulatory follicles is a critical mediator of oocyte development. Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) participate in the modulation of certain target genes in the ovary, possibly influencing the Cox-2 gene expression. However, this relationship has not been characterized in canines. This study aimed to examine the possible relationships among BMP-15, GDF-9, progesterone, and Cox-2 gene expression in granulosa-cumulus cells in dogs. Granulosa cells from antral follicles and their corresponding cumulus-oocyte complexes and follicular fluid (FF) were separately obtained from 56 ovaries collected from adult bitches at estrus (n = 15) and proestrus (n = 13) after ovariohysterectomy. Total RNA extraction was performed in follicular cells, and Cox-2 gene expression was assessed by quantitative PCR analysis. Progesterone, BMP-15, and GDF-9 were determined in the FF samples using ELISA assays. Cumulus-oocyte complexes were subjected to in vitro maturation (IVM) with or without (control) recombinant GDF-9 and BMP-15. After 72 h of culture, Cox-2 transcript analyses were performed in cumulus cells via quantitative PCR. Data were evaluated by ANOVA. An increase (P < 0.05) in Cox-2 messenger RNA levels was observed in follicular cells from follicles at estrus with respect to those at proestrus. However, the levels of BMP-15 and GDF-9 in FF decreased (P < 0.05), whereas progesterone increased (P < 0.05) from the proestrus phase to the estrus phase. The expression of Cox-2 gene in cumulus cells was 4-fold greater (P < 0.01) than that in the control when both growth factors were added to the IVM culture. In conclusion, although BMP-15 together with GDF-9 appears to upregulate the levels of Cox-2 transcripts during IVM, the inverse relationship of these paracrine factors with Cox-2 gene expression and the positive correlation of progesterone with Cox-2 transcripts suggest that the high progesterone levels could be more relevant in the local mechanisms regulating the Cox-2 gene expression.
Asunto(s)
Proteína Morfogenética Ósea 15/metabolismo , Ciclooxigenasa 2/metabolismo , Perros , Factor 9 de Diferenciación de Crecimiento/metabolismo , Folículo Ovárico/metabolismo , Progesterona/metabolismo , ARN Mensajero/metabolismo , Animales , Proteína Morfogenética Ósea 15/genética , Ciclooxigenasa 2/genética , Ciclo Estral/fisiología , Femenino , Factor 9 de Diferenciación de Crecimiento/genética , Progesterona/sangre , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Criopreservación/veterinaria , Perros , Microscopía Electroquímica de Rastreo/veterinaria , Preservación de Semen/veterinaria , Interacciones Espermatozoide-Óvulo , Espermatozoides/fisiología , Acrosoma/ultraestructura , Reacción Acrosómica , Animales , Femenino , Calor , Masculino , Oocitos/fisiología , Oocitos/ultraestructura , Preservación de Semen/métodos , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/ultraestructuraRESUMEN
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.
Asunto(s)
Perros , Oocitos/ultraestructura , Zona Pelúcida/ultraestructura , Animales , Células Cultivadas , FemeninoRESUMEN
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.
Asunto(s)
Acrosina/metabolismo , Perros , Congelación , Capacitación Espermática/fisiología , Espermatozoides/enzimología , Animales , Western Blotting/veterinaria , MasculinoRESUMEN
In vitro gamete derivation based on differentiation of germ cells (GC) from stem cells has emerged as a potential new strategy for the treatment of male infertility. This technology also has potential applications in animal reproduction as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to differentiate into mesodermal lineages. Under the effect of selected bioactive factors, MSC upregulate expression of pluripotent and GC specific-markers revealing their potential for GC differentiation. In addition to the effect of trophic factors, cell-to-cell interaction with Sertoli cells (SC) may be required to guide the sequential differentiation of MSC into GC. Thus, the aim of the present study was to investigate the effect of coculture with SC on the potential for in vitro GC differentiation of bovine fetal MSC (bfMSC) derived from bone marrow (BM-MSC) and adipose tissue (AT-MSC). bfMSC were isolated from male bovine fetuses and SC were collected from adult bull testes. The effect of SC interaction with BM-MSC or AT-MSC was analyzed on the expression of pluripotent factors OCT4 and NANOG, GC genes FRAGILLIS, STELLA and VASA and male GC markers DAZL, PIWIL2, STRA8 and SCP3 at Day 14 of coculture. Flow cytometry analyses detected that the majority (95,5%⯱â¯2.5; Pâ¯<â¯0.05) of the isolated population of SC cultures were positive for SC-specific marker WT1. Levels of mRNA of WT1 in BM-MSC and AT-MSC were lower (Pâ¯<â¯0.05) compared to SC; whereas, WT1 expression was not detected in bovine fetal fibroblasts (FB). Cocultures of BM-MSC and AT-MSC with SC had higher (Pâ¯<â¯0.05) OCT4 mRNA levels compared to monocultures of BM-MSC, AT-MSC and SC. Moreover, cocultures of BM-MSC with SC had higher (Pâ¯<â¯0.05) proportion of cells positive for Oct4 and Nanog compared to monocultures of BM-MSC and SC. Levels of mRNA of DAZL, PIWIL2 and SCP3 were upregulated in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Accordingly, the proportion of cells positive for Dazl were higher (Pâ¯<â¯0.05) in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Changes in gene expression profiles during coculture of SC with AT-MSC suggest that cell-to-cell interaction or bioactive factors provided by SC may induce progression of AT-MSC into early stages of GC differentiation.
Asunto(s)
Bovinos , Diferenciación Celular/fisiología , Técnicas de Cocultivo/veterinaria , Células Germinativas/fisiología , Células Madre Mesenquimatosas/fisiología , Células de Sertoli/fisiología , Animales , Linaje de la Célula , MasculinoRESUMEN
Ovine reproduction efficiency in herds at high altitude (ha) is lower than that at low altitude (la). In ewes, ha effects are due to hypoxia and oxidative stress. Our aim was to establish the effect of antioxidant vitamin supplementation on semen traits and antioxidant status of rams exposed to short or long time ha. A total of 32 rams native to la (~500 m) were used, 16 were kept at la and the other 16 were brought to ha (~3600 m), where they were placed in the same flock as the ha native rams (n=16). Half of the animals in each group were supplemented daily with vitamins C 600 mg and E 450 IU per os, during the entire experimental period, starting the 4th day after animal's arrival at ha (day 0). At days 0, 30 and 60 of treatment, blood and semen samples were collected for evaluation of antioxidant status and semen standard characteristics. Data were compared within each experimental time by analysis of variance using a general linear model. Elevated concentrations of oxidative stress biomarkers were present in blood from animals maintained at ha. Ejaculates from ha exposed rams showed decreased sperm concentration, progressive motility and viability, in addition to decreased antioxidant status in seminal fluid. A total of 30 days of oral supplementation with vitamins C and E prevented some ha negative effects on semen characteristics, mainly in recently ha exposed rams. It is concluded that exposure of rams to ha negatively affects semen quality, where oxidative stress plays a predominant role. These effects are mainly prevented by oral supplementation of vitamins C and E, which constitutes a simple and cheap alternative to improve semen quality of rams when they are moved to ha.
Asunto(s)
Antioxidantes/administración & dosificación , Ácido Ascórbico/administración & dosificación , Suplementos Dietéticos , Semen/efectos de los fármacos , Ovinos/fisiología , Altitud , Animales , Masculino , Estrés Oxidativo/efectos de los fármacos , Análisis de Semen/veterinaria , Preservación de Semen/veterinaria , Espermatozoides/efectos de los fármacosAsunto(s)
Admisión del Paciente/normas , Neumonía/terapia , Calidad de la Atención de Salud , Femenino , Humanos , MasculinoRESUMEN
Experiments were conducted to evaluate in vitro fertilization (IVF) of in vitro matured (IVM) bitch oocytes using dog spermatozoa frozen in three different extenders. Sperm-rich fraction from eight ejaculates of five dogs was frozen in each one of three egg yolk Tris extenders with additional: (A) 1.4 g citric acid and 0.8 g glucose; (B) 0.7 g citric acid and 3.5 g glucose; or (C) 1.4 g citric acid and 0.8 g fructose (all with 5% glycerol in 100 mL milliQ water). Thawed sperm were co-incubated with IVM bitch oocytes for 6 h. Oocytes were fixed and evaluated under an epifluorescence microscope; penetrated oocytes were defined as those having sperm heads in the perivitelline space or in the oocyte cytoplasm. Higher penetration rates (P < 0.05) were obtained in oocytes cultured with spermatozoa frozen in extenders B and C than those frozen in extender A (33.1, 34.2 and 26.4%, respectively).
Asunto(s)
Criopreservación/veterinaria , Perros/fisiología , Fertilización In Vitro/veterinaria , Oocitos/fisiología , Preservación de Semen/veterinaria , Espermatozoides/fisiología , Animales , Femenino , Colorantes Fluorescentes/química , Masculino , Microscopía Fluorescente/veterinaria , Embarazo , Propidio/química , Interacciones Espermatozoide-Óvulo/fisiologíaRESUMEN
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.
Asunto(s)
Bovinos/fisiología , Atresia Folicular/fisiología , Líquido Folicular/metabolismo , Folículo Ovárico/fisiología , Preñez/metabolismo , Esteroides/metabolismo , Animales , Estradiol/metabolismo , Ciclo Estral/metabolismo , Femenino , Histocitoquímica/veterinaria , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Embarazo , Progesterona/metabolismo , Testosterona/metabolismoAsunto(s)
Chlamydophila pneumoniae/aislamiento & purificación , Neumonía Bacteriana/diagnóstico , Streptococcus pneumoniae/aislamiento & purificación , Infecciones por Chlamydophila/diagnóstico , Infecciones por Chlamydophila/microbiología , Humanos , Masculino , Persona de Mediana Edad , Neumonía Bacteriana/microbiología , Neumonía Neumocócica/diagnóstico , Neumonía Neumocócica/microbiologíaRESUMEN
Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potential test were used to follow an incident of seasonal filamentous foaming at the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant. A positive correlation was observed between an increase in foaming potential and the appearance of foam on the surfaces of aeration basins and secondary clarifiers. In addition, during the occurrence of foaming, the mass and activity of Gordonia spp. increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fraction of mixed liquor volatile suspended solids attributed to Gordonia spp. increased from 4% to more than 32% of the total mixed liquor volatile suspended solids. These results indicate that both the activity and biomass level of Gordonia spp. in activated sludge increased relative to the activity aid the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass, and the role of Gordonia spp. in filamentous foaming may be linked more tightly to the physical presence of filamentous microorganisms than to the metabolic activity of the cells.
Asunto(s)
Actinomycetales/aislamiento & purificación , Immunoblotting , Hibridación Fluorescente in Situ , Aguas del Alcantarillado/microbiología , Purificación del Agua , Actinomycetales/genética , Anticuerpos , Biomasa , Sondas de Oligonucleótidos , ARN Ribosómico/genética , Estaciones del AñoRESUMEN
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).