RESUMEN
The fertilizing spermatozoa induce a Ca2+ oscillatory pattern, the universal hallmark of oocyte activation, in all sexually reproducing animals. Assisted reproductive technologies (ARTs) like intracytoplasmic sperm injection (ICSI) bypass the physiological pathway; however, while a normal Ca2+ release pattern occurs in some species, particularly humans, artificial activation is compulsory for ICSI-fertilized oocytes to develop in most farm animals. Unlike the normal oscillatory pattern, most artificial activation protocols induce a single Ca2+ spike, undermining proper ICSI-derived embryo development in these species. Curiously, diploid parthenogenetic embryos activated by the same treatments develop normally at high frequencies and implant upon transfer in the uterus. We hypothesized that, at least in ruminant embryos, the oscillatory calcium waves late in the first cell cycle target preferentially the paternal pronucleus and are fundamentally important for paternal nuclear remodeling. We believe that Ca2+ signaling is central to full totipotency deployment of the paternal genome. Research in this area could highlight the asymmetry between the parental genome reprogramming timing/mechanisms in early development and impact ARTs like ICSI and cloning.
Asunto(s)
Calcio , Semen , Animales , Femenino , Masculino , Humanos , Calcio/metabolismo , Semen/metabolismo , Citoplasma/metabolismo , Fertilización , Espermatozoides/metabolismo , Oocitos/metabolismoRESUMEN
In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.
Asunto(s)
Autofagia , Atresia Folicular , Células de la Granulosa , Femenino , Animales , Atresia Folicular/fisiología , Ovinos/fisiología , Autofagia/fisiología , Células de la Granulosa/fisiología , Ovario , Folículo Ovárico/fisiología , ApoptosisRESUMEN
OBJECTIVES: To evaluate the effectiveness of a personalised rehabilitative programme in improving fatigue and function in rheumatoid arthritis (RA) female patients treated with biologic DMARDs. METHODS: Thirty-eight consecutive female RA in-patients treated with biologics, entered this prospective pilot study. All subjects were in high disease activity (DAS-28>5.1). After baseline (T0) evaluation, a personalised 4-weeks rehabilitative programme was added to standard biologic treatment and all patients were re-evaluated at the end of the rehabilitative treatment (T1), at 3 (T2), 6 (T3) and 9 (T4) month follow-up. Clinical rheumatologic assessment included the DAS-28, TJC, SJC, global health status, HAQ and FACIT. RESULTS: Subjects showed a mean age of 65±3.5 years and a 10±1,1 years mean disease duration. All clinical and laboratory outcomes significantly improved at the different follow-up times as compared to baseline. In particular, a significant improvement in function and fatigue indices (HAQ and FACIT) was found since T1 to T4 as compared to T0. During the follow-up, DAS-28 decreased. Accordingly, about 30% of subjects achieved a moderate disease activity (DAS-28<5.1). CONCLUSIONS: A combined treatment biologics-rehabilitation is effective in improving function and fatigue in female patients with established RA. Fatigue results independent from disease activity.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Terapia por Ejercicio , Fatiga/rehabilitación , Medicina de Precisión , Anciano , Artritis Reumatoide/complicaciones , Artritis Reumatoide/diagnóstico , Terapia Combinada , Evaluación de la Discapacidad , Fatiga/diagnóstico , Fatiga/etiología , Femenino , Humanos , Persona de Mediana Edad , Proyectos Piloto , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Encuestas y Cuestionarios , Factores de Tiempo , Resultado del TratamientoRESUMEN
In recent years, the exposure of organisms to static magnetic fields (SMFs) is continuously increasing. Thus, we investigated the effect of chronic exposure to a 2 mT SMF on in vitro cultured swine granulosa cells (GCs). In particular, the culture expansion (cell viability and doubling time), the cell phenotype (cell morphology and orientation, actin and α-tubulin cytoskeleton), the cell metabolism (intracellular Ca(2+) concentration [Ca(2+)]i and mitochondrial activity) and the cell function (endocrine activity) were assessed. It has been found that the exposure to the field did not affect the cell viability, but the doubling time was significantly reduced (p < 0.05) in exposed samples after 72 h of culture. At the same time, the cell length and thickness significantly changed (p < 0.05), while the cell orientation was unaffected. Evident modifications were induced on actin and α-tubulin cytoskeleton after 3 days of exposure and, simultaneously, a change in [Ca(2+)]i and mitochondrial activity started to become evident. Finally, the SMF exposure of GCs longer than 72 h determined a significant alteration of progesterone and estrogen production (p < 0.05). In conclusion, our results demonstrate that the chronic exposure of swine GCs to a 2 mT SMF exerts a negative effect on cell proliferation, morphology, biochemistry and endocrine function in an in vitro model.
Asunto(s)
Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Campos Magnéticos , Actinas/metabolismo , Animales , Calcio/metabolismo , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Citoesqueleto/metabolismo , Femenino , Hormonas/biosíntesis , Mitocondrias/metabolismo , Esteroides/biosíntesis , Porcinos , Factores de Tiempo , Tubulina (Proteína)/metabolismoRESUMEN
To date, the impossibility of treating resistant forms of bacteria and fungi (AMR) with traditional drugs is a cause for global alarm. We have made the green synthesis of Argirium silver ultra nanoclusters (Argirium-SUNCs) very effective against resistant bacteria (< 1 ppm) and mature biofilm (0.6 ppm). In vitro and preclinical tests indicate that SUNCs are approximately 10 times less toxic in human cells than bacteria. Unique chemical-physical characteristics such as particle size < 2 nm, a core composed of Ag0, and a shell of Ag +, Ag2+ , Ag3+ never observed before in stable form in ultra pure water, explain their remarkable redox properties Otto Cars (Lancet Glob. Health 9:6, 2021). Here we show that Argirium-SUNCs have strong antimicrobial properties also against resistant Aspergillus niger GM31 mycelia and spore inactivation (0.6 ppm). The membrane depolarization is a primary target leading to cell death as already observed in bacteria. Being effective against both bacteria and fungi Argirium-SUNCs represent a completely different tool for the treatment of infectious diseases.
Asunto(s)
Antiinfecciosos , Nanopartículas del Metal , Humanos , Aspergillus niger , Antiinfecciosos/farmacología , Oxidación-Reducción , Bacterias , Antibacterianos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Objective: To assess the effectiveness of home videogame-based exercise (exergaming) as an additional rehabilitative tool in young patients with rheumatoid arthritis (RA). Materials and Methods: After a baseline (T0) evaluation, 40 RA inpatients (18-35 years of age) underwent both a 4-week-lasting traditional rehabilitation program and a training by Nintendo® Wii-Fit™ videogame system. At discharge (T1), subjects were randomly assigned (1:1) to two groups: Group A (experimental group), including subjects who continued Wii-Fit training at home for additional 8 weeks, and Group B (control group), including subjects maintaining their habitual activity during the 8-week follow-up (T2). Measures of disease activity, quality of life, and fatigue were evaluated at each time point. Results: From T0 to T1, a significant improvement in most evaluated outcomes was reported in both study groups. At T2 assessment, only Group A patients experienced a significant improvement of quality of life and fatigue, with a 13.4% reduction in Global Health (GH) values, only a slight increase (4.2%) in Health Assessment Questionnaire (HAQ) score, and a 19.1% Functional Assessment of Chronic Illness Therapy (FACIT) improvement as compared with T1. In contrast, Group B patients reported a 65.8% increase in GH values, a 33% increase in HAQ score, and a 53.4% reduction in FACIT values from T1 to T2. The extended videogame-based home training was an independent predictor of Δ%GH (ß = 0.851; P < 0.001), Δ%HAQ (ß = 0.542; P < 0.001), and Δ%FACIT (ß = -0.505; P < 0.001). Conclusions: Home exergaming may be an effective additional rehabilitative tool in RA, since it allows to maintain the benefits of traditional multidisciplinary rehabilitation.
Asunto(s)
Artritis Reumatoide/rehabilitación , Ejercicio Físico/psicología , Juegos Recreacionales/psicología , Adolescente , Adulto , Artritis Reumatoide/psicología , Femenino , Humanos , Masculino , Modalidades de Fisioterapia/normas , Modalidades de Fisioterapia/estadística & datos numéricos , Proyectos Piloto , Calidad de Vida/psicologíaRESUMEN
In mammals, apoptosis has been accepted as the type of programmed cell death (PCD) that occurs in ovarian follicles undergoing atresia. Results of recent studies, however, indicate autophagy may be an alternative mechanism involved in follicle depletion through independent or tandem actions with apoptosis. Western blotting and immunofluorescence procedures were used in the present study to investigate the abundances of LC3B protein in freshly collected granulosa cells (GCs), cumulus cells (CCs), and oocytes to evaluate whether autophagy is an important process of antral follicle atresia in sexually mature sows. Furthermore, apoptosis was analyzed using annexin V and TUNEL assays in the same cellular cohorts to evaluate the correlation between the two processes. Immunostaining results indicate autophagy was induced in the majority of GCs, CCs, and oocytes from early and advanced stage atretic follicles. The quantitative results of western blot analysis indicate there is a progressive increase (Pâ¯<⯠0.05) in abundance of autophagy-related protein (LC3B-II) in these cells compared with cells in non-atretic follicles. Furthermore, there is confirmation that apoptosis occurs in the GCs of atretic follicles, thus indicating that in pigs apoptosis and autophagy are processes in GCs that regulate PCD and as a consequence antral follicle depletion. There was a greater abundance of LC3B-II in CCs and oocytes of atretic follicles, while apoptosis was not detected. It, therefore, is suggested that in these cells the two processes function independently, with autophagy having a cytoprotective rather than PCD mechanism of action.
Asunto(s)
Autofagia/fisiología , Células del Cúmulo/fisiología , Atresia Folicular/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Oocitos/fisiología , Porcinos , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
Protein kinase C (PKC), an enzyme playing a central role in signal transduction pathways, is activated in fertilized mouse eggs downstream of the fertilization Ca2+ signal, to regulate different aspects of egg activation. Given the presence of Ca2+-independent PKC isoforms within the egg, we investigated whether fertilization triggers PKC stimulation in mouse eggs by activating Ca2+-independent signalling pathways. An increase in PKC activity was detected as early as 10 min after the beginning of insemination, when about 90% of eggs had fused with sperm and the first Ca2+ rise was evident in most of the eggs. A similar level of activity was found 20 min later, when about 60% of eggs had resumed meiosis. When the Ca2+ increase was buffered by an intracellular Ca2+ chelating agent, PKC stimulation was not blocked but only slightly reduced. Confocal microscopy analysis revealed that the increase in PKC activity at fertilization coincided with the translocation of PKCdelta, a Ca2+-independent and diacylglycerol-dependent PKC isoform, to the meiotic spindle. When, in the absence of the Ca2+ signal, metaphase-anaphase transition was inhibited, PKCdelta moved to the meiotic spindle but still maintained a sustained cytoplasmic distribution. In summary, our results indicate that: 1) PKC activation is an early event of egg activation; 2) both Ca2+-dependent and Ca2+-independent pathways contribute to increased PKC activity at fertilization; 3) PKCdelta is one of the isoforms participating in this signalling process.
Asunto(s)
Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Proteína Quinasa C/metabolismo , Transducción de Señal , Animales , Quelantes/farmacología , Ácido Egtácico/farmacología , Activación Enzimática , Femenino , Fertilización , Inmunohistoquímica , Masculino , Meiosis , Ratones , Microscopía Confocal , Isoformas de Proteínas , Proteína Quinasa C/química , Proteína Quinasa C-delta , Espermatozoides/metabolismo , Huso Acromático , Factores de TiempoRESUMEN
OBJECTIVES: To assess the motor control during quiet stance in patients with established ankylosing spondylitis (AS) and to evaluate the effect of visual input on the maintenance of a quiet posture. METHODS: 12 male AS patients (mean age 50.1 ± 13.2 years) and 12 matched healthy subjects performed 2 sessions of 3 trials in quiet stance, with eyes open (EO) and with eyes closed (EC) on a baropodometric platform. The oscillation of the centre of feet pressure (CoP) was acquired. Indices of stability and balance control were assessed by the sway path (SP) of the CoP, the frequency bandwidth (FB1) that includes the 80% of the area under the amplitude spectrum, the mean amplitude of the peaks (MP) of the sway density curve (SDC), and the mean distance (MD) between 2 peaks of the SDC. RESULTS: In severe AS patients, the MD between two peaks of the SDC and the SP of the center of feet pressure were significantly higher than controls during both EO and EC conditions. The MP was significantly reduced just on EC. CONCLUSIONS: Ankylosing spondylitis exerts negative effect on postural stability, not compensable by visual inputs. Our findings may be useful in the rehabilitative management of the increased risk of falling in AS.
Asunto(s)
Movimiento , Equilibrio Postural , Postura , Espondilitis Anquilosante/fisiopatología , Análisis y Desempeño de Tareas , Percepción Visual , Adulto , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Since nerve growth factor (NGF) is produced in vitro by granulosa cells after gonadotropin stimulation, the present research has been designed to investigate whether this neurotropin is involved in the events triggered by the gonadotropin surge that lead the follicle to ovulate a mature oocyte. To this aim, NGF levels in follicular fluid, collected before or 20 hours after the gonadotropin surge, was measured by ELISA. To evaluate whether NGF may have a non-neurotropic effect on follicle cells, the presence of NGF receptors was investigated by immunohistochemistry and further evaluated by analysing the tyrosine-phosphorylation pattern after NGF stimulation in vitro. The effect of NGF on the degree of cumulus expansion, cumulus-oocyte metabolic coupling, and meiotic maturation was finally studied by using the culture of follicle-enclosed oocyte. The results demonstrate that GnRH causes a dramatic rise of NGF in large follicles. Immunohistochemistry revealed a discrete positivity for trkA receptors localised in cumulus cells. Tyrosine phosphorylation pattern confirms that somatic cells are capable to transduce NGF signal. By contrast, all the oocytes examined were negative for trkA and did not change the phosphorylation pattern after NGF. In vitro NGF (100 ng/ml) induced a marked cumulus expansion and a progressive cumulus-oocyte uncoupling similar to that produced by gonadotropins. The addition of NGF also caused the resumption of meiosis in more than 70% of the oocytes analysed with an effect that is only slightly less pronounced than that of gonadotropins (80%). The increase in NGF secretion following gonadotropin surge suggests that this neurotropin may be involved in the control of oocyte maturation.
Asunto(s)
Líquido Folicular/metabolismo , Factores de Crecimiento Nervioso/fisiología , Oocitos/fisiología , Animales , Gonadotropina Coriónica/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnicas In Vitro , Meiosis/efectos de los fármacos , Factores de Crecimiento Nervioso/metabolismo , Oocitos/efectos de los fármacos , Oocitos/crecimiento & desarrollo , OvinosRESUMEN
The in vitro effects of 8-MOP (concentrations of 20, 100 and 500 ng/ml) alone or in combination with UVA on mediator release from human basophils and skin mast cells (HSMC), activated with immunological and non-immunological stimuli, were investigated. With respect to basophils activated with anti-IgE serum, the results of this study show that: (i) 8-MOP alone inhibits histamine, LTC(4), IL-4 and IL-13 release concentration dependently with a maximal effect at 500 ng/ml (a concentration not reached in vivo); and (ii) UVA irradiation (5 J/cm(2)), after 8-MOP incubation, enhances this inhibitory effect on all released mediators, but for IL-4 and IL-13 the percentage inhibition is also significant for the 8-MOP concentrations (20-100 ng/ml) employed in vivo during PUVA treatment. Moreover, histamine release from basophils activated with non-immunological stimuli (FMLP and A23187) is inhibited by 8-MOP, alone or in combination with UVA. With respect to the HSMC activated with anti-IgE serum, the results show that: (i) 8-MOP alone reduces histamine release concentration dependently; and (ii) this inhibitory effect is enhanced by UVA irradiation (5 J/cm(2)). Histamine release from HSMC activated with A23187 is not modified either by 8-MOP alone or by 8-MOP plus UVA.
Asunto(s)
Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Metoxaleno/farmacología , Piel/efectos de los fármacos , Piel/efectos de la radiación , Rayos Ultravioleta , Liberación de Histamina , Humanos , Inmunoglobulina E/metabolismo , Técnicas In Vitro , Radioinmunoensayo , Piel/metabolismoRESUMEN
Hemocytes are a critical component of the mussel defense system and the present study aims at investigating their spreading and oxidative properties during phagocytosis under in vivo experimental stress conditions. The spreading ability was measured by an automated cell analyzer on the basis of the circularity, a parameter corresponding to the hemocyte roundness. The oxidative activity was investigated by micromethod assay, measuring the respiratory burst as expression of the fluorescence generated by the oxidation of specific probe. Following the application of high temperature and exposure to air, there was evidence of negative modulation of spreading and oxidative response, as revealed by a cell roundness increase and fluorescence generation decrease. Therefore, the fall of respiratory burst appeared as matched with the inhibition of hemocyte morphological activation, suggesting a potential depression of the phagocytosis process and confirming the application of the circularity parameter as potential stress marker, both in experimental and field studies.
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Bivalvos/citología , Hemocitos/citología , Estrés Oxidativo , Fagocitosis/efectos de los fármacos , Aire , Animales , Bivalvos/crecimiento & desarrollo , Compuestos Ferrosos/farmacología , Fluorescencia , Calor , Oxidación-Reducción , Estallido Respiratorio/efectos de los fármacosRESUMEN
OBJECTIVE: Diffuse idiopathic skeletal hyperostosis (DISH) is characterized by ossification of different entheseal sites. Several metabolic factors have been suggested to be involved in DISH development. We assessed the prevalence of DISH and its relationship to traditional vascular risk factors in a cohort of patients diagnosed with cardiovascular diseases. METHODS: Among the 521 consecutive patients admitted to the heart diseases rehabilitation program in our Rehabilitative Cardiology Unit, only those (n = 436) with recent coronary artery bypass grafting (CABG), a heart valve replacement (HVR), or congestive heart failure (CHF) were enrolled (45 CHF, 338 CABG, and 53 HVR). All patients underwent a rheumatologic examination, blood sample collections, and chest radiographs. Body mass index (BMI), blood pressure, and information about sex, age, smoking habit, and other vascular risk factors were recorded. DISH was established according to the Resnick and Niwayama criteria. RESULTS: In the setting (77.1% men), the mean ± SD age was 65.44 ± 9.66 years and the overall prevalence of DISH was 30.3%. A logistic regression analysis showed that both age (odds ratio [OR] 1.076, 95% confidence interval [95% CI] 1.044-1.109; P < 0.001) and obesity (OR 2.28, 95% CI 1.33-3.89; P = 0.003) were significant predictors of the presence of DISH. An increasing OR for the presence of DISH was found for increasing tertiles of age and BMI. No difference resulted according to other traditional vascular risk factors. BMI and age directly correlated with C-reactive protein levels. CONCLUSION: The overall prevalence of DISH was 30.3%. This is expected because of the study population. Obese and older individuals exhibit a higher risk of DISH development.
Asunto(s)
Enfermedad de la Arteria Coronaria/epidemiología , Insuficiencia Cardíaca/epidemiología , Hiperostosis Esquelética Difusa Idiopática/epidemiología , Índice de Severidad de la Enfermedad , Anciano , Índice de Masa Corporal , Puente de Arteria Coronaria/estadística & datos numéricos , Enfermedad de la Arteria Coronaria/cirugía , Femenino , Humanos , Hiperlipidemias/epidemiología , Hipertensión/epidemiología , Masculino , Persona de Mediana Edad , Obesidad/epidemiología , Prevalencia , Factores de RiesgoRESUMEN
The present experiments compared the ability of pig oocytes matured either in vivo or in vitro to structurally reorganize the penetrated sperm chromatin into male pronucleus (PN) and to carry out, in parallel, the epigenetic processes of global chromatin methylation and acetylation, 12-14 h after in vitro fertilization (IVF). In addition, PN distribution of histone deacetylase (HDAC), a major enzyme interfacing DNA methylation and histone acetylation, was investigated. The ability of the oocyte to operate an efficient block to polyspermy was markedly affected by maturation. The monospermic fertilization rate was significantly higher for in vivo than for in vitro matured (IVM) oocytes (P < 0.01) which, furthermore, showed a reduced ability to transform the chromatin of penetrated sperm into male PN (P < 0.01). Indirect immunofluorescence analysis of global DNA methylation, histone acetylation and HDAC distribution (HDAC-1, -2 and -3), carried out in monospermic zygotes that reached the late PN stage, showed that IVM oocytes also had a reduced epigenetic competence. In fact, while in about 80% of in vivo matured and IVF oocytes the male PN underwent a process of active demethylation and showed a condition of histone H4 hyperacetylation, only 40% of IVM/IVF zygotes displayed a similar PN remodelling asymmetry. Oocytes that carried out the first part of maturation in vivo (up to germinal vesicle breakdown; GVBD) and then completed the process in vitro, displayed the same PN asymmetry as oocytes matured entirely in vivo. A crucial role of HDAC in the establishment of PN acetylation asymmetry seems to be confirmed by the use of HDAC inhibitors as well as by the abnormal distribution of the enzyme between the two PN in IVM zygotes. Collectively, these data demonstrated that some pig IVM oocytes fail to acquire full remodelling competence which is independent from their ooplasmic ability to morphologically reorganize the sperm nucleus into PN.
Asunto(s)
Ensamble y Desensamble de Cromatina , Oogénesis/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Cigoto/ultraestructura , Acetilación , Animales , Metilación de ADN , Femenino , Fertilización In Vitro/métodos , Técnica del Anticuerpo Fluorescente , Histona Desacetilasas/metabolismo , Histonas , Masculino , Microscopía Confocal , Embarazo , Porcinos , Factores de Tiempo , Transferencia Intrafalopiana del CigotoRESUMEN
Fluo-4 loaded immature oocytes were cooled from 30 degrees C to various lower temperatures between 20 and 10 degrees C and changes in intracellular calcium (Ca(2+)) levels were measured. Pig oocytes cooled to 14 degrees C exhibited a clear biphasic Ca(2+) rise. Lower temperatures produced similar responses, while higher temperatures did not exert any effect. The Ca(2+) response appeared to rely on ryanodine dependent stores as removal of extracellular Ca(2+) and intracytoplasmic injection of heparin did not modify cold-induced Ca(2+) elevation, while procaine or ruthenium red virtually eliminated the response. Confocal analysis of subcellular Ca(2+) distribution during cooling revealed that the ion rises sharply within the nucleus. As Ca(2+) imbalance may activate nuclear endonucleases, DNA integrity of cooled pig oocytes was evaluated by TUNEL and comet assays. Most cooled oocytes showed clear signs of DNA fragmentation. Oocytes injected with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid tetrapotassium salt (BAPTA), a Ca(2+) chelator, maintained their DNA integrity thus confirming that intracellular Ca(2+) is involved in triggering DNA fragmentation. The protective effect exerted by ruthenium red and/or procaine further confirmed this hypothesis. These data show that a moderate and transient cooling is sufficient to cause an intracellular Ca(2+) rise that leads to DNA damage. The addition of inhibitors of ryanodine dependent Ca(2+) stores may represent a valuable protective treatment to reduce chilling injuries.