RESUMEN
The quality of metaphase II oocytes deteriorates rapidly following ovulation as the result of an aging process associated with impaired fertilizing potential, disrupted developmental competence, and increased likelihood of embryonic resorption. Because oxidative stress accelerates the onset of apoptosis in oocytes and influences their capacity for fertilization, this study aimed to characterize the significance of such stress in the postovulatory aging of mouse oocytes in vitro. We investigated the ability of the potent antioxidant melatonin to arrest the aging process when used to supplement oocyte culture medium. This study demonstrated that oxidative stress may occur in oocytes after as little as 8 h in culture and coincides with the appearance of early apoptotic markers such as phosphatidylserine externalization, followed 16 h later by caspase activation (P < 0.05) and morphological evidence of oocyte senescence. Importantly, supplementation of oocyte culture medium with 1 mM melatonin was able to significantly relieve the time-dependent appearance of oxidative stress in oocytes (P < 0.05) and, as a result, significantly delay the onset of apoptosis (P < 0.05). Furthermore, melatonin supplementation extended the optimal window for fertilization of oocytes aged for 8 and 16 h in vitro (P < 0.05) and significantly improved the quality of the resulting embryos (P < 0.01). We conclude that melatonin may be a useful tool in a clinical setting to prevent the time-dependent deterioration of oocyte quality following prolonged culture in vitro.
Asunto(s)
Antioxidantes/farmacología , Senescencia Celular/efectos de los fármacos , Melatonina/farmacología , Oocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Cafeína/farmacología , Evaluación Preclínica de Medicamentos , Femenino , Fertilización In Vitro , Ratones , Ratones Endogámicos C57BL , Inhibidores de FosfodiesterasaRESUMEN
Mature mammalian eggs are ovulated arrested at meiotic metaphase II. Sperm break this arrest by an oscillatory Ca(2+) signal that is necessary and sufficient for the two immediate events of egg activation: cell cycle resumption and cortical granule release. Previous work has suggested that cell cycle resumption, but not cortical granule release, is mediated by calmodulin-dependent protein kinase II (CamKII). Here we find that mouse eggs contain detectable levels of only one CamKII isoform, gamma 3. Antisense morpholino knockdown of CamKIIgamma3 during oocyte maturation produces metaphase II eggs that are insensitive to parthenogenetic activation by Ca(2+) stimulation and insemination. The effect is specific to this morpholino, as a 5-base-mismatch morpholino is without effect, and is rescued by CamKIIgamma3 or constitutively active CamKII cRNAs. Although CamKII-morpholino-treated eggs fail to exit metaphase II arrest, cortical granule exocytosis is not blocked. Therefore, CamKIIgamma3 plays a necessary and sufficient role in transducing the oscillatory Ca(2+) signal into cell cycle resumption, but not into cortical granule release.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Isoenzimas/fisiología , Metafase/fisiología , Oocitos , Animales , Elementos sin Sentido (Genética) , Señalización del Calcio/genética , Ciclo Celular/fisiología , Regulación hacia Abajo , Exocitosis/fisiología , Femenino , Regulación Enzimológica de la Expresión Génica , Técnicas In Vitro , Ratones , Oocitos/citología , Oocitos/fisiología , Partenogénesis/fisiología , ARN ComplementarioRESUMEN
Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).