RESUMEN
SummaryDirect swim-up procedure is widely used to separate the motile competent spermatozoa from the antioxidant-rich semen. Subsequently, spermatozoa become more vulnerable to reactive oxygen species (ROS) due to their cytological characteristics. The effect of vitamin C, a highly concentrated antioxidant in the semen, on direct swim-up-enriched sperm population is not fully investigated. Therefore, the aim of the present study was to assess the effect of vitamin C on sperm functional properties during direct swim-up procedure. Semen samples were collected from 22 participants. Each semen sample was divided into several aliquots. The first portion was overlaid with sperm medium without ascorbic acid (0 µM AA). The second and third fractions were overlaid with sperm medium supplemented with 300 µM and 600 µM AA; respectively. After 1 h of incubation, basic sperm parameters, intracellular ROS levels, acrosome reaction, chromatin integrity, and glucose uptake were assessed. Swim-up without AA significantly increased the percentage of ROS(+) spermatozoa compared with the raw semen (P<0.01). Interestingly, swim-up with 300 µM AA did not increase the percentage of ROS(+) sperm compared with the raw semen. In parallel, the percentage of sperm with altered chromatin integrity was significantly lower in the 300 µM AA group compared with that in the raw semen (P<0.05). These findings suggest that supplementation of vitamin C to sperm medium could be beneficial for direct swim-up-derived spermatozoa.
Asunto(s)
Ácido Ascórbico/farmacología , Separación Celular/métodos , Espermatozoides/fisiología , Reacción Acrosómica , Adulto , Ácido Ascórbico/administración & dosificación , Cromatina/patología , Medios de Cultivo/química , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Glucosa/farmacocinética , Humanos , Masculino , Especies Reactivas de Oxígeno/metabolismo , Semen/fisiología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacosRESUMEN
At mitosis onset, cortical tension increases and cells round up, ensuring correct spindle morphogenesis and orientation. Thus, cortical tension sets up the geometric requirements of cell division. On the contrary, cortical tension decreases during meiotic divisions in mouse oocytes, a puzzling observation because oocytes are round cells, stable in shape, that actively position their spindles. We investigated the pathway leading to reduction in cortical tension and its significance for spindle positioning. We document a previously uncharacterized Arp2/3-dependent thickening of the cortical F-actin essential for first meiotic spindle migration to the cortex. Using micropipette aspiration, we show that cortical tension decreases during meiosis I, resulting from myosin-II exclusion from the cortex, and that cortical F-actin thickening promotes cortical plasticity. These events soften and relax the cortex. They are triggered by the Mos-MAPK pathway and coordinated temporally. Artificial cortex stiffening and theoretical modelling demonstrate that a soft cortex is essential for meiotic spindle positioning.
Asunto(s)
Meiosis/fisiología , Oocitos/metabolismo , Huso Acromático/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Animales , Femenino , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Miosinas/metabolismo , Proteínas Oncogénicas v-mos/metabolismo , Transducción de SeñalRESUMEN
Polar body cytokinesis is the physical separation of a small polar body from a larger oocyte or ovum. This maternal meiotic division shares many similarities with mitotic and spermatogenic cytokinesis, but there are several distinctions, which will be discussed in this review. We synthesize results from many different model species, including those popular for their genetics and several that are more obscure in modern cell biology. The site of polar body division is determined before anaphase, by the eccentric, cortically associated meiotic spindle. Depending on the species, either the actin or microtubule cytoskeleton is required for spindle anchoring. Chromatin is necessary and sufficient to elicit differentiation of the associated cortex, via Ran-based signaling. The midzone of the anaphase spindle serves as a hub for regulatory complexes that elicit Rho activation, and ultimately actomyosin contractile ring assembly and contraction. Polar body cytokinesis uniquely requires another Rho family GTPase, Cdc42, for dynamic reorganization of the polar cortex. This is perhaps due to the considerable asymmetry of this division, wherein the polar body and the oocyte/ovum have distinct fates and very different sizes. Thus, maternal meiotic cytokinesis appears to occur via simultaneous polar relaxation and equatorial contraction, since the polar body is extruded from the spherical oocyte through the nascent contractile ring. As such, polar body cytokinesis is an interesting and important variation on the theme of cell division.
Asunto(s)
Citocinesis/fisiología , Cuerpos Polares/metabolismo , Anafase/fisiología , Animales , Cromatina/metabolismo , Humanos , Transducción de Señal/fisiología , Huso Acromático/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Female meiotic divisions are extremely asymmetric, giving rise to a large oocyte and small degenerating polar bodies, keeping the maternal stores for further embryo development. This asymmetry is achieved via off-center positioning of the division spindle. Mouse oocytes have developed a formin-2-dependent actin-based spindle positioning mechanism that allows the meiotic spindle to migrate towards the closest cortex. Using spinning disk microscopy and FRAP analysis, we studied the changes in the organization of the cytoplasmic F-actin meshwork during the first meiotic division. It is very dense in prophase I, undergoes a significant density drop upon meiosis resumption and reforms progressively later on. This meshwork remodeling correlates with endogenous formin 2 regulation. High formin 2 levels at meiosis I entry induce meshwork maintenance, leading to equal forces being exerted on the chromosomes, preventing spindle migration. Hence, the meshwork density drop at meiosis resumption is germane to the symmetry-breaking event required for successful asymmetric meiotic divisions.
Asunto(s)
Actinas/metabolismo , Meiosis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Oocitos/citología , Huso Acromático/fisiología , Animales , Femenino , Recuperación de Fluorescencia tras Fotoblanqueo , Técnica del Anticuerpo Fluorescente , Immunoblotting , Ratones , Microscopía Confocal , Oocitos/fisiología , Plásmidos/genéticaRESUMEN
Meiotic maturation is characterized by the succession of two asymmetric divisions each giving rise to a small polar body and a large oocyte. These highly asymmetric divisions are characteristic of meiosis in higher organisms. They allow most of the maternal stores to be retained in the oocyte, a vital property for further embryo development. In mouse oocytes, the asymmetry is ensured by the migration and the anchoring of the division spindle to the cortex in meiosis I and by its anchoring to the cortex in meiosis II. In addition, and subsequent to this off-centre positioning of the spindle, a differentiation of the cortex overhanging the chromosomes takes place and is necessary for the extrusion of small polar bodies. In the present review, we will emphasize the role of the actin cytoskeleton in the control of spindle positioning, spindle anchoring to the cortex and cortical differentiation.
Asunto(s)
Citoesqueleto de Actina/metabolismo , División Celular , Ratones/metabolismo , Oocitos/citología , Oocitos/metabolismo , Citoesqueleto de Actina/genética , Animales , Diferenciación Celular , Femenino , Ratones/genética , Transducción de Señal , Huso Acromático/genética , Huso Acromático/metabolismoRESUMEN
Female meiosis in higher organisms consists of highly asymmetric divisions, which retain most maternal stores in the oocyte for embryo development. Asymmetric partitioning of the cytoplasm results from the spindle's "off-center" positioning, which, in mouse oocytes, depends mainly on actin filaments [1, 2]. This is a unique situation compared to most systems, in which spindle positioning requires interactions between astral microtubules and cortical actin filaments [3]. Formin 2, a straight-actin-filament nucleator, is required for the first meiotic spindle migration to the cortex and cytokinesis in mouse oocytes [4, 5]. Although the requirement for actin filaments in the control of spindle positioning is well established in this model, no one has been able to detect them in the cytoplasm [6]. Through the expression of an F-actin-specific probe and live confocal microscopy, we show the presence of a cytoplasmic actin meshwork, organized by Formin 2, that controls spindle migration. In late meiosis I, these filaments organize into a spindle-like F-actin structure, which is connected to the cortex. At anaphase, global reorganization of this meshwork allows polar-body extrusion. In addition, using actin-YFP, our FRAP analysis confirms the presence of a highly dynamic cytoplasmic actin meshwork that is tightly regulated in time and space.