Hyaluronic acid capacitation induces intracellular signals modulated by membrane-associated adenylate cyclase and tyrosine kinase involved in bovine in vitro fertilization.

Heparin is the most commonly used in vitro capacitation inducer in the bovine. However, hyaluronic acid (HA) has been recently used for capacitation induction as well as for other reproductive biotechnologies, such as sperm selection and in vitro fertilization (IVF). Our aim was to induce sperm capacitation with heparin or HA in order to study mAC and TK intracellular signals and their relation with cleavage and blastocyst rates after IVF as well as with the oxidative status of the potential bovine embryos. 2,5-dideoxyadenosine and genistein were used as mAC and TK inhibitors, respectively. Sperm capacitation was analyzed using CTC technique, sperm plasma membrane and acrosome integrity were determined using trypan blue stain and differential interference contrast, and mitochondrial activity was evaluated using fluorochrome JC-1. Cleavage rate was analyzed 48h and blastocyst production 7-8 days after IVF, while cytosolic oxidative activity was determined using RedoxSensor Red CC-1 fluorochrome 7h after IVF. When mAC and TK inhibitors were added to sperm samples, only capacitation decreased significantly both in HA and heparin treated samples (P < 0.05), but plasma membrane and acrosome integrity percentages were not affected in any of these groups (P > 0.05). Sperm mitochondrial membrane potential only decreased in heparin treated samples in the presence of both inhibitors (P < 0.05). Oocytes activated with HA sperm treated samples with the addition of 2,5-dideoxyadenosine and genistein presented a lower cytosolic oxidative status than those activated with sperm treated with HA alone (P < 0.05). On the other hand, oocytes activated with heparin treated sperm samples presented a lower cytosolic oxidative status only in the presence of 2,5-dideoxyadenosine (P < 0.05). Therefore, mAC and TK present a differential participation in heparin and HA sperm induced capacitation and mitochondrial function as well as in IVF.

Morphological, biochemical and functional studies to evaluate bovine oocyte vitrification.

The aim of the present study was to evaluate the effect of vitrification on morphological, biochemical and functional parameters of matured bovine oocytes at different recovery times. To this end, matured bovine oocytes were vitrified using the Cryotech® kit (a minimum-volume system) and then incubated in maturation medium for different post-warming durations (0 h, 3 h or 21 h). Morphology, viability and biochemical parameters were assessed at each time point mentioned above and the recovery of the metaphase plate was analyzed at 2 h, 3 h and 4 h post-warming. The vitrification-warming process did not affect the viability or morphology of oocytes at any time point. However, the recovery of the metaphase plate occurred mostly between 3 and 4 h rather than at 2 h after warming (P < 0.05). Both control and vitrified-warmed oocytes showed changes in cytosolic oxidative activity, quantification of active mitochondria, reactive oxygen species (ROS) levels and redox status at the different time points studied (P < 0.05). However, differences between control and vitrified-warmed oocytes were found only in the quantification of active mitochondria and ROS production (P < 0.05). Finally, in vitro fertilization and embryo culture were carried out as functional studies to establish whether vitrification-warming affected oocyte competence, and a significant decrease was found both in the cleavage rate and embryo development (P < 0.05). We concluded that major improvements in oocyte vitrification, at list with Cryotech® kit, are still needed to avoid variations in oocyte metabolism which could contribute to the reduction in the developmental competence of bovine oocytes.

Effect of the hematopoietic growth factors erythropoietin and kit ligand on bovine oocyte in vitro maturation and developmental competence.

The overall aim of this work was to study the influence of the hematopoietic growth factors erythropoietin (EPO) and kit ligand (KITL) during bovine oocyte in vitro maturation (IVM). The effect of adding different concentrations of EPO or KITL to maturation medium was evaluated analyzing oocyte nuclear maturation, cumulus cells apoptosis, embryo cleavage, reactive oxygen species (ROS) production in matured oocytes and cleaved embryos and the developmental competence to the blastocyst stage. No significant differences were observed in the percentage of oocytes that completed nuclear maturation among treatments, but the percentages of cleaved embryos and blastocysts obtained increased. With the addition of both hematopoietic growth factors the percentage of cumulus cells undergoing apoptosis decreased, the number of blastomeres per cleaved embryo was larger and ROS production per cleaved embryo increased. In conclusion, although the addition of EPO and KITL hematopoietic growth factors during bovine oocyte IVM had no impact on nuclear maturation, it had a positive effect on oocyte cytoplasmic maturation and developmental competence.

The control of reactive oxygen species influences porcine oocyte in vitro maturation.

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H2O2 or O2∙(-) production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O2∙(-) and H2O2 scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O2∙(-) and H2O2 during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.

Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation.

The knowledge concerning redox and reactive oxygen species (ROS)-mediated regulation of early embryo development is scarce and remains controversial. The aim of this work was to determine ROS production and redox state during early in vitro embryo development in sperm-mediated and parthenogenetic activation of bovine oocytes. Sperm-mediated oocyte activation was carried out in IVF-modified synthetic oviductal fluid (mSOF) with frozen-thawed semen. Parthenogenetic activation was performed in TALP plus ionomycin and then in IVF-mSOF with 6-dimethylaminopurine plus cytochalasin B. Embryos were cultured in IVF-mSOF. ROS and redox state were determined at each 2-h interval (7-24 h from activation) by 2',7'-dichlorodihydrofluorescein diacetate and RedoxSensor Red CC-1 fluorochromes respectively. ROS levels and redox state differed between activated and non-activated oocytes (P<0.05 by ANOVA). In sperm-activated oocytes, an increase was observed between 15 and 19 h (P<0.05). Conversely, in parthenogenetically activated oocytes, we observed a decrease at 9 h (P<0.05). In sperm-activated oocytes, ROS fluctuated throughout the 24 h, presenting peaks around 7, 19, and 24 h (P<0.05), while in parthenogenetic activation, peaks were detected at 7, 11, and 17 h (P<0.05). In the present work, we found clear distinctive metabolic patterns between normal and parthenogenetic zygotes. Oxidative activity and ROS production are an integral part of bovine zygote behavior, and defining a temporal pattern of change may be linked with developmental competence.