Oxidative stress caused from in vitro culture contributes to inadequate oocyte maturation which l... more Oxidative stress caused from in vitro culture contributes to inadequate oocyte maturation which leads to a poor embryo development. Therefore, it is important to protect oocytes and embryos against oxidative stress. This study was aimed at evaluating the effect of Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone), an antioxidant with various pharmacologic activities, on nuclear and cytoplasmic maturation of pig oocytes as well as on steroidogenesis of cumulus cells (CCs). Another objective was to determine the influence of Embelin on developmental competence of pig oocytes as well as the expression levels of three key genes (Nanog, Sox2 and Oct4) involved in the control of pluripotency in parthenogenetically activated embryos. Embelin (0, 10, 20 and 40 μM) was added during in vitro maturation of cumulus oocyte complexes; media of both the first and the second day of culture were collected and assayed for progesterone and estradiol-17β. At the end of the maturation period, the oocytes were fixed (to determine nuclear maturation) or partenogenically activated to evaluate cytoplasmic maturation and genes expression. Embelin did not exert any effect on the proportion of MII oocytes, steroidogenesis of CCs, percentage of embryos that developed to blastocyst stage and the number of blastomeres/blastocyst. Moreover, no significant differences of Oct4, Nanog and Sox2 transcripts were detected in blastocyst stage embryos. In conclusion, Embelin did not influence the reproductive parameters assessed, confirming that it is not possible to predict whether the beneficial effect exerted by an antioxidant in a particular tissue could be present also in another one.
Three experiments were designed to evaluate the effects of vitrification using Cryotop method on ... more Three experiments were designed to evaluate the effects of vitrification using Cryotop method on MII porcine oocyte viability, chromosomes configuration, meiotic spindle morphology and in vitro fertilization; to do this, in vitro matured oocytes were subjected to the cryoprotectant treatment excluding the plunging into liquid nitrogen, the whole vitrification/warming/rehydration procedure or no treatment (control). In experiment 1 viable oocytes were not reduced by either cryoprotectants or vitrification when they were evaluated immediately after warming and cryoprotectant dilution. However, after a 2 h incubation, the survival rate significantly decreased (P<0.05). In experiment 2 cryoprotectant exposure significantly (P<0.05) influenced spindle morphology even if chromosome organization did not vary, while vitrification significantly (P<0.05) increased oocytes with damaged spindles and chromosomes displaced from the metaphase plate. No significant improvements in these parameters were observed after 2 h of incubation but, on the contrary, the rate of oocytes with normal chromosome configuration was reduced. In experiment 3 significant differences among the three groups in the fertilization rate but not in the percentages of monospermy fertilization were recorded; in addition, exposure to cryoprotectants and vitrification significantly (P<0.05) increased degenerated oocyte rate. Overall, these findings confirm that porcine oocytes at MII stage are very sensitive to vitrification, which reduces the rate of viable oocytes and alters microtubule organization, thus impairing fertilization; in addition, incubation of oocytes for 2 h after devitrification seems to be detrimental rather than ameliorative. Further improvements of the current protocol will be necessary in order to optimize the Cryotop method for vitrifying pig matured oocytes.
We determined HSP70 and HSP90 expression by Western blot and their cellular distribution by immun... more We determined HSP70 and HSP90 expression by Western blot and their cellular distribution by immunofluorescence in ejaculated, capacitated and acrosome-reacted boar spermatozoa. The expression of both HSPs was not different among the three types of sperms. HSP90 fluorescence was localized in the midpiece of the three groups of sperms, while permeabilized ejaculated sperms showed the HSP70 signal in a cone-shaped region in the equatorial segment. This localization underwent several rearrangements following capacitation and acrosome reaction (AR). In non-permeabilized cells the HSP70 signal was present only in few ejaculated sperms, while an increased percentage of positive cells was recorded after capacitation and AR. Our results indicate that during capacitation and AR a relocalization of HSP70 occurs. This protein is probably translocated from the inner to the outer leaflet of the membrane suggesting a possible role in sperm-oocyte interaction
Recent evidences (Matwee et al., Mol Hum Reprod 7:829, 2001) suggest that heat shock protein 70 (... more Recent evidences (Matwee et al., Mol Hum Reprod 7:829, 2001) suggest that heat shock protein 70 (Hsp70) could exert an important effect during bovine gamete interaction. As Hsp70 has been identified in extracts from boar semen (Huang et al., Anim Reprod Sci 63:231, 2000), the aim of this study was to examine the possible role of this protein during in vitro fertilization in pig. In vitro matured oocytes were fertilized in presence of 2, 5 and 10 \ub5g/ml of anti-Hsp70 monoclonal antiserum or 10 \ub5g/ml mouse myeloma ascites IgG1 (MOPC21). After 20 h the oocytes were fixed and stained with lacmoid to evaluate the nuclear status. The presence of anti Hsp70 antibody induced a clear dose-dependent inhibition of in vitro fertilization: the fertilization rate was in fact significantly lower (P<0.05) in the presence of 10μg/ml of anti-Hsp70 as compared to control (49.8\ub16.6 vs 83.6\ub14.2%). The inhibition is unlikely due to non specific binding, since the exposure to MOPC21 did not induce similar effects (fertilization rate 84.5\ub13.4%). Moreover, the addition of a FITC-conjugated sheep anti-mouse antibody to spermatozoa collected from IVF wells with 10μg/ml of anti-Hsp70 antibody and spotted onto glass slides, induced a thick post-equatorial band green fluorescence, thus confirming the binding in vivo of anti-Hsp70 antibodies to sperm cells. These results strongly suggest that Hsp70 could play an important role in sperm-egg interaction during in vitro fertilization in swine species
Oxidative stress caused from in vitro culture contributes to inadequate oocyte maturation which l... more Oxidative stress caused from in vitro culture contributes to inadequate oocyte maturation which leads to a poor embryo development. Therefore, it is important to protect oocytes and embryos against oxidative stress. This study was aimed at evaluating the effect of Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone), an antioxidant with various pharmacologic activities, on nuclear and cytoplasmic maturation of pig oocytes as well as on steroidogenesis of cumulus cells (CCs). Another objective was to determine the influence of Embelin on developmental competence of pig oocytes as well as the expression levels of three key genes (Nanog, Sox2 and Oct4) involved in the control of pluripotency in parthenogenetically activated embryos. Embelin (0, 10, 20 and 40 μM) was added during in vitro maturation of cumulus oocyte complexes; media of both the first and the second day of culture were collected and assayed for progesterone and estradiol-17β. At the end of the maturation period, the oocytes were fixed (to determine nuclear maturation) or partenogenically activated to evaluate cytoplasmic maturation and genes expression. Embelin did not exert any effect on the proportion of MII oocytes, steroidogenesis of CCs, percentage of embryos that developed to blastocyst stage and the number of blastomeres/blastocyst. Moreover, no significant differences of Oct4, Nanog and Sox2 transcripts were detected in blastocyst stage embryos. In conclusion, Embelin did not influence the reproductive parameters assessed, confirming that it is not possible to predict whether the beneficial effect exerted by an antioxidant in a particular tissue could be present also in another one.
Three experiments were designed to evaluate the effects of vitrification using Cryotop method on ... more Three experiments were designed to evaluate the effects of vitrification using Cryotop method on MII porcine oocyte viability, chromosomes configuration, meiotic spindle morphology and in vitro fertilization; to do this, in vitro matured oocytes were subjected to the cryoprotectant treatment excluding the plunging into liquid nitrogen, the whole vitrification/warming/rehydration procedure or no treatment (control). In experiment 1 viable oocytes were not reduced by either cryoprotectants or vitrification when they were evaluated immediately after warming and cryoprotectant dilution. However, after a 2 h incubation, the survival rate significantly decreased (P<0.05). In experiment 2 cryoprotectant exposure significantly (P<0.05) influenced spindle morphology even if chromosome organization did not vary, while vitrification significantly (P<0.05) increased oocytes with damaged spindles and chromosomes displaced from the metaphase plate. No significant improvements in these parameters were observed after 2 h of incubation but, on the contrary, the rate of oocytes with normal chromosome configuration was reduced. In experiment 3 significant differences among the three groups in the fertilization rate but not in the percentages of monospermy fertilization were recorded; in addition, exposure to cryoprotectants and vitrification significantly (P<0.05) increased degenerated oocyte rate. Overall, these findings confirm that porcine oocytes at MII stage are very sensitive to vitrification, which reduces the rate of viable oocytes and alters microtubule organization, thus impairing fertilization; in addition, incubation of oocytes for 2 h after devitrification seems to be detrimental rather than ameliorative. Further improvements of the current protocol will be necessary in order to optimize the Cryotop method for vitrifying pig matured oocytes.
We determined HSP70 and HSP90 expression by Western blot and their cellular distribution by immun... more We determined HSP70 and HSP90 expression by Western blot and their cellular distribution by immunofluorescence in ejaculated, capacitated and acrosome-reacted boar spermatozoa. The expression of both HSPs was not different among the three types of sperms. HSP90 fluorescence was localized in the midpiece of the three groups of sperms, while permeabilized ejaculated sperms showed the HSP70 signal in a cone-shaped region in the equatorial segment. This localization underwent several rearrangements following capacitation and acrosome reaction (AR). In non-permeabilized cells the HSP70 signal was present only in few ejaculated sperms, while an increased percentage of positive cells was recorded after capacitation and AR. Our results indicate that during capacitation and AR a relocalization of HSP70 occurs. This protein is probably translocated from the inner to the outer leaflet of the membrane suggesting a possible role in sperm-oocyte interaction
Recent evidences (Matwee et al., Mol Hum Reprod 7:829, 2001) suggest that heat shock protein 70 (... more Recent evidences (Matwee et al., Mol Hum Reprod 7:829, 2001) suggest that heat shock protein 70 (Hsp70) could exert an important effect during bovine gamete interaction. As Hsp70 has been identified in extracts from boar semen (Huang et al., Anim Reprod Sci 63:231, 2000), the aim of this study was to examine the possible role of this protein during in vitro fertilization in pig. In vitro matured oocytes were fertilized in presence of 2, 5 and 10 \ub5g/ml of anti-Hsp70 monoclonal antiserum or 10 \ub5g/ml mouse myeloma ascites IgG1 (MOPC21). After 20 h the oocytes were fixed and stained with lacmoid to evaluate the nuclear status. The presence of anti Hsp70 antibody induced a clear dose-dependent inhibition of in vitro fertilization: the fertilization rate was in fact significantly lower (P<0.05) in the presence of 10μg/ml of anti-Hsp70 as compared to control (49.8\ub16.6 vs 83.6\ub14.2%). The inhibition is unlikely due to non specific binding, since the exposure to MOPC21 did not induce similar effects (fertilization rate 84.5\ub13.4%). Moreover, the addition of a FITC-conjugated sheep anti-mouse antibody to spermatozoa collected from IVF wells with 10μg/ml of anti-Hsp70 antibody and spotted onto glass slides, induced a thick post-equatorial band green fluorescence, thus confirming the binding in vivo of anti-Hsp70 antibodies to sperm cells. These results strongly suggest that Hsp70 could play an important role in sperm-egg interaction during in vitro fertilization in swine species
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