Vitrification of germinal vesicle (GV) stage oocytes has been shown to be closely associated with decreased rates of meiosis maturation and increased rates of aneuploidy. However, little is known about the effects of melatonin on these events in mice vitrified GV oocytes. In this study, the effects of melatonin on meiosis maturation potential and the incidence rate of aneuploidy in mouse vitrified oocytes were analyzed by supplementing in vitro maturation (IVM) solution with melatonin at different concentrations. This study, for the first time, showed that the mitochondrial heat production was markedly increased in vitrified oocytes (P < 0.05), which compromised the first polar body extrusion (PBE) of vitrified oocytes (73.3% vs. 85.1%, P < 0.05). However, 10-11 mol/L melatonin could significantly decrease mitochondrial heat production and ROS level (9.1 vs. 12.0 pixels, P < 0.05), meanwhile increase ATP level (1.1 vs. 0.88 pmol, P < 0.05) and mtDNA copies (107438 vs. 67869, P < 0.05), which rescued the abnormal chromosome alignment (32% vs. 69%, P < 0.05) and reduced the incidence of aneuploidy (15.6% vs. 38.5%, P < 0.05) in vitrified oocytes. The meiosis maturation ability of vitrified oocytes with melatonin supplementation was similar to that of fresh ones (83.4% vs. 85.1%, P > 0.05). Collectively, our data revealed that melatonin has a protective action against vitrification-induced injuries of oocytes meiosis maturation.

This study was conducted to determine the effect of meiotic status, cumulus cells and cytoskeleton stabilizer on ovine oocyte vitrification. Oocytes at various developmental stages including GV (germinal vesicle), GVBD (GV breakdown), MI (metaphase I) and MII (metaphase II) were vitrified using open pulled straw (OPS) method. After warming, the survival rates were determined based on the morphological appearance and 3′,6′-diacetyl fluorescein staining. The developmental potential of treated oocytes was evaluated by their ability to undergo successful in vitro fertilization (IVF) and support embryo development in culture after in vitro maturation. In the first experiments, we evaluated the effect of meiosis status on oocytes vitrification. Survival rates of oocytes after warming were not different among all groups. However, significantly higher proportion of cleavages and blastocysts were obtained from vitrified MII oocytes than those from vitrified immature oocytes. Next, we selected MII oocytes to determine the influence of cumulus cells on vitrification and the results showed that survival rates were not affected by the absence of cumulus cells. Furthermore, the cleavage rates and blastocyst rates were not different with or without cumulus cells. Lastly, we examined the effect of cytoskeleton stabilizer on MII oocyte vitrification. Compared with the vehicle treated controls, pretreatment with Taxol significantly improved the survival rates (81.91% vs. 66.00%), cleavage rates ((52.29% vs.34.25%) and blastocyst rates (9.72% vs. 4.86%). Pretreatment of MII oocytes with another cytoskeleton stabilizer Cytochalansin B had no effect on oocyte survival and in vitro embryo development. Collectively, the meiotic status affected the developmental potential of oocytes after vitrification. MII stage oocytes showed better resistance to cryopreservation compared with the oocytes at immatured stages. Taxol treatment prior to vitrification was beneficial to vitrified/warmed ovine matured oocytes.

This study was conducted to systematically assess the reproductive performance of transgenic TLR4 ewes.In the TLR4 transgenic founders (F 0 ) and their positive offspring (F 1 ), hematological and reproductive parameters and the global DNA methylation level in oocytes at various stages were analyzed.The values of the physiological and biochemical parameters determined from the blood samples did not differ significantly between the transgenic and wild-type ewes.Moreover, the transgenic ewes showed reproductive traits similar to the wildtype ewes.These traits included characteristics of puberty, the estrus cycle, estrus duration, gestation, the pregnancy rate and the superovulation response.Additionally, no significant differences were found between transgenic and wild-type ewes in the DNA methylation level of the oocytes at various stages.In summary, the preliminary evidence presented in this paper demonstrates that the presence of the TLR4 transgene did not affect the reproductive performance in sheep.

This study was conducted to investigate the effects of Trichostatin A (TSA) on cumulus expansion during mouse oocyte maturation. TSA treatment inhibited cumulus expansion and significantly reduced the cumulus expansion index (CEI) (p<0.05). To determine the underlying mechanism, the expression levels of several key factors that play crucial roles in cumulus expansion including components of extracellular matrix (ECM) (Has2, Ptgs2, Ptx3, and Tnfaip6) and Growth differentiation factor 9 (GDF9) were measured in control and TSA treated samples by real-time PCR. The effect of TSA on ERK phosphorylation (p-ERK1/2) in cumulus cells and GDF9 protein level in fully grown oocytes (FGOs) were detected by Western blotting. The expression levels of the ECM genes were significantly decreased (p<0.05) by TSA treatment while GDF9 expression did not response to TSA (p>0.05). TSA treatment blocked the activation of ERK1/2 (p<0.05) and had no significant effect on GDF9 protein expression (p>0.05). Collectively, these results suggested that TSA treatment altered ECM gene expression and blocked ERK1/2 activation to inhibit cumulus expansion in the mouse.

Cytokeratin (CK) is a type of the cytoskeleton that increases cell stabilization during oocyte maturation. This study was conducted to investigate the effect of vitrification on the distribution and expression of CK during mouse oocyte maturation. Germinal vesicle (GV) oocytes were randomly allocated into three groups: (1) untreated (fresh), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (CPA exposure), or (3) vitrified using the open-pulled straw (OPS) method (vitrification). The oocytes were then incubated for 0 h, 6h (metaphase I (MI) stage), and 10h (metaphase II (MII) stage). The CK distribution in the oocytes at the GV, MI, and MII stages was observed by immunofluorescence, and the expression at the MII stage oocytes derived from vitrified GV oocytes was detected by Western blotting. The CK distribution in the GV oocytes (88.5%) displayed a cortical pattern in the fresh group, whereas a granular pattern was mainly found at the MI (86.7%) and MII (93.5%) stages. In the CPA exposure group, 90.3% of the GV oocytes were observed to display the cortical pattern, and 69.2% of the MI oocytes and 92.9% of the MII oocytes showed the granular pattern. In the vitrification group, most oocytes (GV, 88.9%; MI, 100%; MII, 93.3%) exhibited the cortical pattern. The CK fluorescence intensities of the MII stage oocytes in both the fresh (59.27) and CPA exposure (60.05) group were significantly higher than that of the vitrification (26.53) group (p<0.05). Western blotting showed that the CK expression in the MII oocytes derived from vitrified GV oocytes was significantly lower (p<0.05) than in the control. In conclusion, OPS vitrification affects the normal CK distribution pattern during oocyte maturation and results in decreased CK expression in MII oocytes derived from vitrified GV oocytes.

SUMMARY The aim of this study was to determine the effects of trans ‐10, cis ‐12 conjugated linoleic acid (t10c12 CLA) supplementation on oocyte maturation and embryo development in pigs. Compared with the control, supplementation of 50 µM t10c12 CLA to in vitro maturation (IVM) medium significantly increased the proportion of oocytes at the metaphase‐II (MII) stage and subsequent parthenogenetic embryo development in terms of cleavage rate, blastocyst formation rate, and cell numbers in blastocysts. The t10c12 CLA‐treated oocytes resumed meiotic maturation and progressed to the MII stage significantly faster than those of control. The expression of phosphorylated mitogen‐activated protein kinase 3/1 (p‐MAPK3/1) and cyclooxygenase‐2 (COX2) in cumulus oocyte complexes (COCs) at 5, 10, and 22 hr of IVM were significantly increased in the t10c12 CLA‐treatment group. The level of p‐MAPK3/1 in t10c12 CLA‐treated MII oocytes was also higher ( P < 0.05) than that of control. Moreover, t10c12 CLA supplementation partially overcame the negative effects of U0126 on cumulus expansion and nuclear maturation, and completely recovered COX2 protein levels in the presence of U0126. Treatment of COCs with NS398 also significantly suppressed cumulus expansion and nuclear maturation, which was overcome by t10c12 CLA. Yet, this simulatory effect of t10c12 CLA was blocked in the presence of both U0126 and NS398. The t10c12 CLA treatment significantly reduced reactive oxygen species level and increased glutathione concentrations in MII oocyte. In conclusion, supplementation of t10c12 CLA during porcine oocyte maturation exerts its beneficial effects on nuclear and cytoplasmic maturation, which contributes to enhancing subsequent embryo development. Mol. Reprod. Dev. 81: 20–30, 2014. © 2013 Wiley Periodicals, Inc .

This study was designed to evaluate DNA methylation and the expression of DNA methyltransferases (Dnmt1, Dnmt3a, Dnmt3b and Dnmt3L) in metaphaseII (MII) oocytes and the DNA methylation of pre-implantation embryos during mouse aging to address whether such aging-related changes are associated with decreased reproductive potential in aged mice.