Xiangwei Fu

L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs.

The objective was to determine whether adding L-carnitine in IVM/IVC medium enhanced maturation and developmental competence of porcine oocytes in vitro. Oocyte maturation rates did not differ significantly among groups supplemented with 0, 0.25, 0.5, or 1 mg/mL of L-carnitine added during IVM (although 2 mg/mL of L-carnitine reduced maturation rate). Compared with control oocytes, those treated with 0.5 mg/mL of L-carnitine during IVM had greater (P < 0.05) rates of blastocyst formation after parthenogenetic activation, and these blastocysts had less (P < 0.05) apoptosis. Adding 0.5 mg/mL of L-carnitine during IVM also significantly reduced intracellular reactive oxygen species (ROS), and increased glutathione (GSH) concentrations. With or without glucose supplementation, 0.5 mg/mL of L-carnitine in the IVM medium significantly hastened nuclear maturation of oocytes. Moreover, supplementing the IVM medium with either glucose or L-carnitine increased (P < 0.05) percentages of oocytes that reached the metaphase II (MII) stage, relative to a control group. Final maturation rates in IVM medium containing either glucose or L-carnitine were not significantly different. Adding L-carnitine (0 to 2 mg/mL) to IVC medium for activated porcine oocytes did not significantly affect development. However, 0.5 mg/mL of L-carnitine in IVC medium significantly reduced reactive oxygen species levels and apoptosis in activated blastocysts, although glutathione concentrations were not significantly altered. In conclusion, adding L-carnitine during IVM/IVC improved developmental potential of porcine oocytes, and also the quality of parthenogenetic embryos, probably by accelerating nuclear maturation, and preventing oxidative damage and apoptosis.

Mitochondrial behaviors in the vitrified mouse oocyte and its parthenogenetic embryo: effect of Taxol pretreatment and relationship to competence

OBJECTIVE To investigate the effect of Taxol pretreatment on mitochondrial behaviors in vitrified mouse mature oocytes and their parthenogenetic embryos. DESIGN Experimental animal study. SETTING University research laboratory and state key laboratory. ANIMAL(S) Sexually mature female Kunming white strain mice. INTERVENTION(S) Taxol before vitrification group (Tax). Oocytes were pretreated with M(2) containing 1 mmol/L Taxol for 2 minutes at 37C and then vitrified-warmed using the OPS vitrification procedure. Both ED solution and EDFS30 solution contained 1 mmol/L Taxol. MAIN OUTCOME MEASURE(S) Mitochondrial behaviors examined by fluorescence microscopy technology and fluorescence recovery after photobleaching (FRAP) technology. RESULT(S) In the control group, mitochondria were homogeneously distributed, in slow movement in oocytes, and perinuclearly distributed in 42.6% (n = 115) of their parthenogenetic two-cell embryos. Mitochondria from the toxicity group showed similar localization and movement to those of the control group, but not in the vitrification group. The perinuclear mitochondrial localization pattern of two-cell embryos was statistically significantly lower in both the toxicity (27.2%) and vitrification groups (19.8%) than in the control group. After parthenogenetic activation, the blastocyst formation rate of oocytes in the treated groups (28.1 to 48.6%) was statistically significantly lower than that of control (61.2%), but the rate of Taxol group (47.9%) was statistically significantly higher than that in the vitrification group (28.1%). CONCLUSION(S) Taxol pretreatment before vitrification helps to reduce the mitochondrial disturbance induced by vitrification in oocytes and their parthenogenetic early-stage embryo.

Positive effects of Taxol pretreatment on morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrification of in vitro matured porcine oocytes

This study was designed to determine the effects of Taxol pretreatment on the morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrified porcine oocytes matured in vitro. The result showed that: (1) the rate of normal mitochondria distribution in fresh group (92.85%) was significantly higher (P<0.05) than that in other three groups (toxicity, 72.48%; vitrification, 50.83%; Taxol+vitrification, 69.98%) and Taxol pretreatment significantly (P<0.05) increased the ratio of normal mitochondria distribution in vitrified oocytes; (2) lipid droplets in vitrified oocytes got cracked, resulting in a great number of smaller lipid droplets (diameter <5 microm). The number of lipid droplets (5-10 microm in diameter) in vitrified oocytes pretreated with Taxol was higher (P<0.05) than that in the oocytes without Taxol pretreatment (81.87+/-13.63 vs. 64.27+/-13.72); (3) both toxicity and vitrification cause the difference in the ultrastructure of mitochondria and lipid droplets. Mitochondria were well maintained in the form of typical round and ellipse shape with smooth surface and clear outline and lipid droplets existed in the form of integrity in Taxol pretreatment group. In conclusion, Taxol pretreatment has positive effects on vitrified porcine oocytes matured in vitro in terms of morphology, distribution and ultrastructure of mitochondria and lipid droplets.

Changes in acetylation on lysine 12 of histone H4 (acH4K12) of murine oocytes during maternal aging may affect fertilization and subsequent embryo development

OBJECTIVE To compare acH4K12 levels in oocytes during mouse aging and then assess how such changes might affect the developmental potential of oocytes. DESIGN Experimental animal study. SETTING State key laboratory and university research laboratory. ANIMAL(S) Kunming white strain mice. INTERVENTION(S) Oocytes obtained from TSA treated group or aging mouse group were fertilized and the formation of pronuclei and subsequently developmental potential in vitro or in vivo were assessed. MAIN OUTCOME MEASURE(S) AcH4K12 levels in oocytes were assessed using fluorescence staining, and confocal microscopy and oocyte developmental potentials were determined by in vitro or in vivo methods. RESULT(S) The AcH4K12 levels in oocytes statistically significantly increased during mouse aging. When histone acetylation of oocytes of young mice was artificially increased by trichostatin A (TSA) treatment, the acH4K12 levels in male and female pronuclei in fertilized oocytes showed statistically significant changes. About 38.9% of TSA-treated oocytes failed to form pronuclei or formed morphologically abnormal pronuclei 6 hours after fertilization, which statistically significantly decreased the blastocyst rate of TSA-treated oocytes when compared with the control group (41.5% vs. 60.5%). A similar reduction in blastocyst development was also observed when oocytes collected in older mice were compared with younger mice (17.3% vs. 69.4%). CONCLUSION(S) The AcH4K12 levels in oocytes statistically significantly increased during the aging process in mice, and such changes may affect the acetylation patterns and morphology of pronuclei during fertilization and lead to a reduction in oocyte developmental potential.

Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2-PN) embryos.

The present study was designed to investigate the effect of vitrification on mitochondrial distribution, membrane potential (Δψ) and microtubule distribution in mouse 2‐PN embryos, as well as to document the relationship between mitochondrial distribution and developmental ability of those embryos. Mitochondrial distribution was examined by fluorescence microscopy technology. Results indicated that: (1) The rate of mitochondrial ring formation around pronuclei in vitrified 2‐PN embryos was significantly lower than in fresh ones (67.3 ± 3.0% vs. 84.9 ± 3.1%) (P < 0.05). (2) Blastocyst development rate of vitrified 2‐PN embryos without mitochondrial rings (61.7 ± 4.5%) was significantly lower than that of vitrified embryos with mitochondrial rings (82.1 ± 2.8%). (3) Following staining by 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbo‐cyanine iodide (JC‐1), most red‐colored mitochondria (high Δψ) were distributed peripherally around pronuclei and along cell membranes of fresh 2‐PN embryos. Conversely, red‐colored mitochondria were greatly diminished in vitrified embryos, with green mitochondria (low Δψ) evenly distributed throughout the cytoplasm. The proportion of fresh 2‐PN embryos with obvious aggregation of high Δψ mitochondria (84.2 ± 2.2%) was significantly higher than that of vitrified embryos (26.7 ± 3.0%) (P < 0.05). (4) The proportion of fresh embryos with microtubules distributed around pronuclei (83.5 ± 3.4%) was similar to that of vitrified embryos (74.7 ± 2.5%). In conclusion, vitrification affected mitochondrial distribution and decreased the mitochondrial membrane potential in mouse 2‐PN embryos, events which may affect subsequent developmental viability of such embryos. Mol. Reprod. Dev. 76: 1056–1063, 2009.

OPS vitrification of mouse immature oocytes before or after meiosis: the effect on cumulus cells maintenance and subsequent development.

Cryopreservation can cause cumulus cell damage around the immature oocytes, which may result in poor subsequent development. To evaluate the effect of the meiosis stage on the cumulus cell cryoinjury and determine the suitable stage for cryopreservation in immature oocytes, mouse oocytes at germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages were vitrified using open pulled straw (OPS) method. Cumulus cells damage was scored immediately after thawing by double-fluorescent staining. The survival rate of the oocytes was evaluated and the subsequent development of oocytes was assessed through in vitro culture (IVC) and in vitro fertilization (IVF) separately. After vitrification, a higher proportion of cumulus cells of GV oocytes were damaged than those of GVBD and untreated control groups. The survival rate of vitrified GVBD oocytes (94.1%) was significantly higher (p < 0.05) than that of GV oocytes (85.4%). Oocytes vitrified at GVBD stage (55.7%) showed similar cleavage rate compared to those at GV stage (49.2%), but significantly higher (p < 0.05) blastocyst rate (40.9% vs. 27.4%). These results demonstrate that oocytes at GVBD stage remain better cumulus membrane integrity and developmental ability during vitrification than those at GV stage, indicating they are more suitable for immature oocytes cryopreservation in mice.

DNA methylation pattern in mouse oocytes and their in vitro fertilized early embryos: effect of oocyte vitrification

This study was conducted to investigate the pattern of DNA methylation in vitrified-thawed mouse oocytes and their in vitro fertilized early embryos. Firstly, mouse oocytes at metaphase II (MII) stage of meiosis were allocated randomly into three groups: (1) untreated (control); (2) exposed to vitrification solution without being plunged into liquid nitrogen (toxicity); or (3) vitrified by open-pulled straw (OPS) method (vitrification). Oocytes from all three groups were fertilized subsequently in vitro. The level of DNA methylation in the MII oocytes and their early embryos was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Developmental rates to 2-cell embryos (62.28%) and blastocysts (43.68%) of the vitrified-thawed oocytes were lower (P < 0.01) than those of fresh oocytes (81.47%, 61.99%) and vitrification solution treated (79.20%, 60.04%) oocytes. DNA methylation (as reflected by 5-MeC fluorescence intensity) in the vitrification group was less (P < 0.01) for MII oocyte and 2- to 8-cell stages compared with that in the control and toxicity groups. Accordingly, a reduction in global genomic methylation due to vitrification of MII oocytes may result in compromised in vitro developmental potential in early mouse embryos.

trans-10, cis-12 conjugated linoleic acid enhances in vitro maturation of porcine oocytes

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.

NUCLEAR MATURATION AND EMBRYO DEVELOPMENT OF PORCINE OOCYTES VITRIFIED BY CRYOTOP: EFFECT OF DIFFERENT STAGES OF IN VITRO MATURATION

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were cultured in IVM medium supplemented with 1mM dibutyryl cAMP (dbcAMP) for 22 h and then for an additional 22 h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0-22, 26, 32, 38 and 44 h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44 h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44 h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44 h, but significantly decreased survival rate of oocytes matured for 0 or 22 h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P<0.05) than that in the control. At 2h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44 h of IVM were similar but were higher (P<0.05) than those of oocytes vitrified at 0 or 22 h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38 h of IVM did not differ from the control and were higher (P<0.05) than those of oocytes vitrified at 22, 26 or 32 h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44 h did not differ, but all were lower (P<0.05) than those matured 0 h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.

The Extracellular Calcium-Sensing Receptor (CASR) Regulates Gonadotropins-Induced Meiotic Maturation of Porcine Oocytes

ABSTRACT Gonadotropins and epidermal growth factor (EGF) play crucial roles in promoting oocyte maturation. The regulatory network downstream of these key factors is not well understood. The present study was designed to investigate the role of the calcium-sensing receptor (CASR) in porcine oocyte in vitro maturation. CASR expression was up-regulated in oocytes matured in gonadotropin-containing medium. Cortical distribution of CASR was enhanced with gonadotropins but not EGF. Supplementation of a CASR agonist (NPS R-568) in the gonadotropin (FSH and/or LH)-containing maturation medium significantly enhanced oocyte nuclear maturation. Addition of NPS2390, a CASR antagonist, compromised oocyte nuclear maturation. Furthermore, increased cortical distribution and decreased expression of CASR was observed after the NPS R-568 treatment. Oocytes treated with NPS R-568 had higher concentration of CYCLIN B1, decreased reactive oxygen species, and increased glutathione levels, indicative of advanced cytoplasmic maturation. In contrast, NPS2390 treatment compromised oocyte cytoplasmic maturation. A higher blastocyst formation rate after parthenogenetic activation was observed when oocytes were matured in the presence of the CASR agonist, NPS R-568. MAPK3/1 phosphorylation was increased during in vitro maturation and after NPS R-568 treatment, and decreased following CASR antagonist supplementation. Taken together, our data showed that the CASR is a gonadotropin-regulated factor that promotes porcine oocyte maturation in a MAPK-dependent manner.