Guang-Bin Zhou

Melatonin exists in porcine follicular fluid and improves in vitro maturation and parthenogenetic development of porcine oocytes.

Abstract:  This study focused on the effect of melatonin on in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Melatonin was measured in porcine follicular fluid of follicles of different sizes in the same ovary. Melatonin exists in follicular fluid, and the concentration is approximately 10−11 m. Its concentration decreased as the diameter of follicle increased, which suggests an effect of melatonin on oocyte maturation. Therefore, immature oocytes were cultured in vitro in maturation medium supplemented with melatonin (10−11, 10−9, 10−7, 10−5 and 10−3 m) or without melatonin. The oocytes at maturation stage were collected and activated. The parthenogenetic embryos were cultured and observed in medium supplemented with or without melatonin. Fresh immature oocytes without melatonin treatment were used as control. When only maturation medium was supplemented with 10−9 m melatonin, the cleavage rate, blastocyst rate and the cell number of blastocyst (70 ± 4.5%, 28 ± 2.4% and 50 ± 6.5%) were significantly higher (P < 0.05) than that of controls; when only culture medium was supplemented with melatonin, the highest cleavage rate, blastocyst rate and the cell number of blastocyst was observed at 10−7 m melatonin, which were significantly higher than that of controls (P < 0.05). The best results (cleavage rates 79 ± 8.4%, blastocyst rates 35 ± 6.7%) were obtained when both the maturation and culture medium were supplemented with 10−9 m melatonin respectively (P < 0.05). In conclusion, exogenous melatonin at the proper concentration may improve the in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Further research is needed to identify the effect of melatonin on in vitro and in vivo oocyte maturation and embryo development in porcine.

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.

Effects of Melatonin on In Vitro Development of Mouse Two-Cell Embryos Cultured in HTF Medium

Melatonin is capable of improving the developmental capacity of ovine, porcine and bovine embryos in vitro. However, whether melatonin possesses similar benefits to the in vitro mouse embryonic development has yet to be determined. In this study, we assessed the effects of various concentrations of melatonin (10–13 to 10–3 M) on the in-vitro development of mouse embryos cultured in HTF medium for 96 hr; embryos cultured without melatonin were used as control. The in vitro development of mouse two-cell embryos significantly benefited from treatment with melatonin in a concentration-dependent manner. The effects of melatonin on the rates of blastocyst formation, hatching/hatched blastocysts and cell number per blastocyst were bi-phasic; all significantly increased by melatonin at 10–13 to 10–5 M and decreased by melatonin at 10–3 M. Maximal benefit of melatonin on in vitro mouse 2-cell embryo development was achieved at a concentration of 10–9 M. In comparison to control, 10–9 M melatonin increased blastocyst formation rate from 48.08 ± 5.25% to 82.08 ± 2.34% (p < 0.05), hatched blastocyst rate from 25.65 ± 11.79% to 66.47 ± 4.94% (p < 0.05), and cell number per blastocyst 62.71 ± 5.97 to 77.91 ± 10.63 (p < 0.05). Thus, our datas demonstrated firstly that melatonin has beneficial effects on the in vitro development of 2-cell mouse embryos cultured in HTF medium.

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.

Positive effects of Forskolin (stimulator of lipolysis) treatment on cryosurvival of in vitro matured porcine oocytes

In order to examine its effect on oocyte lipid content and cryosurvival, Forskolin was added to the medium for in vitro maturation of porcine oocytes. Treatments were control (IVM without Forskolin during the 42 h incubation period), addition of 10 μM Forskolin for the entire 42 h (0-42) and addition of 10 μM Forskolin between 24 and 42 h only (24-42). In Experiment 1, treatments did not differ significantly in cleavage rate, but the blastocyst formation rate was lower in the 0-42 group than for control and 24-42 group oocytes (17, 32 and 40%, respectively; P < 0.05). It was shown in Experiment 2 that Forskolin treatment from 0-42 h and from 24-42 h significantly reduced lipid content of oocytes compared to that of control cells (65 and 99 vs. 140 μm(2) intensity of fluorescence, respectively; P < 0.05). In Experiment 3, the percentage of oocyte survival after cryopreservation and thawing was significantly higher in both Forskolin treatment groups than in control oocytes (72% for 0-42, 65% for 24-42 and 52% for control; P < 0.05). However, Forskolin treatment did not increase cleavage rates of vitrified in vitro matured porcine oocytes (Control group 28%, 0-42 h group 0%, 24-42 h group 26.67%). Addition of Forskolin affected the nuclear maturation of porcine oocytes. The percentage of PBE (polar body extrusion) were significantly reduced in the 0-42 h group (0-42 h group 42.00 ± 2.08 vs. Control group 79.70 ± 2.82 and 24-42 h group 70.60 ± 2.83; P < 0.05). The 24-42 h group showed similar nuclear status to that of the Control group. We propose that delipation engendered by incubation with 10 μM Forskolin during 24-42 hours of maturation increased cryosurvival of in vitro-maturated porcine oocytes and that attendant chemical lipolysis did not impair their further development as it may have done in oocytes incubated with Forskolin for the full 42 h.

Conventional Freezing, Straw, and Open-Pulled Straw Vitrification of Mouse Two Pronuclear (2-PN) Stage Embryos

Little is known on the cryopreservation of mouse pronuclear (PN) stage embryos. In the present experiment the mouse 2-PN stage embryos were cryopreserved by conventional freezing, straw, or open-pulled straw (OPS) vitrificaiton methods. The conventional freezing solution was 1.5 mol/L ethylene glycol (EG), and vitrification solutions were EFS30 (30% EG, Ficoll, and sucrose), EFS40 (40% EG, Ficoll, and sucrose), EDFS30 (15% EG, 15%dimethyl sulfoxide [DMSO], Ficoll, and sucrose), or EDFS40 (20% EG, 20%DMSO, Ficoll, and sucrose). The blastocyst rate of 2-PN stage embryos cryopreserved by conventional method (30.4%) was lower than those vitrified by straw method with EDFS (56.9% to 69.1%), by OPS method (66.0% to 85.7%), and that of control (80.8%) (P < 0.05). With a given vitrificaiton solution EFS30, EFS40, EDFS30, or EDFS40, the blastocyst rate of embryos vitrified by the OPS method (66.7%, 66.0%, 85.7%, or 76.9%) was higher than that of those vitrified by the straw method (46.8%, 43.8%, 69.1%, or 56.9%) (P < 0.05). When mouse 2-PN-stage embryos were vitrified with EDFS30 by straw or OPS method, the highest blastocyst rate was achieved (69.1% or 85.7%) and was similar to that of the control, respectively. The embryos transfer results revealed that the full-term development of blastocysts derived from 2-PN stage embryos vitrified by OPS method with EDFS30 (19.9%) was similar to that of the control (23.5%), and higher than that of those cryopreserved by conventional freezing (9.3%) (P < 0.05). The present research demonstrates that the OPS method, especially with EDFS30, is more effective in cryopreserving mouse 2-PN embryos.

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.

Quantitative Investigations on the Effects of Exposure Durations to the Combined Cryoprotective Agents on Mouse Oocyte Vitrification Procedures

Vitrification by using two-step exposures to combined cryoprotective agents (CPAs) has become one of the most common methods for oocyte cryopreservation. By quantitatively examining the status of oocytes during CPA additions and dilutions, we can analyze the degree of the associated osmotic damages. The osmotic responses of mouse MII oocyte in the presence of the combined CPAs (ethylene glycol, EG, and dimethyl sulfoxide, DMSO) were recorded and analyzed. A two-parameter model was used in the curve-fitting calculation to determine the values of hydraulic conductivity (Lp) and permeability (Ps) to the combined CPAs at 25°C and 37°C. The effects of exposure durations and the exposure temperatures on the cryopreservation in terms of frozen-thawed cell survival rates and subsequent development were examined in a series of cryopreservation experiments. Mouse MII oocytes were exposed to pretreatment solution (PTS) and vitrification solution (VS) at specific temperatures. The PTS used in our experiment was 10% EG and 10% DMSO dissolved in modified PBS (mPBS), and the VS was EDFS30 (15% EG, 15% DMSO, 3 × 10−3 M Ficoll, and 0.35 M sucrose in mPBS).The accumulative osmotic damage (AOD) and intracellular CPA concentrations were calculated under the different cryopreservation conditions, and for the first time, the quantitative interactions between survival rates, subsequent development rates, and values of AOD were investigated.

Open-pulled Straw (OPS) Vitrification of Mouse Hatched Blastocysts

This study was first employed to investigate the developmental potential of mouse hatched blastocyts (HBs) vitrified by a two-step open-pulled straw (OPS) method. HBs were obtained by culture of morulae in vitro. First, the embryos were placed in four cryprotectant solutions—that is, 10% ethylene glycol (EG), 10%E + 10%D (10% EG and 10% dimethyl sulphoxide (DMSO) in mPBS), EFS30 (30% EG, Ficoll, and sucrose) and EDFS30 (15% EG, 15% DMSO, Ficoll, and sucrose)—at 25°C for 0.5 to 10 min, respectively, to determine their optimal survival after rapid dilution in 0.5 M sucrose. Secondly, based on the above best survival, the embryos were plunged into liquid nitrogen after first pretreatment in 10%E for 0.5 min and then 0.5 min equilibration in EFS30 (Group 1), or 10%E + 10%D and EDFS30 for 0.5 min, respectively (Group 2). When warming, three methods were used to dilute the cryoprotectants from the vitrified embryos. The embryos were assessed by the re-expansion of the blastocoel or development to term. The result showed that all the vitrified-warmed HBs got high in vitro survival rates (83.7% to 98.9%). The highest in vitro survival rates (87.8% in Group 1, 98.9% in Group 2) were obtained when the vitrified embryos were diluted first in 0.3 M sucrose for 5 min, then in 0.15 M sucrose for 2 min (method C). When the vitrified embryos diluted with method C were transferred, their survival rate in vivo (35.5% to 42.2% of the total) were similar to (P > 0.05) that of control (45.7%). These results demonstrate OPS method was highly efficient for the cryopreservation of mouse HBs.