OBJECTIVEThis study was aimed to determine the effects of n-hexane on the maturation of mouse oocytes.

METHODSCell culture was used to observe the maturation of mouse oocytes and CLSM was employed to determine their apoptosis.

RESULTSGerminal vesicle breakdown (GVBD) and extrusion of the first polar body in mouse oocytes were significantly inhibited by n-hexane. After fertilization, the number of eggs in the mouse was significantly reduced by n-hexane. Mitochondrial membrane potentials (ΔΨm) were altered in mouse oocytes that were leading to apoptosis of the oocytes.

CONCLUSIONN-hexane might have affected the maturation of oocytes, causing alteration of ΔΨm and leading to apoptosis which maybe one of the most important mechanisms.

Animals ; Apoptosis ; drug effects ; Environmental Pollutants ; toxicity ; Female ; Fertilization ; drug effects ; Hexanes ; toxicity ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Mice, Inbred ICR ; Oocytes ; drug effects ; growth & development

OBJECTIVETo study the effect of epidermal growth factor(EGF) and different concentrations of gonadotrophin (Gn) on the in vitro maturation of human oocytes.

METHODSEGF was added to the vitro culture medium in order to observe the effect of Gn combined with or without EGF on the result of in vitro maturation. The concentrations of hCG and FSH were changed respectively to observe the difference between the results.

RESULTSAdding EGF to the culture medium improved the maturation rate of oocyte significantly (P < 0.05). There was no difference between the results with different concentrations of hCG and FSH in the culture medium.

CONCLUSIONEGF can improve the results of the in vitro maturation of human oocytes by increasing the maturation rate significantly. Increasing the concentration of Gn does not influence the results of in vitro maturation.

Cells, Cultured ; Dose-Response Relationship, Drug ; Epidermal Growth Factor ; pharmacology ; Female ; Fertilization ; drug effects ; Gonadotropins ; pharmacology ; Humans ; Oocytes ; drug effects ; physiology

Parthenogenetic activation is a procedure that an oocyte at meiosis II stage is activated into mitosis by some chemical or physical stimulation other than a sperm and the embryo is formed in the absence of any contribution from a male gamete. The activation of oocyte is the result of calcium ion oscillations and deactivation of some cytokines such as maturation promoting factor, mitogen-activated protein kinase and cytostatic factor. Parthenogenetic activation is artificially induced by various kinds of physical and/or chemical methods. The main activation method of human oocyte is chemical methods. The rates of activation and cleavage depend on the age, origin,and culture conditions of the oocyte.
Adenine ; analogs & derivatives ; pharmacology ; Animals ; Calcium ; metabolism ; Cycloheximide ; pharmacology ; Cytokines ; metabolism ; Female ; Humans ; Oocytes ; drug effects ; growth & development ; metabolism ; Parthenogenesis ; drug effects

This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blastocyst formation after PA was significantly increased by rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) relative to the control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM relative to untreated oocytes (20.3% vs. 14.3%, 0.05 < p < 0.1), while no improvement was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin relative to the control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs.
Animals ; Embryonic Development/*drug effects ; Female ; In Vitro Oocyte Maturation Techniques/veterinary ; Nuclear Transfer Techniques/*veterinary ; Oocytes/growth & development ; *Parthenogenesis ; Sirolimus/*pharmacology ; Sus scrofa/*growth & development/metabolism

Supplementation of beta-mercaptoethanol (beta-ME) in in vitro maturation (IVM) medium was shown to improve embryo development and quality in several species. Epidermal growth factor (EGF) was also shown to improve IVM of human oocyte and embryo development after in vitro fertilization (IVF). The effect of these two compounds were suggested to be mediated through the synthesis of glutathione (GSH) which is known to play an important role in protecting the cell or embryos from oxidative damage. Thus, it is suggested that supplementation of canine IVM medium with beta-ME or EGF may be of benefit due to its positive role in IVM of various mammalian oocytes and embryo development, including cattle, pigs, rodents and humans. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with beta-ME or EGF on IVM of canine oocytes. As results, a significantly higher percentage of oocytes progressed to metaphase II (MII) stage in 50 or 100 microM of beta-ME supplemented oocytes collected from the follicular stage. The maturation rate to metaphase I (MI) stage was also significantly higher in oocytes collected from follicular stage and cultured with 25 or 100 microM compared to other experimental groups. After IVM culture, oocytes recovered from dogs with the follicular stage and matured in TCM-199 supplemented with 20 ng/ml EGF yielded better oocyte maturation to MII phase compared to other groups. Taken together, supplementation of beta-ME (50 or 100 microM) or EGF (20 ng/ml) improved IVM of canine oocytes to MII stage.
Animals ; Benzimidazoles/chemistry ; Dogs/*physiology ; Epidermal Growth Factor/*pharmacology ; Estrus/*physiology ; Female ; Fluorescent Dyes/chemistry ; Meiosis/drug effects/physiology ; Mercaptoethanol/*pharmacology ; Microscopy, Ultraviolet/veterinary ; Oocytes/drug effects/growth&development/*physiology ; Ovary/drug effects/*physiology

OBJECTIVETo study the effects of the co-administration of growth hormone (GH) and aspirin to women with suboptimal response to GnRHa/FSH hyperstimulation protocol during in vitro fertilization and embryo transfer (IVF-ET) cycles.

METHODSForty cases of poor ovarian response in previous IVF-ET cycles were randomly divided into 2 groups: the studied group of GH and aspirin (n = 20), and the control group without GH or aspirin (n = 20).

RESULTSThe co-administration of GH and aspirin significantly increased the rates of retrieved oocytes (P < 0.01), promoted the maturation of oocytes (P < 0.01) and improve the fertilization rates (P < 0.05). However, there were no statistically differences between the two groups in the number of replaced embryos (P > 0.05) and the pregnancy rate (P > 0.05).

CONCLUSIONThe co-administration of GH and aspirin to poor ovarian responders is effective to increase the rates of retrieved oocytes, promote the maturation of oocytes and improve the fertilization rate in IVF-ET.

Aspirin ; administration & dosage ; Embryo Transfer ; Female ; Fertilization in Vitro ; Growth Hormone ; administration & dosage ; Humans ; Infertility, Female ; drug therapy ; physiopathology ; Oocytes ; cytology ; drug effects ; physiology ; Ovulation Induction ; Pregnancy ; Treatment Outcome

OBJECTIVETo investigate the sex chromosomes and the expression of insulin-like growth factor-II (IGF-II) on activated human unfertilized oocytes after intracytoplasmic sperm injection(ICSI) with calcium ionophore A23187 and puromycin.

METHODSAll 95 discarded oocyes that showed no evidence of fertilization at 16-18 h after in vitro maturation and intracytoplasmic sperm injection cycles (IVM-ICSI)/conventional ICSI were exposed to calcium ionophore A23187 (5 micromol/L) for 5 min and then were incubated with puromycin (10 microg/mL) for 4 h. After activation, the oocytes were cultured in vitro for 3-5 days. The sex chromosome analysis was performed by dual color fluorescence in situ hybridization. The expression of IGF-II on the activated embryos, normal embryos, and parthenotes was examined.

RESULTSThe combination of calcium ionophore A23187 with puromycin could activate the unfertilized oocytes 22 h after ICSI. The activated rate, cleavage rate, and quality of activated embryos of the IVM-ICSI group were similar to those of ICSI group, respectively. Sex chromosome analysis indicated that 8 male and 5 female embryos had been derived from two pronucleus and a second polar body. The expression of IGF-II on activated embryos and normal embryos was high and similar, which was much stronger than that of parthenotes.

CONCLUSIONThe combination of calcium ionophore A23187 with puromycin could effectively activate unfertilized oocytes 22 h after ICSI. Moreover, the unfertilized oocytes activated by calcium ionophore A23187 and puromycin had normal sex chromosomes and expression of IGF-II like the normal embryos. These suggest that oocyte activation may be considered as a remedial measure in the presence of total or nearly total fertilization failure in ICSI.

Calcimycin ; pharmacology ; Chromosomes, Human, X ; genetics ; Chromosomes, Human, Y ; genetics ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Insulin-Like Growth Factor II ; metabolism ; Ionophores ; pharmacology ; Oocytes ; cytology ; drug effects ; metabolism ; Puromycin ; pharmacology ; Sperm Injections, Intracytoplasmic ; methods

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 microm) were isolated by micro-dissection and cultured in 0 (control), 10(-3), 10(-5), 10(-7), and 10(-9) M SNP. To examine the reversible effect of SNP, PFs were cultured with 10(-5) M SNP + 1 mM Nomega-nitro-L-arginine methyl ester (L-NAME) or 1.0 microg hemoglobin (Hb). The results showed that greater concentrations of SNP (10(-3), 10(-5), 10(-7) M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10(-9) M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.
Animals ; *Apoptosis ; Buffaloes/*physiology ; Estradiol/biosynthesis ; Female ; Follicle Stimulating Hormone/metabolism ; NG-Nitroarginine Methyl Ester/pharmacology ; Nitrates/pharmacology ; Nitric Oxide/*metabolism ; Nitric Oxide Donors/pharmacology ; Nitrites/pharmacology ; Nitroprusside/pharmacology ; Oocytes/cytology/drug effects/growth & development/metabolism ; Ovarian Follicle/*cytology/drug effects/growth & development/*metabolism ; Progesterone/biosynthesis

For parthenogenetic activation as a model system of nuclear transfer, microinjection and electroporation as activation treatments in bovine metaphase II oocytes were administered to each of three groups as follows: control group (treatments with Ca2+, Mg2+ -free PBS+100 micro M EGTA), IP3 group (control+25 micro M IP3) and IP3+ ryanodine group (control+25 micro M IP3+10 mM ryanodine). In experiments using microinjection, no significant differences were observed between any of the developmental stages of the electroporation experiment. For electroporation, cleavage rates were significantly higher in the IP3+ryanodine group than in the IP3 or control group (85.6% vs 73.7% or 67.6%, respectively). In the subsequent stages of embryonic development, such as morula and blastocyst formation, the IP3 and ryanodine group exhibited significantly higher rates of morula fomation than the IP3 or control groups (40.6% vs 24.2% or 16.7%, respectively). Similarly, the rate of blastocyst formation in the IP3+ryanodine group was significantly higher than the control group (16.3% vs 6.9%) but did not differ significantly from the IP3 group (16.3% vs 9.5%). In nuclear transfer, activation was performed at 30 hpm by microinjection and elecroporation with 25 micro M IP3+ 10 mM ryanodine followed by 6-DMAP treatment. No significant differences were observed at any stage of embryonic development and none of the embryos activated by electroporation reached either the morula or blastocyst stage. However, 3.8% and 1.9% of embryos activated by microinjection sucessfully developed to the morula and blastocyst stages, respectively. In conclusion, activation treatments using IP3 and ryanodine are able to support the development of bovine parthenogenetic and reconstructed embryos.
Adenine/administration & dosage/*analogs & derivatives/pharmacology ; Animals ; Cattle/*embryology/physiology ; Cell Fusion ; Electroporation/veterinary ; Embryonic and Fetal Development/*drug effects ; Enzyme Inhibitors/administration & dosage/pharmacology ; Female ; Inositol 1,4,5-Trisphosphate/administration & dosage/*pharmacology ; Microinjections/veterinary ; Nuclear Transfer Techniques ; Oocytes/drug effects/growth & development ; Parthenogenesis/*drug effects ; Protein Kinase Inhibitors ; Ryanodine/administration & dosage/*pharmacology ; Skin/cytology

OBJECTIVETo evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level.

METHODSImmature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level.

RESULTSIn dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 micromol/L SOV and control groups (P < 0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 micromol/L. In incontinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 micromol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P < 0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed.

CONCLUSIONDynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or M I to anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.

Animals ; Cells, Cultured ; Cyclin B ; genetics ; metabolism ; Cyclin B1 ; Dyneins ; antagonists & inhibitors ; Female ; Gene Expression Regulation ; Meiosis ; drug effects ; Mice ; Mice, Inbred BALB C ; Oocytes ; drug effects ; growth & development ; metabolism ; RNA, Messenger ; analysis ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Vanadates ; pharmacology