Background:Irreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation.Antioxidants were always used to antagonist the oxidative stress caused by vitrification....Background:Irreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation.Antioxidants were always used to antagonist the oxidative stress caused by vitrification.However,the comprehensive mechanism underlying the protective role of antioxidants has not been studied.Procyanidin B2(PCB2)is a potent natural antioxidant and its functions in response to vitrification are still unknown.In this study,the effects of PCB2 on vitrified-thawed oocytes and subsequent embryo development were explored,and the mechanisms underlying the protective role of PCB2 were systematically elucidated.Results:Vitrification induced a marked decline in oocyte quality,while PCB2 could improve oocyte viability and further development after parthenogenetic activation.A subsequent study indicated that PCB2 effectively attenuated vitrification-induced oxidative stress,rescued mitochondrial dysfunction,and improved cell viability.Moreover,PCB2 also acts as a cortical tension regulator apart from strong antioxidant properties.Increased cortical tension caused by PCB2 would maintain normal spindle morphology and promote migration,ensure correct meiosis progression and finally reduce the aneuploidy rate in vitrified oocytes.Further study reveals that ATP biosynthesis plays a crucial role in cortical tension regulation,and PCB2 effectively increased the cortical tension through the electron transfer chain pathway.Additionally,PCB2 would elevate the cortical tension in embryo cells at morula and blastocyst stages and further improve blastocyst quality.What's more,targeted metabolomics shows that PCB2 has a beneficial effect on blastocyst formation by mediating saccharides and amino acids metabolism.Conclusions:Antioxidant PCB2 exhibits multi-protective roles in response to vitrification stimuli through mitochondria-mediated cortical tension regulation.展开更多
Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and...Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.Methods Oocytes were treated with different concentrations of ZEA(3,5,8 and 10μmol/L)and isorhamnetin(5,10,20 and 30μmol/L)for 44 h at 39℃.ZEA(5μmol/L)and isorhamnetin(10μmol/L)were selected for subsequent studies.Polar body exclusion rate,apoptosis rate and apoptosis related proteins,ROS levels and SOD2 protein,mitochondrial membrane potential and distribution,endoplasmic reticulum distribution and proteins expression,and PI3K,Akt and p-Akt proteins expression of oocytes were detected.In addition,the effect of PI3K antagonist(LY294002)on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.Results Our findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes.Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes.Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production.Moreover,isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA.Changing the expression of endoplasmic reticulum stress-related marker proteins(CHOP,GRP78)and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress.Mechanistically,isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.Conclusions Collectively,these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway,which enhances meiotic maturation and mitochondrial function,and inhibits early apoptosis,oxidative stress and endoplasmic reticulum stress in porcine oocytes.Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.展开更多
Background:Elevated ambient temperature-caused heat stress is a major concern for livestock production due to its negative impact on animal feed intake,growth,reproduction,and health.Particularly,the germ cells are ex...Background:Elevated ambient temperature-caused heat stress is a major concern for livestock production due to its negative impact on animal feed intake,growth,reproduction,and health.Particularly,the germ cells are extremely sensitive to the heat stress.However,the effective approach and strategy regarding how to protect mammalian oocytes from heat stress-induced defects have not been determined.Methods:Germinal vesicle(GV)porcine oocytes were cultured at 41.5℃ for 24 h to induce heat stress,and then cultured at 38.5℃ to the specific developmental stage for subsequent analysis.Nicotinamide mononucleotide(NMN)was dissolved in water to 1 mol/L for a stock solution and further diluted with the maturation medium to the final concentrations of 10μmol/L,20μmol/L,50μmol/L or 100μmol/L,respectively,during heat stress.Immunostaining and fluorescence intensity quantification were applied to assess the effects of heat stress and NMN supplementation on the key processes during the oocyte meiotic maturation.Results:Here,we report that NMN supplementation improves the quality of porcine oocytes under heat stress.Specifically,we found that heat stress resulted in oocyte maturation failure by disturbing the dynamics of meiotic organelles,including the cytoskeleton assembly,cortical granule distribution and mitochondrial function.In addition,heat stress induced the production of excessive reactive oxygen species(ROS)and DNA damage,leading to the occurrence of apoptosis in oocytes and subsequent embryonic development arrest.More importantly,we validated that supplementation of NMN during heat stress restored the meiotic defects during porcine oocyte maturation.Conclusions:Taken together,our study documents that NMN supplementation is an effective approach to improve the quality of oocytes under heat stress by promoting both nuclear and cytoplasmic maturation.展开更多
[Objectives] This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer( JIVET). [Methods]Ten Dorper × small-tailed ...[Objectives] This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer( JIVET). [Methods]Ten Dorper × small-tailed Han lambs aged 5 to 10 weeks were induced to superovulate via i. p. injection of pregnant mare's serum gonadotropin( PMSG). The oocytes were matured in basal maturation solution or modified maturation solution,which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then,the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum( ESS) or fertilization medium II containing 3 mg/ml bull serum albumin( BSA). Finally,the number of oocytes,oocyte maturation rate and cleavage rate of the lambs of different ages were determined. [Results]The average number of oocytes recovered per lamb was( 111. 00 ± 16. 97),( 139. 50 ± 28. 99),( 108. 50 ± 17. 68) and( 42. 00 ± 11. 31) for5-,7-,8-and 10-week-old Dorper × small-tailed Han lambs,respectively. The number of oocytes obtained from 5-,7-and 8-week-old lambs was significantly higher than that from 10-week-old lambs( P < 0. 05),but there was no significant difference among 5-,7-and 8-week-old lambs( P > 0. 05). The maturation rate of oocytes cultured in modified maturation solution was 3. 64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II( P < 0. 01). [Conclusions] The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks,adding a certain amount of cysteine into oocyte maturation solution,and a certain amount of ESS into fertilization medium.展开更多
Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical f...Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 μs, with a blastocyst development rate of (20.12 ± 8.18)% (P ＞ 0.05). No significant difference was found between treatments of multiple pulses and a single pulse ( P ＞ 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35)% ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) %, P ＞ 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 ± 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79,P ＜ 0.01 ). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2 (5 % CO2:7%O2:88%N2) also showed no significant difference from those in high O2 (5% CO2 in air) [ (20.78 ± 8.80) % and 17.00 ± 6.12 vs. (25.30 ± 7.55) % and 18.01 ± 6.79, P ＞ 0.05 ]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.展开更多
Objective:To explore the effects of different MⅡstage oocytes zona pellucida birefringence on pregnancy outcome.Methods:A total of 46 couples with infertile which induced by single cause received in-vitro fertilizati...Objective:To explore the effects of different MⅡstage oocytes zona pellucida birefringence on pregnancy outcome.Methods:A total of 46 couples with infertile which induced by single cause received in-vitro fertilization treatment were analyzed retrospectively,and randomly divided into the high zona birefringence(HZB)/HZB group.HZB/low zona birefringeuce(LZB) group and LZB/LZB group according to different oocytes zona pellucida birefringence.Intracytoplasmic sperm injection outcome was analyzed and compared.Results:The proportion of HZB oocytes, implantation rate and the pregnancy rate were decreased in three groups(HZB/HZB group】HZB/ LZB group】LZB/LZB group)(P【0.05).But there was no significantly different between the number of oocytes and fertilization rate of these groups(P】0.05).Logistic regression analysis showed that factors allecl MⅡstage oocytes zona pellucida birefringence were age.basal FSH level and the LH level on the day of HCG injection.Age and KSH levels were negatively correlated with the single oocyte zona pellucida birefringence:While the LH level on the day of hCC injection was positively correlated with the single oocylc zona pellucida birefringence.Conclusions:The primary influence factors on MⅡstage oocytes zona pellucida are age.basal FSH level and the LH level on the day of hCG injection.The birefringence value of zona pellucida can affect the pregnancy outcome.展开更多
Purpose: Evaluate the effect of preincubation of oocytes prior to IVF or ICSI cycles with embryo transfer at blastocyst stage. Methods: Retrospective non randomized study based on secondary analysis of data. Setting: ...Purpose: Evaluate the effect of preincubation of oocytes prior to IVF or ICSI cycles with embryo transfer at blastocyst stage. Methods: Retrospective non randomized study based on secondary analysis of data. Setting: Laboratory of Assisted Reproduction at the Alcivar Hospital. Patients: One hundred-eighteen cycles of IVF and ICSI were analyzed in the present study. The evaluated groups were formed for those patients whose oocytes, after retrieval, were inseminated at 1-3 h (Group I) or 4-6 h (Group II). Results: There was no difference in fertilization rate (83.6% and 78.1%), Day 3 cleavage rate (95.1% and 97.1%), and blastocyst formation (31.1% and 39.1%) for groups I and II respectively. Clinical pregnancy rates (PR: 53.0% vs 22.9%) and implantation rates (IR: 38.1% vs 13.0%) were significantly higher in group II versus group I, respectively (P < 0.05). Conclusions: Preincubation of oocytes before insemination is a factor which raises the PR and IR after the blastocyst transfer.展开更多
Objective:To establish a new way to collect superovulated oocytes or zygotes repeatedlyfrom an individual mouse.Methods:Superovulations were induced by injection PMSG and hCG in Kunming strainmice.The ampullaes of ovi...Objective:To establish a new way to collect superovulated oocytes or zygotes repeatedlyfrom an individual mouse.Methods:Superovulations were induced by injection PMSG and hCG in Kunming strainmice.The ampullaes of oviduct of all anaesthetised mouse were put in a specially designed'U'sink and released.The second and third times of PMSG injection were made on the sixth day andeleventh day after the first superovulation injection.The capacity of development was examinedby in vitro culture of parthenogenesis activation oocytes.Results:Development to blastocyst stage was not significantly different between the first andsecond time collection.The percentage of blastocyst stage in CD and Sr^(++) treatment was signifi-cantly higher(P<0.05)than the oocytes treated in CB and Sr^(++).Conclusion:This method enables us to collect oocytes or zygotes repeatedly from one individ-ual mouse in an interval as short as 5 days and without influence on the quality of oocytes.展开更多
Background: In order to assess the chromosomal status in embryos obtained from vitrified and fresh donated oocytes, preimplantational genetic diagnostic (PGD) was performed after biopsy of one blastomere at day 3. MET...Background: In order to assess the chromosomal status in embryos obtained from vitrified and fresh donated oocytes, preimplantational genetic diagnostic (PGD) was performed after biopsy of one blastomere at day 3. METHODS: A total of 249 oocytes were obtained from 23 oocyte donors, 80 oocytes were used in the vitrified group and 151 oocytes were used in the fresh group. Nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) were investigated by fluorescence in situ hybridization (FISH) analysis in 56 and 121 embryos from vitrified and fresh group respectively. Fertilization, cleavage rate, embryo quality and chromosomal abnormality rate were compared between groups evaluated. Results: Vitrified oocytes showed a survival rate of 97.5%. There was no significant difference in the fertilization rate (82.7% and 91.4%), Day 2 cleavage rate (90.3% and 87.7%) or blastocyst formation rate (31.1% and 44.6%) for the vitrified and fresh groups respectively. Chromosomal abnormality rate (66.1% versus 71.9%), percentage of abnormal blastocysts (61.1% versus 64.8%) and percentage of abnormalities for each analyzed chromosome were similar for the vitrified group compared with the control group. Conclusions: The rates of chromosomal abnormalities in embryos from vitrified oocytes are similar to those published previously;and comparable to those observed in embryos from fresh oocytes. These results confirm that the developmental competence and chromosomal status of embryos obtained from vitrified oocytes is not affected by the vitrification procedure, and they preserve the potential to be fertilized and to develop in to blastocyst stage similar to embryos from fresh oocytes.展开更多
Objective:To evaluate changes of feline(Felis catus)oocytes proteins during in vitro maturation by using the proteomic approach.Methods:Immature oocytes(germinal vesicle)isolated from female cats were cultured and col...Objective:To evaluate changes of feline(Felis catus)oocytes proteins during in vitro maturation by using the proteomic approach.Methods:Immature oocytes(germinal vesicle)isolated from female cats were cultured and collected at 0 h and 24 h.After collection,oocytes were investigated into immature(germinal vesicle)and mature(metaphaseⅡ)stages.The qualitative profiles of the proteins at the immature and mature stages were determined by one-dimensional electrophoresis and liquid chromatography-mass spectrometry.Results:Our data revealed that following 24 h in vitro maturation the maturation rate(metaphaseⅡstage)was 58.7%.Eighty-one of the 260 proteins analyzed were differentially expressed between the germinal vesicle stage and the metaphaseⅡ-arrest stage.Proteomic analysis of germinal vesicle and metaphaseⅡoocytes showed abundant expression of proteins involved in transportation(10%),indicating that this was a major characteristic of germinal vesicle oocytes.Similarly,analysis of the proteome of metaphaseⅡoocytes indicated that cell cycle proteins were overexpressed.Interestingly,proteins involved in DNA repair and apoptosis were only expressed in germinal vesicle oocytes and proteins involved in fertilization were only expressed in metaphaseⅡoocytes.Conclusions:The overexpression of certain proteins in germinal vesicle and metaphaseⅡis necessary for oocyte development and maturation.Our findings provide a valuable resource for further investigations into protein expression in oocytes at different developmental stages.展开更多
Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inw...Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inward current in the injected oocytes wa展开更多
Introduction: The roles of genetic, epigenetic, metabolic and other environmental factors such as nutrition and stress, are becoming evident for a successful and healthy pregnancy. This raises the possibility and ques...Introduction: The roles of genetic, epigenetic, metabolic and other environmental factors such as nutrition and stress, are becoming evident for a successful and healthy pregnancy. This raises the possibility and question, if and how, we improve the probability of pregnancy and of a healthy fetus? The present study examined the role of metabolic, antioxidant and minerals and the results suggest that these factors may positively influence the oocyte quality and the pregnancy rate. Methods: CD1 female mice aged 15 and 5 weeks were divided into four groups of ten each and treated by intragastric gavage daily for 3 weeks. G1: Vehicle;G2: Carnitines (L-carnitine 0.4 mg and acetyl-L-carnitine 0.12 mg/mouse);G3: Microelements (Zinc 4 ng, Copper 0.8 ng, Iron 7 ng/mouse);G4: G3+G2. At the end of the treatment period superovulation was induced and oocytes were collected to assess their quantity and quality. Further, in vitro fertilization (IVF) experiments were performed to assess the preimplantation embryo development. The birth success rate was also analyzed in old and young female. The mice were in vivo fertilized. qRT-PCR were performed to analyze a possible modulation in key genes of the reproductive process. Results: The number of oocytes was significantly higher in groups 2 and 4 compared to the control group. The oocyte number in group 3 was not affected. The level of degraded oocytes was 29.1% and 19.3% (group 2 and 4) versus 34.3% (control). Concomitantly, the numbers of embryos arriving to successful birth were also increased in G4, both in the old and young group of mice. Preliminary analysis of genes affected evidenced that AMH was up regulated in the ovary and KITL in the uterus in group 2. Conclusion: Results showed that L-carnitine, acetyl-L-carnitine and micronutrients were able to improve both oocytes quality and success rate of pregnancy. Further studies are planned to further examine ways to improve pregnancy and fetal health.展开更多
Parthenogenesis is a form of asexual reproduction found in females, where growth and development of embryos occurs without fertilization by a male. Parthenogenesis occurs naturally in aphids, Daphnia, rotifers, nemato...Parthenogenesis is a form of asexual reproduction found in females, where growth and development of embryos occurs without fertilization by a male. Parthenogenesis occurs naturally in aphids, Daphnia, rotifers, nematodes and some other invertebrates but can also be induced efficiently in mammalian oocytes by providing appropriate stimuli invitro. Recently, parthenogenesis has attracted wide attention because of the role of activated oocytes in the field of research that have been described such as intra cytoplasmic sperm injection, cloning by nuclear transfer, somatic cell cloning, investigating culture conditions etc. & potential for deriving pluripotent stem cell lines and their differentiation into various cell lines that can be utilized for various tissue engineering applications. The parthenogenetically activated oocytes possess maternal genome and can developed in to either haploid, diploid or polyploidy embryos with the help of it we can analyze the possible role of all the genes involved in imprinting processes as well as the role the paternal genome plays during early embryo development by comparing them with fertilized embryos. Several methods are able to induce parthenogenetic activation through the elevation of cytoplasmic free calcium in oocytes. But one common, universal method or activation agents has not been developed for all species because the process is highly specific for each species. Therefore, activation step for each species need to be optimized accordingly. This review describes the general method of activation of mammalian oocytes and their genomic imprinting analysis.展开更多
The present work was designed to examine the effect of the presence or absence of cumulus cells on the efficiency of vitrification of immature cattle oocytes. In our experiment, we had two groups: group 1, immature ca...The present work was designed to examine the effect of the presence or absence of cumulus cells on the efficiency of vitrification of immature cattle oocytes. In our experiment, we had two groups: group 1, immature cattle oocytes with cumulus cells and group 2, immature cattle oocytes without cumulus cells. The two groups underwent vitrification using 20% ethylene glycol and 20% DMSO, and then thawed, and in vitro matured in TCM-199 medium and examined after 22 hours for assessment of nuclear maturation. Higher survival rate (p < 0.05) after thawing was observed in group 1 (84.6%) than group 2 (57.8%). After in-vitro maturation, the rate of MII oocytes was significantly higher (p < 0.05) in group 1 (74.4%) than group 2 (47.7%). In conclusion, the cumulus cells are very important in increasing the survivability and developmental rate of vitrified-thawed immature cattle oocytes.展开更多
Objective: To investigate the gonadotrophin drug Luveris? (L) and Gonal-F? (G) effects on bovine immature oocyte in vitro maturation culture. Methods: In total, 2000 bovine cumulus-oocyte complexes (COCs) were purchas...Objective: To investigate the gonadotrophin drug Luveris? (L) and Gonal-F? (G) effects on bovine immature oocyte in vitro maturation culture. Methods: In total, 2000 bovine cumulus-oocyte complexes (COCs) were purchased commercially and cultured in media with ovulation stimulation drugs Gonal-F and Luveris (0, 5, 10, 20, 40 IU/mL), individually, for maturation in vitro. 2000 Oocytes were divided evenly into two groups, Luveris and Gonal-F groups, individually, 1000 Oocytes. After 24 and 48h culture, cumulus expansion was assessed under dissecting microscopes. Oocyte maturation (GV, MI, MII) was determined by examining nuclear maturation and polar body formation under inverted microscopes. Results: Luveris and Gonal-F enhanced oocyte maturation in vitro in a dose-dependently manner. Culture media supplemented with L or G significantly improved MⅡ Oocyte maturation rates after culture for 24 h and 48 h in comparison with the control group. Oocyte maturation rates were 33.3% and 37.5%(20 IU/mL), individually, 33.3% and 53.6% (40 IU/mL);20 IU/mL G was 15.1% and 16.2%;40 IU/mL G was 38.9% and 39.5%, respectively. Conclusion: LH (Luveris) can promote prophase bovine immature oocytes developmental competence in vitro, which is mostly likely stimulated by FSH(Gonal-F ). Furthermore, LH can delay oocytes GVBD that is very useful for IVF clinical medication guidance.展开更多
The clomiphene citrate (CC), a nonsteroidal triphenylethylene compound, is a first line of medicine used for the induction of ovulation in anovulatory women worldwide. In spite of high ovulation induction with the use...The clomiphene citrate (CC), a nonsteroidal triphenylethylene compound, is a first line of medicine used for the induction of ovulation in anovulatory women worldwide. In spite of high ovulation induction with the use of CC, the pregnancy rate is much lower. Such a discrepancy could be due to the peripheral anti-estrogenic effect of CC, particularly at the level of ovary, endometrium and cervical mucus. CC induces ovulation by binding to the estrogen receptors and generates hypoestrogrnic state in hypothalamus leading to release of pituitary gonadotropins. CC may have a direct effect at the level of ovary but the molecular mechanism remains unclear. Animal studies suggest that the CC induces apoptosis in granulosa cells and results hypoestrogenic state in the ovary. Reduced estradiol 17β level in the ovary affects development and maturation of oocyte leading to oocyte apoptosis. Further, CC increases hydrogen peroxide (H2O2) level and thereby bax protein expression and DNA fragmentation in cumulus-granulosa cells as well as in oocytes. The exogenous supplementation of either estradiol 17β or melatonin reduces H2O2 level in ovary, delays meiotic cell cycle progression in oocyte and protects oocyte apoptosis. Hence, supplementation of estradiol 17β or melatonin along with CC could be beneficial to protect granulosa cell as well as oocyte apoptosis and inhibit deterioration of oocyte quality. Thus, maintenance of oocyte quality may overcome the adverse effect caused due to CC treatment during infertility management.展开更多
Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chro...Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.展开更多
Starfish oocytes with intact germinal vesicles (GVs) were cut along desired planes with glass needles or ligated using silk thread loops into two parts and allowed to mature in vitro, and inseminated. The experimental...Starfish oocytes with intact germinal vesicles (GVs) were cut along desired planes with glass needles or ligated using silk thread loops into two parts and allowed to mature in vitro, and inseminated. The experimental results showed that (1) only the parts with GVs or partial GV contents (PGVCs) cleaved, those without any GV materials did not; but nucleated and non-nucleated fragments cut from mature eggs were able to divide; (2) the development of animal parts of oocytes containing GVs or PGVGs was like that of animal fragments of matured oocytes with female pronuclei; most of them gave rise to permanent blastulae. and just a few formed ectodermal vesicles with a little primary mesenchyme; (3) a large part of vegetal fragments with GVs or PGVCs. and the vegetal parts of mature eggs without female pronuclei developed into small but normal embryos; (4) the fragments containing GVs or PGVCs obtained from the oocytes along a plane parallel to the animal-vegetal (A-V) axis developed as normally as the展开更多
We studied the effect of different storage conditions of porcine ovaries (time, temperature) on the characteristics of the follicular fluid, immature oocyte quality, meiotic competence and in vitro fertilization of oo...We studied the effect of different storage conditions of porcine ovaries (time, temperature) on the characteristics of the follicular fluid, immature oocyte quality, meiotic competence and in vitro fertilization of oocytes. Ovaries were stored for 2, 4 or 6 h at 3 different temperatures (15°C, 25°C or 35°C). As the storage time increased, pH and glucose concentration of the follicular fluid, percentage of live immature oocytes at germinal vesicle stage, oxidative activity and maturation rate decreased. At higher temperatures, pH and glucose concentration decreased, but oxidative activity and oocyte maturation rate increased. Lactate concentration and immature oocyte ROS production increased as storage time and temperature increased. The ovary storage for longer than 2 h at 25°C and 35°C resulted in low pH of the follicular fluid and high ROS level in immature oocytes. Such conditions seem to damage oocytes and impair their meiotic competence. A decrease in the oxidative activity caused by long time and/or low storage temperature may imply a decrease in oocyte vitality. In conclusion, in the porcine species, the transport of ovaries at 25°C and 35°C for 2 h are the best conditions to maintain adequate oocyte quality, meiotic competence and in vitro fertilization rates.展开更多
Bovine oocytes cultured in vitro for 6 hours or 22 hours were cryopreserved in different vitrification solutions (EFS40, EFS50, EDFS30 or EDFS40) by the two-step method with OPS (open pulled straw).The best results we...Bovine oocytes cultured in vitro for 6 hours or 22 hours were cryopreserved in different vitrification solutions (EFS40, EFS50, EDFS30 or EDFS40) by the two-step method with OPS (open pulled straw).The best results were achieved by using EDFS30 to cryopreserve the oocytes either for in vitro fertilization or for chemical activation. The blastocyst rates were 12% and 17% in 6 hour and 22 hour cultures respectively following in vitro fertilization. If frozen-thawed oocytes were continued in culture up to 24 hours, and were activated by chemicals, the blastocyst rates were 22% and 24% in 6-hour and 22-hour groups respectively.There were no statistical differences between frozen and fresh oocytes (P ＞ 0.05).展开更多
基金National Key Research and Development Program Topics,Grant/Award Number:2021YFD1200402Chinese Universities Scientific Fund,Grant/Award Number:2021TC061+6 种基金Natural Science Foundation of Hebei province,Grant/Award Number:H2020206254Special Program for Training and Guiding Outstanding Young and Middle-aged Talents,Grant/Award Number:SKLSGIHP2021A01National Natural Science Foundation of China,Grant/Award Number:81901562&31372307Key research and development projects in Hebei province,Grant/Award Number:18226604DProgram of Young and Middle-aged Scientific and technological Innovation Leaders of the Xinjiang Production and Construction Corps,Grant/Award Number:2018CB025Xinghuo program of the First Hospital of Hebei Medical University,Grant/Award Number:XH202005The Central Guidance on Local Science and Technology Development Fund of Hebei Province,Grant/Award Number:226Z7713G。
文摘Background:Irreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation.Antioxidants were always used to antagonist the oxidative stress caused by vitrification.However,the comprehensive mechanism underlying the protective role of antioxidants has not been studied.Procyanidin B2(PCB2)is a potent natural antioxidant and its functions in response to vitrification are still unknown.In this study,the effects of PCB2 on vitrified-thawed oocytes and subsequent embryo development were explored,and the mechanisms underlying the protective role of PCB2 were systematically elucidated.Results:Vitrification induced a marked decline in oocyte quality,while PCB2 could improve oocyte viability and further development after parthenogenetic activation.A subsequent study indicated that PCB2 effectively attenuated vitrification-induced oxidative stress,rescued mitochondrial dysfunction,and improved cell viability.Moreover,PCB2 also acts as a cortical tension regulator apart from strong antioxidant properties.Increased cortical tension caused by PCB2 would maintain normal spindle morphology and promote migration,ensure correct meiosis progression and finally reduce the aneuploidy rate in vitrified oocytes.Further study reveals that ATP biosynthesis plays a crucial role in cortical tension regulation,and PCB2 effectively increased the cortical tension through the electron transfer chain pathway.Additionally,PCB2 would elevate the cortical tension in embryo cells at morula and blastocyst stages and further improve blastocyst quality.What's more,targeted metabolomics shows that PCB2 has a beneficial effect on blastocyst formation by mediating saccharides and amino acids metabolism.Conclusions:Antioxidant PCB2 exhibits multi-protective roles in response to vitrification stimuli through mitochondria-mediated cortical tension regulation.
基金supported Key Industry Innovation Chain of Shaanxi Province (2018ZDCXL-NY-02-06)。
文摘Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.Methods Oocytes were treated with different concentrations of ZEA(3,5,8 and 10μmol/L)and isorhamnetin(5,10,20 and 30μmol/L)for 44 h at 39℃.ZEA(5μmol/L)and isorhamnetin(10μmol/L)were selected for subsequent studies.Polar body exclusion rate,apoptosis rate and apoptosis related proteins,ROS levels and SOD2 protein,mitochondrial membrane potential and distribution,endoplasmic reticulum distribution and proteins expression,and PI3K,Akt and p-Akt proteins expression of oocytes were detected.In addition,the effect of PI3K antagonist(LY294002)on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.Results Our findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes.Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes.Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production.Moreover,isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA.Changing the expression of endoplasmic reticulum stress-related marker proteins(CHOP,GRP78)and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress.Mechanistically,isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.Conclusions Collectively,these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway,which enhances meiotic maturation and mitochondrial function,and inhibits early apoptosis,oxidative stress and endoplasmic reticulum stress in porcine oocytes.Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.
基金supported by the National Natural Science Foundation of China(31900592)the Natural Science Foundation of Jiangsu Province(BK20190526).
文摘Background:Elevated ambient temperature-caused heat stress is a major concern for livestock production due to its negative impact on animal feed intake,growth,reproduction,and health.Particularly,the germ cells are extremely sensitive to the heat stress.However,the effective approach and strategy regarding how to protect mammalian oocytes from heat stress-induced defects have not been determined.Methods:Germinal vesicle(GV)porcine oocytes were cultured at 41.5℃ for 24 h to induce heat stress,and then cultured at 38.5℃ to the specific developmental stage for subsequent analysis.Nicotinamide mononucleotide(NMN)was dissolved in water to 1 mol/L for a stock solution and further diluted with the maturation medium to the final concentrations of 10μmol/L,20μmol/L,50μmol/L or 100μmol/L,respectively,during heat stress.Immunostaining and fluorescence intensity quantification were applied to assess the effects of heat stress and NMN supplementation on the key processes during the oocyte meiotic maturation.Results:Here,we report that NMN supplementation improves the quality of porcine oocytes under heat stress.Specifically,we found that heat stress resulted in oocyte maturation failure by disturbing the dynamics of meiotic organelles,including the cytoskeleton assembly,cortical granule distribution and mitochondrial function.In addition,heat stress induced the production of excessive reactive oxygen species(ROS)and DNA damage,leading to the occurrence of apoptosis in oocytes and subsequent embryonic development arrest.More importantly,we validated that supplementation of NMN during heat stress restored the meiotic defects during porcine oocyte maturation.Conclusions:Taken together,our study documents that NMN supplementation is an effective approach to improve the quality of oocytes under heat stress by promoting both nuclear and cytoplasmic maturation.
基金Supported by Special Fund for National Hair Sheep Industrial Technology System(CARS-39-24)Science and Technology Development Program of Shanxi Province(20120311024-1)+2 种基金Science and Technology Innovation Team Project of Shanxi Province(201705D131028-20)Financial Support of Agriculture of Shanxi Province(NYGX2015-03)Talent Project for Science and Technology Development in Outlaying Poor Areas,Frontier Ethnic Minority Areas and Old Revolutionary Base Areas of Shanxi Province,China(2017Sy128)
文摘[Objectives] This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer( JIVET). [Methods]Ten Dorper × small-tailed Han lambs aged 5 to 10 weeks were induced to superovulate via i. p. injection of pregnant mare's serum gonadotropin( PMSG). The oocytes were matured in basal maturation solution or modified maturation solution,which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then,the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum( ESS) or fertilization medium II containing 3 mg/ml bull serum albumin( BSA). Finally,the number of oocytes,oocyte maturation rate and cleavage rate of the lambs of different ages were determined. [Results]The average number of oocytes recovered per lamb was( 111. 00 ± 16. 97),( 139. 50 ± 28. 99),( 108. 50 ± 17. 68) and( 42. 00 ± 11. 31) for5-,7-,8-and 10-week-old Dorper × small-tailed Han lambs,respectively. The number of oocytes obtained from 5-,7-and 8-week-old lambs was significantly higher than that from 10-week-old lambs( P < 0. 05),but there was no significant difference among 5-,7-and 8-week-old lambs( P > 0. 05). The maturation rate of oocytes cultured in modified maturation solution was 3. 64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II( P < 0. 01). [Conclusions] The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks,adding a certain amount of cysteine into oocyte maturation solution,and a certain amount of ESS into fertilization medium.
基金the National Natural Science Foundation of China(30100133) Hongkong Wiss Biotechnical Center.
文摘Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 μs, with a blastocyst development rate of (20.12 ± 8.18)% (P ＞ 0.05). No significant difference was found between treatments of multiple pulses and a single pulse ( P ＞ 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35)% ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) %, P ＞ 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 ± 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79,P ＜ 0.01 ). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2 (5 % CO2:7%O2:88%N2) also showed no significant difference from those in high O2 (5% CO2 in air) [ (20.78 ± 8.80) % and 17.00 ± 6.12 vs. (25.30 ± 7.55) % and 18.01 ± 6.79, P ＞ 0.05 ]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.
文摘Objective:To explore the effects of different MⅡstage oocytes zona pellucida birefringence on pregnancy outcome.Methods:A total of 46 couples with infertile which induced by single cause received in-vitro fertilization treatment were analyzed retrospectively,and randomly divided into the high zona birefringence(HZB)/HZB group.HZB/low zona birefringeuce(LZB) group and LZB/LZB group according to different oocytes zona pellucida birefringence.Intracytoplasmic sperm injection outcome was analyzed and compared.Results:The proportion of HZB oocytes, implantation rate and the pregnancy rate were decreased in three groups(HZB/HZB group】HZB/ LZB group】LZB/LZB group)(P【0.05).But there was no significantly different between the number of oocytes and fertilization rate of these groups(P】0.05).Logistic regression analysis showed that factors allecl MⅡstage oocytes zona pellucida birefringence were age.basal FSH level and the LH level on the day of HCG injection.Age and KSH levels were negatively correlated with the single oocyte zona pellucida birefringence:While the LH level on the day of hCC injection was positively correlated with the single oocylc zona pellucida birefringence.Conclusions:The primary influence factors on MⅡstage oocytes zona pellucida are age.basal FSH level and the LH level on the day of hCG injection.The birefringence value of zona pellucida can affect the pregnancy outcome.
文摘Purpose: Evaluate the effect of preincubation of oocytes prior to IVF or ICSI cycles with embryo transfer at blastocyst stage. Methods: Retrospective non randomized study based on secondary analysis of data. Setting: Laboratory of Assisted Reproduction at the Alcivar Hospital. Patients: One hundred-eighteen cycles of IVF and ICSI were analyzed in the present study. The evaluated groups were formed for those patients whose oocytes, after retrieval, were inseminated at 1-3 h (Group I) or 4-6 h (Group II). Results: There was no difference in fertilization rate (83.6% and 78.1%), Day 3 cleavage rate (95.1% and 97.1%), and blastocyst formation (31.1% and 39.1%) for groups I and II respectively. Clinical pregnancy rates (PR: 53.0% vs 22.9%) and implantation rates (IR: 38.1% vs 13.0%) were significantly higher in group II versus group I, respectively (P < 0.05). Conclusions: Preincubation of oocytes before insemination is a factor which raises the PR and IR after the blastocyst transfer.
文摘Objective:To establish a new way to collect superovulated oocytes or zygotes repeatedlyfrom an individual mouse.Methods:Superovulations were induced by injection PMSG and hCG in Kunming strainmice.The ampullaes of oviduct of all anaesthetised mouse were put in a specially designed'U'sink and released.The second and third times of PMSG injection were made on the sixth day andeleventh day after the first superovulation injection.The capacity of development was examinedby in vitro culture of parthenogenesis activation oocytes.Results:Development to blastocyst stage was not significantly different between the first andsecond time collection.The percentage of blastocyst stage in CD and Sr^(++) treatment was signifi-cantly higher(P<0.05)than the oocytes treated in CB and Sr^(++).Conclusion:This method enables us to collect oocytes or zygotes repeatedly from one individ-ual mouse in an interval as short as 5 days and without influence on the quality of oocytes.
文摘Background: In order to assess the chromosomal status in embryos obtained from vitrified and fresh donated oocytes, preimplantational genetic diagnostic (PGD) was performed after biopsy of one blastomere at day 3. METHODS: A total of 249 oocytes were obtained from 23 oocyte donors, 80 oocytes were used in the vitrified group and 151 oocytes were used in the fresh group. Nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) were investigated by fluorescence in situ hybridization (FISH) analysis in 56 and 121 embryos from vitrified and fresh group respectively. Fertilization, cleavage rate, embryo quality and chromosomal abnormality rate were compared between groups evaluated. Results: Vitrified oocytes showed a survival rate of 97.5%. There was no significant difference in the fertilization rate (82.7% and 91.4%), Day 2 cleavage rate (90.3% and 87.7%) or blastocyst formation rate (31.1% and 44.6%) for the vitrified and fresh groups respectively. Chromosomal abnormality rate (66.1% versus 71.9%), percentage of abnormal blastocysts (61.1% versus 64.8%) and percentage of abnormalities for each analyzed chromosome were similar for the vitrified group compared with the control group. Conclusions: The rates of chromosomal abnormalities in embryos from vitrified oocytes are similar to those published previously;and comparable to those observed in embryos from fresh oocytes. These results confirm that the developmental competence and chromosomal status of embryos obtained from vitrified oocytes is not affected by the vitrification procedure, and they preserve the potential to be fertilized and to develop in to blastocyst stage similar to embryos from fresh oocytes.
基金supported by a grant from the Royal Golden Jubilee,RGJ,Thailand(Grant number:PHD/0350/2551).
文摘Objective:To evaluate changes of feline(Felis catus)oocytes proteins during in vitro maturation by using the proteomic approach.Methods:Immature oocytes(germinal vesicle)isolated from female cats were cultured and collected at 0 h and 24 h.After collection,oocytes were investigated into immature(germinal vesicle)and mature(metaphaseⅡ)stages.The qualitative profiles of the proteins at the immature and mature stages were determined by one-dimensional electrophoresis and liquid chromatography-mass spectrometry.Results:Our data revealed that following 24 h in vitro maturation the maturation rate(metaphaseⅡstage)was 58.7%.Eighty-one of the 260 proteins analyzed were differentially expressed between the germinal vesicle stage and the metaphaseⅡ-arrest stage.Proteomic analysis of germinal vesicle and metaphaseⅡoocytes showed abundant expression of proteins involved in transportation(10%),indicating that this was a major characteristic of germinal vesicle oocytes.Similarly,analysis of the proteome of metaphaseⅡoocytes indicated that cell cycle proteins were overexpressed.Interestingly,proteins involved in DNA repair and apoptosis were only expressed in germinal vesicle oocytes and proteins involved in fertilization were only expressed in metaphaseⅡoocytes.Conclusions:The overexpression of certain proteins in germinal vesicle and metaphaseⅡis necessary for oocyte development and maturation.Our findings provide a valuable resource for further investigations into protein expression in oocytes at different developmental stages.
文摘Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inward current in the injected oocytes wa
文摘Introduction: The roles of genetic, epigenetic, metabolic and other environmental factors such as nutrition and stress, are becoming evident for a successful and healthy pregnancy. This raises the possibility and question, if and how, we improve the probability of pregnancy and of a healthy fetus? The present study examined the role of metabolic, antioxidant and minerals and the results suggest that these factors may positively influence the oocyte quality and the pregnancy rate. Methods: CD1 female mice aged 15 and 5 weeks were divided into four groups of ten each and treated by intragastric gavage daily for 3 weeks. G1: Vehicle;G2: Carnitines (L-carnitine 0.4 mg and acetyl-L-carnitine 0.12 mg/mouse);G3: Microelements (Zinc 4 ng, Copper 0.8 ng, Iron 7 ng/mouse);G4: G3+G2. At the end of the treatment period superovulation was induced and oocytes were collected to assess their quantity and quality. Further, in vitro fertilization (IVF) experiments were performed to assess the preimplantation embryo development. The birth success rate was also analyzed in old and young female. The mice were in vivo fertilized. qRT-PCR were performed to analyze a possible modulation in key genes of the reproductive process. Results: The number of oocytes was significantly higher in groups 2 and 4 compared to the control group. The oocyte number in group 3 was not affected. The level of degraded oocytes was 29.1% and 19.3% (group 2 and 4) versus 34.3% (control). Concomitantly, the numbers of embryos arriving to successful birth were also increased in G4, both in the old and young group of mice. Preliminary analysis of genes affected evidenced that AMH was up regulated in the ovary and KITL in the uterus in group 2. Conclusion: Results showed that L-carnitine, acetyl-L-carnitine and micronutrients were able to improve both oocytes quality and success rate of pregnancy. Further studies are planned to further examine ways to improve pregnancy and fetal health.
文摘Parthenogenesis is a form of asexual reproduction found in females, where growth and development of embryos occurs without fertilization by a male. Parthenogenesis occurs naturally in aphids, Daphnia, rotifers, nematodes and some other invertebrates but can also be induced efficiently in mammalian oocytes by providing appropriate stimuli invitro. Recently, parthenogenesis has attracted wide attention because of the role of activated oocytes in the field of research that have been described such as intra cytoplasmic sperm injection, cloning by nuclear transfer, somatic cell cloning, investigating culture conditions etc. & potential for deriving pluripotent stem cell lines and their differentiation into various cell lines that can be utilized for various tissue engineering applications. The parthenogenetically activated oocytes possess maternal genome and can developed in to either haploid, diploid or polyploidy embryos with the help of it we can analyze the possible role of all the genes involved in imprinting processes as well as the role the paternal genome plays during early embryo development by comparing them with fertilized embryos. Several methods are able to induce parthenogenetic activation through the elevation of cytoplasmic free calcium in oocytes. But one common, universal method or activation agents has not been developed for all species because the process is highly specific for each species. Therefore, activation step for each species need to be optimized accordingly. This review describes the general method of activation of mammalian oocytes and their genomic imprinting analysis.
文摘The present work was designed to examine the effect of the presence or absence of cumulus cells on the efficiency of vitrification of immature cattle oocytes. In our experiment, we had two groups: group 1, immature cattle oocytes with cumulus cells and group 2, immature cattle oocytes without cumulus cells. The two groups underwent vitrification using 20% ethylene glycol and 20% DMSO, and then thawed, and in vitro matured in TCM-199 medium and examined after 22 hours for assessment of nuclear maturation. Higher survival rate (p < 0.05) after thawing was observed in group 1 (84.6%) than group 2 (57.8%). After in-vitro maturation, the rate of MII oocytes was significantly higher (p < 0.05) in group 1 (74.4%) than group 2 (47.7%). In conclusion, the cumulus cells are very important in increasing the survivability and developmental rate of vitrified-thawed immature cattle oocytes.
基金National Natural Science Foundation of China.Project No:81260124.
文摘Objective: To investigate the gonadotrophin drug Luveris? (L) and Gonal-F? (G) effects on bovine immature oocyte in vitro maturation culture. Methods: In total, 2000 bovine cumulus-oocyte complexes (COCs) were purchased commercially and cultured in media with ovulation stimulation drugs Gonal-F and Luveris (0, 5, 10, 20, 40 IU/mL), individually, for maturation in vitro. 2000 Oocytes were divided evenly into two groups, Luveris and Gonal-F groups, individually, 1000 Oocytes. After 24 and 48h culture, cumulus expansion was assessed under dissecting microscopes. Oocyte maturation (GV, MI, MII) was determined by examining nuclear maturation and polar body formation under inverted microscopes. Results: Luveris and Gonal-F enhanced oocyte maturation in vitro in a dose-dependently manner. Culture media supplemented with L or G significantly improved MⅡ Oocyte maturation rates after culture for 24 h and 48 h in comparison with the control group. Oocyte maturation rates were 33.3% and 37.5%(20 IU/mL), individually, 33.3% and 53.6% (40 IU/mL);20 IU/mL G was 15.1% and 16.2%;40 IU/mL G was 38.9% and 39.5%, respectively. Conclusion: LH (Luveris) can promote prophase bovine immature oocytes developmental competence in vitro, which is mostly likely stimulated by FSH(Gonal-F ). Furthermore, LH can delay oocytes GVBD that is very useful for IVF clinical medication guidance.
文摘The clomiphene citrate (CC), a nonsteroidal triphenylethylene compound, is a first line of medicine used for the induction of ovulation in anovulatory women worldwide. In spite of high ovulation induction with the use of CC, the pregnancy rate is much lower. Such a discrepancy could be due to the peripheral anti-estrogenic effect of CC, particularly at the level of ovary, endometrium and cervical mucus. CC induces ovulation by binding to the estrogen receptors and generates hypoestrogrnic state in hypothalamus leading to release of pituitary gonadotropins. CC may have a direct effect at the level of ovary but the molecular mechanism remains unclear. Animal studies suggest that the CC induces apoptosis in granulosa cells and results hypoestrogenic state in the ovary. Reduced estradiol 17β level in the ovary affects development and maturation of oocyte leading to oocyte apoptosis. Further, CC increases hydrogen peroxide (H2O2) level and thereby bax protein expression and DNA fragmentation in cumulus-granulosa cells as well as in oocytes. The exogenous supplementation of either estradiol 17β or melatonin reduces H2O2 level in ovary, delays meiotic cell cycle progression in oocyte and protects oocyte apoptosis. Hence, supplementation of estradiol 17β or melatonin along with CC could be beneficial to protect granulosa cell as well as oocyte apoptosis and inhibit deterioration of oocyte quality. Thus, maintenance of oocyte quality may overcome the adverse effect caused due to CC treatment during infertility management.
基金supported by the National Natural Science Foundation of China(No.30930065 and No.31271605)
文摘Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.
文摘Starfish oocytes with intact germinal vesicles (GVs) were cut along desired planes with glass needles or ligated using silk thread loops into two parts and allowed to mature in vitro, and inseminated. The experimental results showed that (1) only the parts with GVs or partial GV contents (PGVCs) cleaved, those without any GV materials did not; but nucleated and non-nucleated fragments cut from mature eggs were able to divide; (2) the development of animal parts of oocytes containing GVs or PGVGs was like that of animal fragments of matured oocytes with female pronuclei; most of them gave rise to permanent blastulae. and just a few formed ectodermal vesicles with a little primary mesenchyme; (3) a large part of vegetal fragments with GVs or PGVCs. and the vegetal parts of mature eggs without female pronuclei developed into small but normal embryos; (4) the fragments containing GVs or PGVCs obtained from the oocytes along a plane parallel to the animal-vegetal (A-V) axis developed as normally as the
文摘We studied the effect of different storage conditions of porcine ovaries (time, temperature) on the characteristics of the follicular fluid, immature oocyte quality, meiotic competence and in vitro fertilization of oocytes. Ovaries were stored for 2, 4 or 6 h at 3 different temperatures (15°C, 25°C or 35°C). As the storage time increased, pH and glucose concentration of the follicular fluid, percentage of live immature oocytes at germinal vesicle stage, oxidative activity and maturation rate decreased. At higher temperatures, pH and glucose concentration decreased, but oxidative activity and oocyte maturation rate increased. Lactate concentration and immature oocyte ROS production increased as storage time and temperature increased. The ovary storage for longer than 2 h at 25°C and 35°C resulted in low pH of the follicular fluid and high ROS level in immature oocytes. Such conditions seem to damage oocytes and impair their meiotic competence. A decrease in the oxidative activity caused by long time and/or low storage temperature may imply a decrease in oocyte vitality. In conclusion, in the porcine species, the transport of ovaries at 25°C and 35°C for 2 h are the best conditions to maintain adequate oocyte quality, meiotic competence and in vitro fertilization rates.
文摘Bovine oocytes cultured in vitro for 6 hours or 22 hours were cryopreserved in different vitrification solutions (EFS40, EFS50, EDFS30 or EDFS40) by the two-step method with OPS (open pulled straw).The best results were achieved by using EDFS30 to cryopreserve the oocytes either for in vitro fertilization or for chemical activation. The blastocyst rates were 12% and 17% in 6 hour and 22 hour cultures respectively following in vitro fertilization. If frozen-thawed oocytes were continued in culture up to 24 hours, and were activated by chemicals, the blastocyst rates were 22% and 24% in 6-hour and 22-hour groups respectively.There were no statistical differences between frozen and fresh oocytes (P ＞ 0.05).
