This study aimed to optimization of the in vitro fertilization system in Cỏ goat oocytes to achieve the maximum possible blastocyst development rate. In Experiment 1, we assessed the effects of IVF media on the in vit...This study aimed to optimization of the in vitro fertilization system in Cỏ goat oocytes to achieve the maximum possible blastocyst development rate. In Experiment 1, we assessed the effects of IVF media on the in vitro fertilization of Cỏ goat oocytes. There was no significant difference in the cleavage, blastocyst, or hatching rates between TALP-Fert and BO-IVF media. Experiment 2 was performed to assess the concentration of sperm in the in vitro fertilization of Cỏ goat oocytes. The matured Cỏ goat oocytes were fertilized in BO-IVF for four sperm concentrations: 5 × 105, 1 × 106, 2 × 106 and 3 × 106 sperm/ml. The blastocyst rate of 2 × 106 sperm/ml and 3 × 106 sperm/ml groups was higher than that of 5 × 105 sperm/ml and 1 × 106 sperm/ml groups (P Experiment 3 was performed to assess the IVF duration on the in vitro fertilization of Cỏ goat oocytes. The matured Cỏ goat oocytes were fertilized in BO-IVF with sperm concentration of 3 × 106 sperm/ml for 18, 20, 22 and 24 h. The cleavage, blastocyst, and hatching blastocyst rates of 18 h group were lower than those of 20, 22 and 24 h groups (P 0.05). In conclusion, the matured Cỏ goat oocytes were fertilized in BO-IVF with sperm concentration of 3 × 106 sperm/ml for 20 hours, which is suitable for the in vitro Cỏ goat embryo production.展开更多
Meiotic resumption in mammalian oocytes involves nuclear and organelle structural changes,notably the chromatin configuration transition from a non-surrounding nucleolus(NSN)to surrounding nucleolus(SN)in germinal ves...Meiotic resumption in mammalian oocytes involves nuclear and organelle structural changes,notably the chromatin configuration transition from a non-surrounding nucleolus(NSN)to surrounding nucleolus(SN)in germinal vesicle oocytes.In the current study,we found that nuclear speckles(NSs),a subnuclear structure mainly composed of serine-arginine(SR)proteins,changed from a diffuse spotted distribution in mouse NSN oocytes to an aggregated pattern in SN oocytes.We also found that the SR protein-specific kinase 1(SRPK1),an enzyme that phosphorylates SR proteins,co-localized with NSs at the SN stage,and that NSN oocytes failed to transition to SN oocytes after the inhibition of SRPK1 activity.Furthermore,the typical structure of the chromatin ring around the nucleolus in SN oocytes collapsed after treatment with an SRPK1 inhibitor.Mechanistically,phosphorylated SR proteins were found to be related to chromatin as shown by a salt extraction experiment,and in situ DNaseⅠassay showed that the accessibility of chromatin was enhanced in SN oocytes when SRPK1 was inhibited,accompanied by a decreased repressive modification on histone and the abnormal recurrence of a transcriptional signal.In conclusion,our results indicated that SRPK1-regulated phosphorylation of SR proteins was involved in the NSN-SN transition and played an important role in maintaining the condensed nucleus of SN oocytes via interacting with chromatin.展开更多
Oocyte cryopreservation is an essential procedure in assisted reproductive technologies,aimed at preserving fertility,particularly for women undergoing IVF treatment or at risk of ovarian damage due to radiation,chemo...Oocyte cryopreservation is an essential procedure in assisted reproductive technologies,aimed at preserving fertility,particularly for women undergoing IVF treatment or at risk of ovarian damage due to radiation,chemotherapy,or surgery.Despite its growing use,the survival and fertilization rates of cryopreserved oocytes remain suboptimal,largely due to cryo-induced oxidative stress.The generation of Reactive Oxygen Species(ROS)during freezing and thawing causes considerable damage to key cellular components,including proteins,lipids,DNA,and mitochondria.This oxidative stress compromises oocyte quality and reduces developmental potential.To address these challenges,the use of additives-especially antioxidants-has shown significant promise in mitigating oxidative damage.Enzymatic antioxidants such as Superoxide Dismutase(SOD)and Catalase(CAT),along with non-enzymatic antioxidants like glutathione,melatonin,and resveratrol,have demonstrated the ability to neutralize ROS and improve oocyte viability and developmental outcomes.Recent studies highlight the potential of Mitoquinone(MitoQ),a mitochondria-targeted antioxidant,to effectively counteract mitochondrial ROS and enhance cellular defense mechanisms during cryopreservation.This review explores the cellular mechanisms of cryodamage,the role of oxidative stress in oocyte cryopreservation,and the potential of various antioxidant strategies to enhance oocyte survival and function.Developing effective antioxidant supplementation approaches may significantly improve the outcomes of cryopreservation in reproductive medicine.展开更多
Background Polystyrene nanoplastics(PS-NPs)are becoming increasingly prevalent in the environment with great advancements in plastic products,and their potential health hazard to animals has received much attention.Se...Background Polystyrene nanoplastics(PS-NPs)are becoming increasingly prevalent in the environment with great advancements in plastic products,and their potential health hazard to animals has received much attention.Several studies have reported the toxicity of PS-NPs to various tissues and cells;however,there is a paucity of information about whether PS-NPs exposure can have toxic effects on mammalian oocytes,especially livestock.Herein,porcine oocytes were used as the model to investigate the potential effects of PS-NPs on mammalian oocytes.Results The findings showed that different concentrations of PS-NPs(0,25,50 and 100μg/m L)entering into porcine oocytes could induce mitochondrial stress,including a significant decrease in mitochondrial membrane potential(MMP),and the destruction of the balance of mitochondrial dynamic and micromorphology.Furthermore,there was a marked increase in reactive oxygen species(ROS),which led to oocyte lipid peroxidation(LPO).PS-NPs exposure induced abnormal intracellular iron overload,and subsequently increased the expression of transferrin receptor(TfRC),solute carrier family 7 member 11(SLC7a11),and acyl-CoA synthetase long-chain family member 4(ACSL4),which resulted in ferroptosis in oocytes.PS-NPs also indu ced oocyte maturation failure,cytoskeletal dysfunction and DNA damage.Cotreatment with 5μmol/L ferrostatin-1(Fer-1,an inhibitor of ferroptosis)alleviated the cellular toxicity associated with PS-NPs exposure during porcine oocyte maturation.Conclusions In conclusion,PS-NPs caused ferroptosis in porcine oocytes by increasing oxidative stress and altering lipid metabolism,leading to the failure of oocyte maturation.展开更多
[Objective] The aim was to optimize Yanbian cow oocytes mature in vitro and cleavage system after nuclear transfer based on uniform design. [Method] Oocytes were recovered by aspiration method, and oocytes were mature...[Objective] The aim was to optimize Yanbian cow oocytes mature in vitro and cleavage system after nuclear transfer based on uniform design. [Method] Oocytes were recovered by aspiration method, and oocytes were matured in vitro （IVM） with different conditions, and then carried out nucleus transfer, fusion, activation and in vitro culture （IVC） of embryo. Effects of ovary storage temperature, maturation time and follicular diameter size on in vitro maturation and cleavage rates of cow oocytes were compared. [ Result] The best conditions of IVM of Yanbian cow oocytes was that： the oocytes of 8 mm diameter were matured in vitro for 24 hours when the ovaries were stored at 26℃ or 31 ℃. The best cleave conditions after nucleus transfer of oocytes was that： the oocytes of 6 mm or 8 mm diameter were cultured in vitro for 24 hours when the ovaries were stored at 26℃. [ Conclusion] The result has some reference to Yanbian cow and other cow breeding and population expanding propagation.展开更多
[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first...[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first polar body extrusion rate, oocyte glutathione （GSH） content, positive rate of brilliant cresyl blue （BCB） staining and development potential of activated oocytes or fertilized oocytes were employed as main indicators to investigate the effects of follicular mural granulosa cell （MGC） coculture on cytoplasmic maturation of cumulus cell-removal oocytes （Denuded Oocyte, DO）. [Result] According to in vitro maturation results, compared with DO group, the first polar body extrusion rate of porcine oocytes in DO＋MGC group was not significantly different, but the nuclear maturation process was improved and was more similar to that in COC （cumulus-oocyte complex） group. Detection of GSH content in mature oocytes showed that there was no significant difference between DO＋ MGC group （optical density of 1 053.67） and COC group （optical density of 1 426.00） or between DO＋MGC group and COC＋GC group （optical density of 1 541.00）, however, GSH content in mature oocytes of DO group （optical density of 724.67） was significantly lower than that of COC group and COC＋GC group （P0.05）. Detection of glucose-6-phosphate dehydrogenase （G6PDH） activity showed that there was no significant difference in BCB positive oocyte rate between DO ＋MGC group （88.26% ） and COC group （92.75%） or between DO＋MGC group and DO group （82.86% ）, however, BCB positive oocyte rate of DO group was significantly lower than that of COC group （P0.05）. Furthermore, the cleavage rate and blastocyst rate of activated mature oocytes derived from DO ＋MGC group （94.98% and 43.67% , respectively） were significantly higher than those from DO group （52.54% and 8.97%, respectively） （P0.05）, and were not significantly different compared with those from COC group （97.11% and 38.30%, respectively）. In addition, the cleavage rate of fertilized oocytes derived from DO＋MGC group （72.65%） showed no significant difference compared with that from DO group （63.59%）, but the blastocyst rate of DO＋MGC group was significantly higher than that of DO group （9.88%） （P0.05）. [Conclusion] MGC coculture can significantly improve the in vitro cytoplasmic maturation quality of denuded porcine oocytes, thereby enhancing the subsequent developmental potential.展开更多
[Objective] The research aimed to enhance culture efficiencies of oocyte and embryo of goat in vitro and to explore serum-free culture system in vitro.[Method] At present,the conventional solutions of oocyte maturatio...[Objective] The research aimed to enhance culture efficiencies of oocyte and embryo of goat in vitro and to explore serum-free culture system in vitro.[Method] At present,the conventional solutions of oocyte maturation and embryo development in vitro were always added into 1% ITS(Insulin-transferrin-selenium) or using 1% ITS to replace FBS in 2 kinds culture solutions for conducting in vitro cultures of goat oocyte and parthenogenetic embryo.The influences of ITS on their developments were detected.[Result] ITS in maturation liquid of oocytes could not increase oocytes maturation rate but significantly increased blastocyst rate (58.06% vs. 48.19%)of parthenogenetic embryo.If FBS in maturation liquid of oocytes was replaced by ITS, the maturation rate, cleavage rate and blastocyst rate were basically unchanged.Adding ITS into embryo medium could increase blastocyst rate (68.30% vs. 56.82%)of parthenogenetic embryo of goat.If FBS in embryo medium was replaced by ITS,the cleavage rate didn’t change basically,while the blastocyst rate in ITS was obviously lower than that in FBS group(42.33% vs.56.82%).[Conclusion] ITS could promote maturation of oocyte in vitro and early embryonic development, in addition,ITS could replace serum in maturation medium of oocyte as serum-free culture system for conducting relevant researches.展开更多
Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to ex...Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to explore the relationship between oocyte maturation and GDF-9 gene expression. Preantral follicles of lO-day old mice were isolated from the ovaries and were cultured for 12 days. Oocytes from day 2 （D2）, D4, D6, D8, DIO, D12 cultured in vitro were named the in vitro group and oocytes of day 12 （D12）, D14, D16, D18, D20, D22 grown in vivo were named the in vivo group. Follicle survival, antrum formation and maturation rate were 89.5%, 51.8% and 56.6% respectively in follicles cultured in vitro. After RT-PCR and agarose gel electrophoresis, relative mRNA abundance of GDF-9 was measured in each group of oocytes. At day 8 - 12, the GDF-9 gene expression level of oocytes in vitro was significantly lower than that in vivo （P 〈 0.05）. We conclude that M Ⅱ oocytes can be obtained from in vitro culture of preantral follicles. The GDF- 9 gene expression of oocytes varies at different growth stages in vivo. The low expression of GDF-9 in oocytes cuhured in vitro may be the cause of their low developmental capacity.展开更多
[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oo...[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).展开更多
Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15...Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.展开更多
Azoospermia, cryptozoospermia and necrospermia can markedly decrease the ability of males to achieve pregnancy in fertile females. However, patients with these severe conditions still have the option to be treated by ...Azoospermia, cryptozoospermia and necrospermia can markedly decrease the ability of males to achieve pregnancy in fertile females. However, patients with these severe conditions still have the option to be treated by intracytoplasmic sperm injection （ICSI） to become biological fathers. This study analyzed the fertilization ability and the developmental viabilities of the derived embryos after ICSI treatment of the sperm from these patients compared with in vitro fertilization （IVF） treatment of the proven-fertile donor sperm on sibling oocytes as a control. On the day of oocyte retrieval, the number of sperm suitable for ICSI collected from two ejaculates or testicular sperm extraction was lower than the oocytes, and therefore, excess sibling oocytes were treated by IVF with donor sperm. From 72 couples （73 cycles）, 1117 metaphase Ⅱ oocytes were divided into 512 for ICSI and 605 for IVF. Compared with the control, husbands＇ sperm produced a lower fertilization rate in nonobstructive azoospermia （65.4% vs 83.2%; P〈 0.001）, crytozoospermia （68.8% vs 75.5%; P〈 0.05） and necrospermia （65.0% vs 85.2%; P〈 0.05）. The zygotes derived in nonobstructive azoospermia had a lower cleavage rate （96.4% vs 99.4%; P 〈 0.05）, but the rate of resultant good-quality embryos was not different. Analysis of the rates of cleaved and good-quality embryos in crytozoospermia and necrospermia did not exhibit a significant difference from the control. In conclusion, although the sperm from severe male infertility reduced the fertilization ability, the derived embryos had potential developmental viabilities that might be predictive for the expected clinical outcomes.展开更多
The significance of the performance of conventional in vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) using sibling oocytes from couples with subfertile male or unexplained infertility was evaluat...The significance of the performance of conventional in vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) using sibling oocytes from couples with subfertile male or unexplained infertility was evaluated. A total of 410 sibling oocyte cumulus-corona complexes (OCCC) from 21 couples with subfertile male (group A) and 11 unexplained infertile couples (group B) were randomly divided, in order of retrieval, into two groups inseminated either by conventional IVF or by ICSI. The treatment outcomes and the influence of infertility factors on fertilization in each group were compared. The results showed that although the two pronuclear (2PN) fertilization rate per injected sibling oocytes was significantly higher after ICSI (group A: 68.2 %±28.8 %; group B: 66.2 %±24.9 %) than after conventional IVF (group A: 41.8 %±32.7 %; group B: 40.1 %±22.1 %), the other variables studied included: the fertilization rates of per allocated sibling oocytes IVF/ICSI, the fertilization rates of sibling oocytes IVF/ICSI after excluding failed IVF fertilization cycles, as well as the cleavage rates of normal fertilization were not statistically significant (P>0.05). Similarly, though the total fertilization failure rate in the IVF group (group A: 42.9 %; group B: 36.4 %) was significantly higher than in the ICSI group (group A: 4.8 %; group B: 0), we did not cancel cycles due to the normal fertilization of sibling oocytes. Embryo transfer was possible in all 32 couples. There were 10 clinical pregnancies in the two groups. We also discovered a possible association between some semen parameters and sperm functions of group A, and women age and duration of infertility of group B and fertilization. It is suggested that adoption of the split IVF/ICSI technology in the above cases may help eliminate fertilization failures. This is also a useful method to investigate the effect of single factor on the employment of assisted reproductive technology.展开更多
Somatic nuclei can be reprogrammed into a pluripotent state by nuclear transfer, cell fusion and expression of transcription factors. However, these reprogramming processes are very inefficient, which has greatly hind...Somatic nuclei can be reprogrammed into a pluripotent state by nuclear transfer, cell fusion and expression of transcription factors. However, these reprogramming processes are very inefficient, which has greatly hindered efforts to elucidate the underlying molecular mechanisms. Here, we report a new reprogramming strategy that combines the advantages of all three reprogramming methodologies into one process. We injected nuclei from cumulus cells into intact MII oocytes. Following activation, 80% of the reconstructed embryos developed to the blastocyst stage, and tetraploid （4N） embryonic stem （ES） cell lines were generated at a rate of 30% per reconstructed oocyte. We also generated triploid （3N） ES cells after injection of somatic nuclei into activated oocytes. 4N and 3N ES cells expressed pluripotent markers and differentiated into cell types of three embryonic germ layers in vivo. Moreover, all ES cells generated histocompatible, differentiated cells after being engrafted in immunocompetent B6D2F1 mice, showing that ES cells derived from this reprogramming strategy might serve as a source of genetically tailored tissues for transplantation. Thus, we have established a simple and highly efficient reprogramming procedure that provides a system for investigating the molecular mechanisms involved in somatic reprogramming.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
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
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 us, 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 17.0016.12 vs. (25.30 ± 7.55)% and 18.0116.79, 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
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
Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the...Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope （LSCM） was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional （3D） image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM. The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of MⅡocytes.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
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
文摘This study aimed to optimization of the in vitro fertilization system in Cỏ goat oocytes to achieve the maximum possible blastocyst development rate. In Experiment 1, we assessed the effects of IVF media on the in vitro fertilization of Cỏ goat oocytes. There was no significant difference in the cleavage, blastocyst, or hatching rates between TALP-Fert and BO-IVF media. Experiment 2 was performed to assess the concentration of sperm in the in vitro fertilization of Cỏ goat oocytes. The matured Cỏ goat oocytes were fertilized in BO-IVF for four sperm concentrations: 5 × 105, 1 × 106, 2 × 106 and 3 × 106 sperm/ml. The blastocyst rate of 2 × 106 sperm/ml and 3 × 106 sperm/ml groups was higher than that of 5 × 105 sperm/ml and 1 × 106 sperm/ml groups (P Experiment 3 was performed to assess the IVF duration on the in vitro fertilization of Cỏ goat oocytes. The matured Cỏ goat oocytes were fertilized in BO-IVF with sperm concentration of 3 × 106 sperm/ml for 18, 20, 22 and 24 h. The cleavage, blastocyst, and hatching blastocyst rates of 18 h group were lower than those of 20, 22 and 24 h groups (P 0.05). In conclusion, the matured Cỏ goat oocytes were fertilized in BO-IVF with sperm concentration of 3 × 106 sperm/ml for 20 hours, which is suitable for the in vitro Cỏ goat embryo production.
基金National Natural Science Foundation of China(Grant Nos.32070838 and 82301874)Open Fund of State Key Laboratory of Reproductive Medicine,Nanjing Medical University(Grant No.SKLRM K202102)。
文摘Meiotic resumption in mammalian oocytes involves nuclear and organelle structural changes,notably the chromatin configuration transition from a non-surrounding nucleolus(NSN)to surrounding nucleolus(SN)in germinal vesicle oocytes.In the current study,we found that nuclear speckles(NSs),a subnuclear structure mainly composed of serine-arginine(SR)proteins,changed from a diffuse spotted distribution in mouse NSN oocytes to an aggregated pattern in SN oocytes.We also found that the SR protein-specific kinase 1(SRPK1),an enzyme that phosphorylates SR proteins,co-localized with NSs at the SN stage,and that NSN oocytes failed to transition to SN oocytes after the inhibition of SRPK1 activity.Furthermore,the typical structure of the chromatin ring around the nucleolus in SN oocytes collapsed after treatment with an SRPK1 inhibitor.Mechanistically,phosphorylated SR proteins were found to be related to chromatin as shown by a salt extraction experiment,and in situ DNaseⅠassay showed that the accessibility of chromatin was enhanced in SN oocytes when SRPK1 was inhibited,accompanied by a decreased repressive modification on histone and the abnormal recurrence of a transcriptional signal.In conclusion,our results indicated that SRPK1-regulated phosphorylation of SR proteins was involved in the NSN-SN transition and played an important role in maintaining the condensed nucleus of SN oocytes via interacting with chromatin.
基金Anhui Province Clinical Medical Research Translation Special Program(No.2204295107020002).
文摘Oocyte cryopreservation is an essential procedure in assisted reproductive technologies,aimed at preserving fertility,particularly for women undergoing IVF treatment or at risk of ovarian damage due to radiation,chemotherapy,or surgery.Despite its growing use,the survival and fertilization rates of cryopreserved oocytes remain suboptimal,largely due to cryo-induced oxidative stress.The generation of Reactive Oxygen Species(ROS)during freezing and thawing causes considerable damage to key cellular components,including proteins,lipids,DNA,and mitochondria.This oxidative stress compromises oocyte quality and reduces developmental potential.To address these challenges,the use of additives-especially antioxidants-has shown significant promise in mitigating oxidative damage.Enzymatic antioxidants such as Superoxide Dismutase(SOD)and Catalase(CAT),along with non-enzymatic antioxidants like glutathione,melatonin,and resveratrol,have demonstrated the ability to neutralize ROS and improve oocyte viability and developmental outcomes.Recent studies highlight the potential of Mitoquinone(MitoQ),a mitochondria-targeted antioxidant,to effectively counteract mitochondrial ROS and enhance cellular defense mechanisms during cryopreservation.This review explores the cellular mechanisms of cryodamage,the role of oxidative stress in oocyte cryopreservation,and the potential of various antioxidant strategies to enhance oocyte survival and function.Developing effective antioxidant supplementation approaches may significantly improve the outcomes of cryopreservation in reproductive medicine.
基金supported by the National Natural Science Foundation of China(31972759 and 31572589)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)。
文摘Background Polystyrene nanoplastics(PS-NPs)are becoming increasingly prevalent in the environment with great advancements in plastic products,and their potential health hazard to animals has received much attention.Several studies have reported the toxicity of PS-NPs to various tissues and cells;however,there is a paucity of information about whether PS-NPs exposure can have toxic effects on mammalian oocytes,especially livestock.Herein,porcine oocytes were used as the model to investigate the potential effects of PS-NPs on mammalian oocytes.Results The findings showed that different concentrations of PS-NPs(0,25,50 and 100μg/m L)entering into porcine oocytes could induce mitochondrial stress,including a significant decrease in mitochondrial membrane potential(MMP),and the destruction of the balance of mitochondrial dynamic and micromorphology.Furthermore,there was a marked increase in reactive oxygen species(ROS),which led to oocyte lipid peroxidation(LPO).PS-NPs exposure induced abnormal intracellular iron overload,and subsequently increased the expression of transferrin receptor(TfRC),solute carrier family 7 member 11(SLC7a11),and acyl-CoA synthetase long-chain family member 4(ACSL4),which resulted in ferroptosis in oocytes.PS-NPs also indu ced oocyte maturation failure,cytoskeletal dysfunction and DNA damage.Cotreatment with 5μmol/L ferrostatin-1(Fer-1,an inhibitor of ferroptosis)alleviated the cellular toxicity associated with PS-NPs exposure during porcine oocyte maturation.Conclusions In conclusion,PS-NPs caused ferroptosis in porcine oocytes by increasing oxidative stress and altering lipid metabolism,leading to the failure of oocyte maturation.
文摘[Objective] The aim was to optimize Yanbian cow oocytes mature in vitro and cleavage system after nuclear transfer based on uniform design. [Method] Oocytes were recovered by aspiration method, and oocytes were matured in vitro （IVM） with different conditions, and then carried out nucleus transfer, fusion, activation and in vitro culture （IVC） of embryo. Effects of ovary storage temperature, maturation time and follicular diameter size on in vitro maturation and cleavage rates of cow oocytes were compared. [ Result] The best conditions of IVM of Yanbian cow oocytes was that： the oocytes of 8 mm diameter were matured in vitro for 24 hours when the ovaries were stored at 26℃ or 31 ℃. The best cleave conditions after nucleus transfer of oocytes was that： the oocytes of 6 mm or 8 mm diameter were cultured in vitro for 24 hours when the ovaries were stored at 26℃. [ Conclusion] The result has some reference to Yanbian cow and other cow breeding and population expanding propagation.
基金Supported by National Natural Science Foundation of China (30871431)Outstanding Youth Fund of Heilongjiang Province (JC200905)~~
文摘[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first polar body extrusion rate, oocyte glutathione （GSH） content, positive rate of brilliant cresyl blue （BCB） staining and development potential of activated oocytes or fertilized oocytes were employed as main indicators to investigate the effects of follicular mural granulosa cell （MGC） coculture on cytoplasmic maturation of cumulus cell-removal oocytes （Denuded Oocyte, DO）. [Result] According to in vitro maturation results, compared with DO group, the first polar body extrusion rate of porcine oocytes in DO＋MGC group was not significantly different, but the nuclear maturation process was improved and was more similar to that in COC （cumulus-oocyte complex） group. Detection of GSH content in mature oocytes showed that there was no significant difference between DO＋ MGC group （optical density of 1 053.67） and COC group （optical density of 1 426.00） or between DO＋MGC group and COC＋GC group （optical density of 1 541.00）, however, GSH content in mature oocytes of DO group （optical density of 724.67） was significantly lower than that of COC group and COC＋GC group （P0.05）. Detection of glucose-6-phosphate dehydrogenase （G6PDH） activity showed that there was no significant difference in BCB positive oocyte rate between DO ＋MGC group （88.26% ） and COC group （92.75%） or between DO＋MGC group and DO group （82.86% ）, however, BCB positive oocyte rate of DO group was significantly lower than that of COC group （P0.05）. Furthermore, the cleavage rate and blastocyst rate of activated mature oocytes derived from DO ＋MGC group （94.98% and 43.67% , respectively） were significantly higher than those from DO group （52.54% and 8.97%, respectively） （P0.05）, and were not significantly different compared with those from COC group （97.11% and 38.30%, respectively）. In addition, the cleavage rate of fertilized oocytes derived from DO＋MGC group （72.65%） showed no significant difference compared with that from DO group （63.59%）, but the blastocyst rate of DO＋MGC group was significantly higher than that of DO group （9.88%） （P0.05）. [Conclusion] MGC coculture can significantly improve the in vitro cytoplasmic maturation quality of denuded porcine oocytes, thereby enhancing the subsequent developmental potential.
文摘[Objective] The research aimed to enhance culture efficiencies of oocyte and embryo of goat in vitro and to explore serum-free culture system in vitro.[Method] At present,the conventional solutions of oocyte maturation and embryo development in vitro were always added into 1% ITS(Insulin-transferrin-selenium) or using 1% ITS to replace FBS in 2 kinds culture solutions for conducting in vitro cultures of goat oocyte and parthenogenetic embryo.The influences of ITS on their developments were detected.[Result] ITS in maturation liquid of oocytes could not increase oocytes maturation rate but significantly increased blastocyst rate (58.06% vs. 48.19%)of parthenogenetic embryo.If FBS in maturation liquid of oocytes was replaced by ITS, the maturation rate, cleavage rate and blastocyst rate were basically unchanged.Adding ITS into embryo medium could increase blastocyst rate (68.30% vs. 56.82%)of parthenogenetic embryo of goat.If FBS in embryo medium was replaced by ITS,the cleavage rate didn’t change basically,while the blastocyst rate in ITS was obviously lower than that in FBS group(42.33% vs.56.82%).[Conclusion] ITS could promote maturation of oocyte in vitro and early embryonic development, in addition,ITS could replace serum in maturation medium of oocyte as serum-free culture system for conducting relevant researches.
文摘Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to explore the relationship between oocyte maturation and GDF-9 gene expression. Preantral follicles of lO-day old mice were isolated from the ovaries and were cultured for 12 days. Oocytes from day 2 （D2）, D4, D6, D8, DIO, D12 cultured in vitro were named the in vitro group and oocytes of day 12 （D12）, D14, D16, D18, D20, D22 grown in vivo were named the in vivo group. Follicle survival, antrum formation and maturation rate were 89.5%, 51.8% and 56.6% respectively in follicles cultured in vitro. After RT-PCR and agarose gel electrophoresis, relative mRNA abundance of GDF-9 was measured in each group of oocytes. At day 8 - 12, the GDF-9 gene expression level of oocytes in vitro was significantly lower than that in vivo （P 〈 0.05）. We conclude that M Ⅱ oocytes can be obtained from in vitro culture of preantral follicles. The GDF- 9 gene expression of oocytes varies at different growth stages in vivo. The low expression of GDF-9 in oocytes cuhured in vitro may be the cause of their low developmental capacity.
基金Supported by School Program of Henan Institute of Science and Technology(20060516)~~
文摘[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).
文摘Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.
基金The work was supported by grants from the Shanghai Committee of Science and Technology, China （Grant No. 09411964200）, the Major State Basic Research Development Program of China （973 Program, No. 2014CB943104） and the National Natural Science Foundation of China （81270744）.
文摘Azoospermia, cryptozoospermia and necrospermia can markedly decrease the ability of males to achieve pregnancy in fertile females. However, patients with these severe conditions still have the option to be treated by intracytoplasmic sperm injection （ICSI） to become biological fathers. This study analyzed the fertilization ability and the developmental viabilities of the derived embryos after ICSI treatment of the sperm from these patients compared with in vitro fertilization （IVF） treatment of the proven-fertile donor sperm on sibling oocytes as a control. On the day of oocyte retrieval, the number of sperm suitable for ICSI collected from two ejaculates or testicular sperm extraction was lower than the oocytes, and therefore, excess sibling oocytes were treated by IVF with donor sperm. From 72 couples （73 cycles）, 1117 metaphase Ⅱ oocytes were divided into 512 for ICSI and 605 for IVF. Compared with the control, husbands＇ sperm produced a lower fertilization rate in nonobstructive azoospermia （65.4% vs 83.2%; P〈 0.001）, crytozoospermia （68.8% vs 75.5%; P〈 0.05） and necrospermia （65.0% vs 85.2%; P〈 0.05）. The zygotes derived in nonobstructive azoospermia had a lower cleavage rate （96.4% vs 99.4%; P 〈 0.05）, but the rate of resultant good-quality embryos was not different. Analysis of the rates of cleaved and good-quality embryos in crytozoospermia and necrospermia did not exhibit a significant difference from the control. In conclusion, although the sperm from severe male infertility reduced the fertilization ability, the derived embryos had potential developmental viabilities that might be predictive for the expected clinical outcomes.
文摘The significance of the performance of conventional in vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) using sibling oocytes from couples with subfertile male or unexplained infertility was evaluated. A total of 410 sibling oocyte cumulus-corona complexes (OCCC) from 21 couples with subfertile male (group A) and 11 unexplained infertile couples (group B) were randomly divided, in order of retrieval, into two groups inseminated either by conventional IVF or by ICSI. The treatment outcomes and the influence of infertility factors on fertilization in each group were compared. The results showed that although the two pronuclear (2PN) fertilization rate per injected sibling oocytes was significantly higher after ICSI (group A: 68.2 %±28.8 %; group B: 66.2 %±24.9 %) than after conventional IVF (group A: 41.8 %±32.7 %; group B: 40.1 %±22.1 %), the other variables studied included: the fertilization rates of per allocated sibling oocytes IVF/ICSI, the fertilization rates of sibling oocytes IVF/ICSI after excluding failed IVF fertilization cycles, as well as the cleavage rates of normal fertilization were not statistically significant (P>0.05). Similarly, though the total fertilization failure rate in the IVF group (group A: 42.9 %; group B: 36.4 %) was significantly higher than in the ICSI group (group A: 4.8 %; group B: 0), we did not cancel cycles due to the normal fertilization of sibling oocytes. Embryo transfer was possible in all 32 couples. There were 10 clinical pregnancies in the two groups. We also discovered a possible association between some semen parameters and sperm functions of group A, and women age and duration of infertility of group B and fertilization. It is suggested that adoption of the split IVF/ICSI technology in the above cases may help eliminate fertilization failures. This is also a useful method to investigate the effect of single factor on the employment of assisted reproductive technology.
文摘Somatic nuclei can be reprogrammed into a pluripotent state by nuclear transfer, cell fusion and expression of transcription factors. However, these reprogramming processes are very inefficient, which has greatly hindered efforts to elucidate the underlying molecular mechanisms. Here, we report a new reprogramming strategy that combines the advantages of all three reprogramming methodologies into one process. We injected nuclei from cumulus cells into intact MII oocytes. Following activation, 80% of the reconstructed embryos developed to the blastocyst stage, and tetraploid （4N） embryonic stem （ES） cell lines were generated at a rate of 30% per reconstructed oocyte. We also generated triploid （3N） ES cells after injection of somatic nuclei into activated oocytes. 4N and 3N ES cells expressed pluripotent markers and differentiated into cell types of three embryonic germ layers in vivo. Moreover, all ES cells generated histocompatible, differentiated cells after being engrafted in immunocompetent B6D2F1 mice, showing that ES cells derived from this reprogramming strategy might serve as a source of genetically tailored tissues for transplantation. Thus, we have established a simple and highly efficient reprogramming procedure that provides a system for investigating the molecular mechanisms involved in somatic reprogramming.
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 us, 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 17.0016.12 vs. (25.30 ± 7.55)% and 18.0116.79, 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.
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Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope （LSCM） was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional （3D） image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM. The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of MⅡocytes.
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.
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.
