Maternal aging can impair the quality and decrease the developmental competence of ovulated oocytes. In this study, compromised germinal vesicle breakdown (GVBD) was found in aged mice oocytes. Furthermore, we observed increased reactive oxygen species (ROS) and mitochondrial Ca2+ levels, along with reduced mitochondrial temperature in aged oocytes. Maternal aging also changed the crotonylation level in oocytes. Forkhead box O3 (FoxO3a), a member of the forkhead protein family involved in the regulation of cell survival and life span reached a peak level in the metaphase II stage. Compared with a younger group, FoxO3a expression increased in aged oocytes. Intracellular localization of FoxO3a changed from the cytoplasm to chromatin in response to aging. The expression of the upstream regulator nicotinamide-phosphoribosyltransferase (Nampt) peaked in the GVBD stage. Moreover, Nampt expression was increased in aged oocytes, and more intense staining of Nampt was found in aged mice ovary. To further study the role of Nampt in mitochondrial function, specific agonist P7C3 and inhibitor FK866 were applied to aged oocytes, and FK866 significantly decreased adenosine triphosphate and mitochondrial membrane potential. In conclusion, mitochondrial dysfunction in aged oocytes was associated with elevated FoxO3a, and suppression of Nampt could further impair mitochondrial function.

为探究春、秋、冬三季以东弗里升奶绵羊为供体进行体内胚胎生产及移植的效果,并为建立高效优质种羊扩繁体系提供理论依据,试验选用99只东弗里升奶绵羊为供体、934只乌珠穆沁羊为受体,在春、秋、冬季进行体内胚胎生产和移植,45 d后进行B超检测.结果表明:左侧子宫回收胚胎数为春季5.92枚,秋季7.76枚,冬季6.33枚,季节之间差异不显著(P＞0.05);右侧子宫回收胚胎数秋季(8.53枚)显著高于春季(5.08枚)和冬季(5.69枚,P＜0.05);回收胚胎数秋季(16.33枚)显著高于春季(10.42枚)和冬季(12.19枚,P＜0.05);可用胚胎数为春季9.08枚,秋季11.56枚,冬季9.36枚,季节之间差异不显著(P＞0.05);单胚移植妊娠率秋季(63.34％)和冬季(58.99％)显著高于春季(32.79％,P＜0.05);双胚移植妊娠率秋季(86.21％)显著高于冬季(50.00％,P＜0.05);妊娠率为秋季(66.21％)略高于春季(51.76％)、冬季(60.71％),季节之间差异不显著(P＞0.05);产羔率为春季(68.18％)略高于秋季(67.83％)、冬季(65.13％),季节之间差异不显著(P＞0.05).说明春、秋、冬三季东弗里升奶绵羊超排和胚胎移植效果相近,其中秋季进行东弗里升奶绵羊体内胚胎生产和移植效果最佳.

Oocyte cryopreservation demonstrates great benefits in the conservation of animal germplasm resources and assisted reproductive technology. However, vitrification causes damages in oocytes, which would lead to the decrease of oocyte quality, and embryonic development post fertilization. Cytoskeleton plays an important role in regulating cell shape, organelle migration, cell division and mechanical signal transduction. Cortical tension is a reflection of the physiological state and contractile ability of cortical cytoskeleton. Appropriate cortical tension is prerequesite for normal oocyte meiosis. In the present study, oocyte cortical tension was examined by evaluating the levels of cortical tension-related protein pERM (Phospho-Ezrin/Radixin/Moesin) and pMRLC (Phospho-Myosin Light Chain 2). We found that the cortical tension of vitrified oocytes was decreased. Increasing cortical tension of vitrified oocytes by adding 10 μg/ml ConA during in vitro culture could significantly improve the polar body extrusion rate and embryo development. Furthermore, increasing the cortical tension could improve spindle positioning, maintain kinetochore-microtubule (KT-MT) attachment, strengthen spindle assembly checkpoint (SAC) activity, and reduce the aneuploidy rate in vitrified oocytes. In conclusion, vitrification induced a remarkable decrease in cortical tension, and increasing the cortical tension could rescue the meiosis defect and improve oocyte quality.

Cryopreservation of embryos is important for long-distance embryo transfer and conservation of genetic resources. Porcine research is important for animal husbandry and biomedical research. However, porcine embryos are difficult to cryopreserve because of their high cytoplasmic lipid content and sensitivity to chilling stress. Vitrification is more efficient than slow freezing, and vitrification is mostly used in embryo cryopreservation. So far, the vitrification process of porcine embryos has been continuously improved, resulting in improved survival rates of warmed embryos and farrowing rates after the transplant procedure. It is worth noting that automatic vitrification has made great progress, which is expected to promote the standardization and application of vitrification. In this article, the vitrification process of porcine embryos at the blastula stage and early development stages is reviewed in detail. In addition, the efficiency of different vitrification systems was compared. In addition, we summarize technology that can improve the survival rate of cryopreserved porcine embryos, such as delipidation methods (including physical delipidation and chemical delipidation) and medium improvements (including chemically defined media and adding antioxidants). Meanwhile, gene expression changes during cryopreservation are also elaborated.

[Ca2+]i is essential for mammalian oocyte maturation and early embryonic development, as those processes are Ca2+ dependent. In the present study, we investigated the effect of [Ca2+]i on in vitro maturation and reprogramming of oocytes in a lower calcium model of oocyte at metaphase II (MII) stage, which was established by adding cell-permeant Ca2+ chelator BAPTA-AM to the maturation medium. Results showed that the extrusion of the first polar body (PB1) was delayed, and oocyte cytoplasmic maturation, including mitochondrial and endoplasmic reticulum distribution, was impaired in lower calcium model. The low-calcium-model oocytes presented a poor developmental phenotype of somatic cell nuclear transfer (SCNT) embryos at the beginning of activation of zygotic genome. At the same time, oxidative stress and apoptosis were observed in the low-calcium-model oocytes; subsequently, an RNA-seq analysis of the lower-calcium-model oocytes screened 24 genes responsible for the poor oocyte reprogramming, and six genes (ID1, SOX2, DPPA3, ASF1A, MSL3, and KDM6B) were identified by quantitative PCR. Analyzing the expression of these genes is helpful to elucidate the mechanisms of [Ca2+]i regulating oocyte reprogramming. The most significant difference gene in this enriched item was ID1. Our results showed that the low calcium might give rise to oxidative stress and apoptosis, resulting in impaired maturation of bovine oocytes and possibly affecting subsequent reprogramming ability through the reduction of ID1.

Intracellular lipids provide energy for oocyte maturation and development. Triglycerides are the main components of cytoplasm lipid droplets, and hydrolysis of triglycerides requires several lipase-mediated steps. The aim of this study was to determine the effects of the β-adrenoceptor agonist isoproterenol (ISO) and the hormone-sensitive lipase (HSL) inhibitor CAY10499 on the IVM of porcine oocytes. ISO (5mg L−1) and CAY10499 (20mg L−1) had positive and negative effects respectively on invitro oocyte maturation and subsequent embryo development. The rates of polar body extrusion, cleavage and blastocyst formation were significantly higher in the ISO-treated group than the control and CAY10499-treated groups. ISO treatment also upregulated intracellular cAMP levels in comparison with the control group, while CAY10499 significantly increased the triglyceride content of matured oocytes when compared with other groups, consistent with the observed decrease in LIPE (HSL) mRNA levels. Furthermore, the inhibitory effects of CAY10499 included decreases in mitochondrial membrane potential and mitochondrial temperature. These results indicate that ISO has a positive effect on the IVM of porcine oocytes, and that intracellular lipid metabolism can be modulated by CAY10499 through inhibition of HSL and is closely related to mitochondrial function.

Oocytes vitrification is frequently applied in assisted reproductive technologies. However, chromosomes segregation was error-prone during meiosis maturation of vitrified oocytes. The fidelity of chromosomes segregation depends on the correct kinetochore-microtubule attachments (KT-MTs). In meiosis I, the Aurora B/C would not spatially separate from the attachment sites upon bivalents stretched. Oocytes lack a mechanism for coordinating bivalent stretching and Aurora B/C inhibition in meiosis I. Thus, the KT-MTs are unstable in oocytes. In this study, we firstly found the incorrect KT-MTs were markedly increased in vitrified oocytes. The Aurora B/C activity in vitrified oocytes was significantly increased when the bivalents were stretched. This Aurora B/C activity could not induce a SAC response, as the SAC protein Mad2 was significantly decreased during MI stage in vitrified oocytes. Thus, the KT-MTs in vitrified oocytes were error-prone. This study, for the first time, revealed the mechanism of the incorrect KT-MTs occurred in vitrified oocytes and provided a theoretical basis for further improvement of oocytes vitrification.

This study investigated the effect of aging on meiosis progression, embryo developmental competence and DNA double-strand breaks (DSBs) in mouse oocytes and resultant early embryos. Germinal vesicle (GV) oocytes were first cultured to monitor the progression of germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) during in vitro maturation (IVM), then the harvested metaphase II (MII) oocytes were parthenogenetically activated to evaluate pronuclear (PN) formation of parthenogenetic embryo and embryo development. The cytoplasmic maturation was examined by measuring the intracellular reactive oxygen species (ROS) and glutathione (GSH). DNA DSBs were examined by immunostaining of pi-H2AX, the marker of DNA DSBs. The results showed that the GVBD rates were similar in oocytes of young and aged mice. Polar body extrusion was significantly delayed in aged mice (P < 0.05), however the rate of polar body extrusion was similar to that of young mice at 16 h of IVM. Moreover, PN formation of parthenogenetic embryo was significantly delayed in aged mice (P < 0.05). Afterward the two groups obtained similar results with respect to the percentages of activated oocytes, 2-cell embryos and blastocysts. The cytoplasmic maturation of MII oocytes and blastocysts in aged mice were significantly compromised to those of young mice (P < 0.05). Furthermore, GV oocytes, 2-cell embryos and blastocysts showed significantly higher relative intensities of pi-H2AX in aged mice (P <0.05). Taken together, our result indicate that aging disturbed oocyte maturation and parthenogenetic embryo development, which could be related to insufficient cytoplasmic maturation and worsening DNA DSBs in oocytes and early embryos.

Extracellular calcium is required for intracellular Ca2+ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium-sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R-568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R-568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation-dependent [Ca2+ ]i rise. When egg activation was conducted in intracellular Ca2+ chelator BAPTA-AM and NPS R-568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca2+ ]i effector p-CAMKII, and the presence of KN-93, an inhibitor of CAMKII, significantly reduced the CASR-mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca2+ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII.

Vitrification of germinal vesicle (GV) stage oocytes has been shown to be closely associated with decreased rates of meiosis maturation and increased rates of aneuploidy. However, little is known about the effects of melatonin on these events in mice vitrified GV oocytes. In this study, the effects of melatonin on meiosis maturation potential and the incidence rate of aneuploidy in mouse vitrified oocytes were analyzed by supplementing in vitro maturation (IVM) solution with melatonin at different concentrations. This study, for the first time, showed that the mitochondrial heat production was markedly increased in vitrified oocytes (P < 0.05), which compromised the first polar body extrusion (PBE) of vitrified oocytes (73.3% vs. 85.1%, P < 0.05). However, 10-11 mol/L melatonin could significantly decrease mitochondrial heat production and ROS level (9.1 vs. 12.0 pixels, P < 0.05), meanwhile increase ATP level (1.1 vs. 0.88 pmol, P < 0.05) and mtDNA copies (107438 vs. 67869, P < 0.05), which rescued the abnormal chromosome alignment (32% vs. 69%, P < 0.05) and reduced the incidence of aneuploidy (15.6% vs. 38.5%, P < 0.05) in vitrified oocytes. The meiosis maturation ability of vitrified oocytes with melatonin supplementation was similar to that of fresh ones (83.4% vs. 85.1%, P > 0.05). Collectively, our data revealed that melatonin has a protective action against vitrification-induced injuries of oocytes meiosis maturation.

Genetic modification provides a means to enhancing disease resistance in animals. In this study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was produced by microinjection for better disease resistance. To compare semen characteristics including sperm quality, seminal plasma biochemical index, sperm DNA methylation and pregnancy rate of three-year old transgenic sheep with TLR4 overexpressed (toll like receptor 4, TLR4) and non-transgenic ram. Sixteen transgenic ram of F0 generation were produced by microinjection of the TLR4 plasmid into the pronucleus of fertilized ova. Seven transgenic sheep of F1 generation was produced by breeding F0 transgenic founders with non-transgenic sheep of the same breed. There were no significant differences between transgenic and control rams for all semen quality parameters, including semen volume, sperm concentration, sperm viability, and percentages of sperm with an intact plasma membrane, acrosomal integrity, and viable sperm with high mitochondrial membrane potential in both F0 and F1 generation. Furthermore, no significant differences were found for seminal plasma concentrations of zinc, neutral alpha-glucosidase, acid phosphatase or fructose, nor for levels of H19 and IGF2R methylation in sperm DNA. In addition, pregnancy rate was also similar between these two groups. In conclusion, there was no evidence that TLR4 overexpression altered the sperm quality, seminal plasma or sperm DNA of transgenic sheep.

Cryopreservation has undergone tremendous advances and is widely used in animal production based on decades of study of cellular permeability, freezability and empirical generalization. Several improvement are particularly important: the cryopreservation protocol has been continuously refined over the years to achieve greater reproductive performance; cryoprotective agents are more effective and less toxic than previously; there has been significant innovation in advanced cryopreservation systems and carriers. Despite this, there are still problems that urgently require practical solutions, such as remedies for cryodamage and encouraging the use of frozen–thawed porcine sperm in pig production.

BACKGROUND:The open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. This study examined the effects of OPS vitrification on embryo development and the reproductive capacity of the transgenic offspring in order to establish a method for preservation of microinjected embryos.METHODS:Ovine pronuclear embryos were microinjected with the exogenous aralkylamine N-acetyltransferase gene (AANAT), frozen by the OPS method, and subsequently thawed for embryo transplantation. Pregnancy rate, lambing rate, survival rate, average birth weight and transgenic positive rate as well as reproduction efficiency and hormone level of the transgenic offspring were investigated to analyze the effect of OPS vitrification on microinjectd pronuclear embryos.RESULTS:No significant differences were observed in the birth rate, lamb survival rate and transgenic positive rate between the frozen and non-frozen AANAT-microinjected pronuclear embryos. The average birth weight of the frozen embryos offspring was greater than that of the non-frozen embryos. Importantly, the transgenic offspring that overexpressed the AANAT gene showed improved ovulation efficiency and lambing rate by regulating their hormone levels.CONCLUSIONS:The OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. In addition, the AANAT+ transgenic offspring exhibited improved reproductive capacity on account of regulation effect of melatonin on reproductive hormone. These data may provide available references for human-assisted reproduction.

Embryo vitrification has advantages in assisted reproduction yet it also induces zona hardening. Laser zona thinning (LZT) is considered as a solution yet its efficacy and security have not been well studied. In this study, we used vitrified-warmed morulae from 2-month-old and 10-month-old ICR female mice as model to investigate the impacts that LZT treatment brings to the in vitro hatching process and implantation by analyzing hatching rate, implantation rate, and blastocyst quality. The results showed that the fully hatched rate was significantly higher after LZT treatment for both young (25.7% vs. 16.2%, P < 0.05) and aged (36.6% vs. 13.2%, P < 0.01) mice. For zona-thinned morulae in young mice, its onset of hatching occurred earlier (28.6% vs. 8.8%, P < 0.01) at D4 and with a greater percentage of U-shaped hatching at D5 (48.3% vs. 33.0%, P < 0.05). LZT treatment did not induce expression change of apoptosis-related genes in all groups (P > 0.05), but for young mice, the total cell number of day 5 blastocyst in zona-thinned group was significantly less than that of the control group (40.6 ± 5.1 vs. 59.9 ± 14.5, P < 0.01). At last, there was an increasing implantation rate in zona-thinned compared to the control group for young (63.8% vs. 52.5%, P > 0.05) and aged (55.6% vs. 47.2%; P > 0.05) mice after embryos were bilaterally transferred in the same recipient. In conclusion, the significant increase of fully hatched rate after LZT treatment is related to the advanced onset of hatching as well as the enhancement of superior hatching structure, and LZT also lead to a better implantation after embryo transfer.

To improve the embryo transfer efficiency of elite species of sheep with high meat quality,a total of 76 Black Suffolk and 27 Dorper ewes were treated by "CIDR+FSH+PMSG" method for superovulation as donors.The 384 small tailed han sheep were treated by "CIDR+PMSG" method for estrus synchronization as receptors.It was explored to test the influence of developmental stages of embryo,frozen processing of the embryo,embryo in vitro residence time and transplantation of lateral corpus luteum number of receptor on the pregnancy rates after embryo transfer.The pregnancy rate of blastocyst transplantation was significantly higher than morula transplantation (P＜0.01) when a single fresh embryo was transplanted.The frozen/thaw blastocyst transplantation pregnancy rate was significantly lower than fresh blastocyst (P＜0.01),but the difference between frozen/thaw morula and fresh morula was not significant(P＞0.05).In addition,the pregnancy rate with transplanted morula would be significantly affected (P＜0.01),the transplanted blastocyst would not be significantly affected(P＞0.05),when the transplanted-side-ovary of receptor had 1 corpus luteum or 2～3 corpus luteums.In conclusion,when morula and blastocyst were tansplanted into uterine horn,higher pregnancy rate could be achieved if a fresh blastocyst or a frozen morula was transfered.Fresh morula should be transplanted to the receptor with 2～3 corpus luteums on ovary transplantation side instead of 1 corpus luteum.

Objective:To study the effects of glucose and Zuogui Pill serum on the in vitro development of mouse embryos in high glucose.Methods:Embryos were cultured in vitro and the embryonic development was inhibited by high level of glucose.Zuogui Pill serum was used to increase the development of embryos.The blastocyst rate,2-cell rate and blastocyst nucleus count were used to evaluate the embryonic development.Results:High level of glucose inhibited the embryonic development,and 40mmol/L was selected as the concentration of high glucose to inhibit the embryo development.Compared with the control group,the blastocyst rate of high glucose was decreased (P＜0.05).Compared with the model group,blastocyst rate of Zuogui Pill serum was increased (P＜0.05).From the blastocyst nucleus counting,the high glucose environment reduced the number of blastocyst cells (P＜0.05,P＜0.01).Zuogui Pill serum further reduced the number of blastocysts,but it could improve the rate of blastocyst.Conclusion:High level of glucose reduces the development of embryo while Zuogui Pill serum promotes the blastocyst rate,and promote embryonic development.

BACKGROUND:Maternal obesity alters oocytes and subsequent fetal metabolism. An increasing number of studies have shown that the endoplasmic reticulums (ER) or mitochondria have important effects on oocyte quality, but there has been no study of the effect of mitochondria-associated ER membranes (MAMs) on oocyte quality. The present study was designed to assess whether the level of MAM and MAM-related proteins were different in oocytes from obese and control mice.RESULTS:First, oocytes from mice with high-fat-diet (HFD)-induced obesity had higher levels (either greater numbers or a higher proportion for the same numbers) of MAM than oocytes from control mice. The abundance of MAM-related proteins in oocytes from obese mice was significantly greater at both the messenger RNA and protein levels, including inositol 1,4,5-trisphosphate receptor, type 1 (IP3R1), inositol 1,4,5-trisphosphate receptor, type 2 (IP3R2) and phosphofurin acidic cluster sorting protein 2 (PACS-2). Further, there was an increase in mitochondrial Ca2+ ([Ca2+]m) which was associated with increased apoptosis and compromised cytoplasmic maturation in oocytes from obese mice. Down-regulation of MAM-related protein IP3R1 in oocytes from obese mice decreased [Ca2+]m and apoptosis and improved cytoplasmic maturation but did not reduce the overall MAM level. However, down-regulating MAM-related protein PACS-2 in oocytes from obese mice did reduce the level of MAM and [Ca2+]m, which decreased the rate of apoptosis and improved cytoplasmic maturation of oocytes from obese mice.CONCLUSIONS:It is possible that enriched MAM could increase [Ca2+]m, and this increase has been found to be associated with increased apoptosis and compromised cytoplasmic maturation in oocytes from obese mice. This finding suggests a novel therapeutic target for obesity-induced oocyte defects.

In vitro maturation (IVM) and vitrification have been widely used to prepare oocytes before fertilization; however, potential effects of these procedures, such as expression profile changes, are poorly understood. In this study, mouse oocytes were divided into four groups and subjected to combinations of in vitro maturation and/or vitrification treatments. RNA-seq and in silico pathway analysis were used to identify differentially expressed genes (DEGs) that may be involved in oocyte viability after in vitro maturation and/or vitrification. Our results showed that 1) 69 genes were differentially expressed after IVM, 66 of which were up-regulated. Atp5e and Atp5o were enriched in the most significant gene ontology term "mitochondrial membrane part"; thus, these genes may be promising candidate biomarkers for oocyte viability after IVM. 2) The influence of vitrification on the transcriptome of oocytes was negligible, as no DEGs were found between vitrified and fresh oocytes. 3) The MII stage is more suitable for oocyte vitrification with respect to the transcriptome. This study provides a valuable new theoretical basis to further improve the efficiency of in vitro maturation and/or oocyte vitrification.

BACKGROUND Antioxidants protect spermatozoa against cell damage during cryopreservation. OBJECTIVE To investigate whether melatonin supplement in the extender may improve the quality of cryopreserved mouse sperm. METHODS Kunming mice sperm frozen in extender R18S3 (18% (w/v) raffinose and 3% (w/v) skim milk) supplemented with melatonin were thawed and evaluated. RESULTS Mouse spermatozoa were cryopreserved in the freezing extender R18S3 that contained melatonin at 0, 0.125, 0.25 and 0.5 mg/mL melatonin. The extender without melatonin supplement was associated with increased formation of reactive oxygen species (ROS) and decreased sperm motility. Melatonin supplement at 0.125 mg/mL significantly increased the progressive motility of sperm in comparison to other melatonin concentration or control. The percentage of thawed viable sperm with ROS was lower in the melatonin-treated groups than in untreated group. Melatonin supplement also increased antiapoptotic gene Bcl-xl expression in the thawed sperm. CONCLUSION Supplement of 0.125 mg/mL melatonin could reduce oxidative damage and apoptosis.

卵母细胞玻璃化冷冻导致的胚胎发育阻滞与其DNA甲基化模式的异常密切相关.本研究以小鼠为模型,旨在探讨玻璃化冷冻对卵母细胞及其形成的早期胚胎中DNA甲基转移酶(DNMTs)表达模式的影响.采用免疫荧光染色结合激光共聚焦显微成像技术检测了DNMT1、DNMT3A和DNMT3B在新鲜和冷冻卵母细胞及其形成的各阶段早期胚胎中的表达与分布,结果表明:在卵母细胞及受精后的合子中冷冻组3种DNMTs的表达均出现了异常.采用实时定量PCR技术检测了新鲜和冷冻卵母细胞及其形成的囊胚中,Dnmt1、Dnmt3a和Dnmt3b的mRNA表达水平,显示在冷冻卵母细胞中3种Dnmts的表达水平均显著下降,至囊胚阶段Dnmt3b的表达水平仍然很低(P＜0.05).研究结果表明卵母细胞玻璃化冷冻导致了小鼠早期胚胎发育过程中DNMTs表达模式的异常,这可能是DNA甲基化异常的重要原因之一.