Nampt affects mitochondrial function in aged oocytes by mediating the downstream effector FoxO3a.

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.

Effect of L-citrulline supplementation on sperm characteristics and hormonal and antioxidant levels in blood and seminal plasma of rams.

With the aim of providing a theoretical basis for the application of L-citrulline (L-Cit) in animal husbandry, the effects of L-Cit on reproductive hormone levels, antioxidant capacity and semen quality of rams were studied by feeding them varying doses of L-Cit. A total of 32 rams were randomly divided into four groups with eight rams each. After all rams were trained to donate sperm normally, the control group was fed a basic diet, whereas the experimental groups I, II and III were provided with feed supplemented with 4, 8 and 12 g/d of L-Cit respectively. The experiment was conducted for 70 days, during which blood samples were collected from the jugular vein on days 0, 15, 30, 45 and 60, and semen samples were collected on days 0, 20, 40 and 60. In the same group, 100 µl of semen was used to test for quality, The rest of the semen sample and blood samples were centrifuged at 600 g for 15 min, and the supernatant and serum, respectively, were used to determine the levels reproductive hormones and antioxidant indices. Ram semen samples were also collected on day 70 and used to study sperm plasma membrane, substitution and mitochondrial membrane potential. Compared with the control group, the groups receiving L-Cit showed an increase in sperm concentration and number of linear motile sperm (p < .01); a decrease in the number of dead sperm (p < .01); an increase in sperm viability, particularly in groups II and III (p < .01); and an increase in sperm mitochondrial membrane potential (p < .01). Moreover, groups I, II and III showed significantly higher levels of serum gonadotropin-releasing hormone (GnRH), glutathione peroxidase (GSH-Px) and nitric oxide (NO) (p < .01). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels increased in groups I (p < .05), II (p < .05) and III (p < .01), whereas testosterone (T), catalase (CAT) and superoxide dismutase (SOD) levels increased in groups I and II (p < .01). Serum total antioxidant capacity (T-A) increased (p < .05), whereas both hydroxyl radical (·OH) and peroxy radical ( O 2 · - ) levels decreased (p < .01). Compared with the control, all groups had significantly higher SOD and GSH-Px in their seminal plasma (p < .01), and groups I, II (p < .05 for both) and III (p < .01) had higher levels of GnRH and FSH. LH, CAT and NO levels increased in group I (p < .05), II and III (p < .01 for both); malondialdehyde levels decreased in groups I, II (p < .05 for both) and group III (p < .01); and O 2 · - levels decreased in groups I, II and III (p < .01). Under our experimental conditions, GnRH, FSH, LH, T, CAT, SOD, T-A, GSH-PX and NO levels in the serum and seminal plasma of rams receiving L-Cit increased, whereas Oestradiol (E2 ), O 2 · - and ·OH levels in the seminal plasma decreased; this improved the semen quality of rams supplemented with L-Cit. Moreover, supplementation with 12 g/d gave the best results.

Dihydroartemisinin exposure impairs porcine ovarian granulosa cells by activating PERK-eIF2α-ATF4 through endoplasmic reticulum stress.

Dihydroartemisinin (DHA) is an artemisinin derivative commonly used in malaria therapy, and a growing number of studies have focused on the potent anticancer activity of DHA. However, the reproductive toxicity of anticancer drugs is a major concern for young female cancer patients. Previous studies have suggested that DHA can cause embryonic damage and affect oocyte maturation. Here, we explored the side effects of DHA exposure on ovarian somatic cells. We exposed porcine granulosa cells to 5 µM and 40 µM DHA for 24 h or 48 h in vitro. DHA inhibited granulosa cell viability in a dose-dependent manner and, in the 48 h treatment group, DHA enhanced the apoptotic rate. We observed that the levels of intracellular calcium, mitochondrial calcium, and ATP concentration were elevated with DHA treatment. In granulosa cells exposed to DHA, the mRNA levels of endoplasmic reticulum stress-related genes GRP78 and ATF4 were increased. Furthermore, analysis of the unfolded protein response signaling pathway showed that the protein levels of P-PERK, P-eIF2α, and ATF4 were upregulated by DHA exposure. These results demonstrate that in granulosa cells, DHA exposure induces endoplasmic reticulum stress that then activates the PERK/eIF2α/ATF4 signaling pathway, thus providing insight into the mechanism underlying DHA-induced reproductive toxicity, and giving reference to DHA use in females.

The extracellular calcium-sensing receptor promotes porcine egg activation via calcium/calmodulin-dependent protein kinase II.

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.

Cytoplasm lipids can be modulated through hormone-sensitive lipase and are related to mitochondrial function in porcine IVM oocytes.

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 ß-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.

Successful vitrification of early-stage porcine cloned embryos.

The objective of this study was to investigate the survival and development of porcine cloned embryos vitrified by Cryotop carrier at the zygote, 2- and 4-cell stages. The quality of resultant blastocysts was evaluated according to their total cell number, apoptotic cell rate, reactive oxygen species (ROS) production, glutathione (GSH) content and mRNA expression levels of genes related to embryonic development. The survival rates of zygotes, 2- and 4-cell embryos after vitrification did not differ from those of their fresh counterparts. Vitrification still resulted in significantly decreased blastocyst formation rates of these early-stage embryos. Moreover, the total cells, apoptotic rate, ROS and GSH levels in resultant blastocysts were unaffected by vitrification. The mRNA expression levels of PCNA, CPT1, POU5F1 and DNMT3B in the blastocysts derived from vitrified early-stage embryos were significantly higher than those in the fresh blastocysts, but there was no change in expression of CDX2 and DNMT3A genes. In conclusion, our data demonstrate that the early-stage porcine cloned embryos including zygotes, 2- and 4-cells can be successfully vitrified, with respectable blastocyst yield and quality.

Hsa_circ_0000231 knockdown inhibits the glycolysis and progression of colorectal cancer cells by regulating miR-502-5p/MYO6 axis.

BACKGROUND: Colorectal cancer (CRC) poses a heavy threat to human health owing to its high incidence and mortality. Circular RNAs (circRNAs) were investigated to participate in the progression of CRC, whereas there was no revenant data on the CRC process regulated by hsa_circ_0000231. This study aimed to explore the effects of hsa_circ_0000231 on CRC progression and underneath regulatory mechanism. METHODS: The expression levels of hsa_circ_0000231, miR-502-5p, and Myosin VI (MYO6) mRNA were detected by quantitative real time polymerase chain reaction (qRT-PCR). Western blot was employed to determine the protein expression levels of MYO6 and proliferating cell nuclear antigen (PCNA). The effects of hsa_circ_0000231 on cell proliferation, apoptosis, migration, and invasive in CRC were determined by cell counting kit-8 proliferation (CCK-8) and colony formation assays, flow cytometry analysis, wound-healing assay, and transwell invasion assay, respectively. Glucose uptake and lactate production were severally illustrated by glucose assay kit and lactate assay kit. The relationship between miR-502-5p and hsa_circ_0000231 or MYO6 was predicted by circular RNA interactome or targetScan online databases, and identified by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. In vivo tumor formation assay was carried out to determine the effects of hsa_circ_0000231 knockdown on tumor growth in vivo. RESULTS: Hsa_circ_0000231 expression was dramatically upregulated while miR-502-5p was obviously downregulated in CRC tissues and cells compared with control groups. Hsa_circ_0000231 knockdown repressed the expression levels of MYO6 and PCNA protein. Functionally, hsa_circ_0000231 knockdown repressed cell glycolysis, proliferation, migration and invasion, and induced cell apoptosis, whereas these effects were decreased by miR-502-5p inhibitor. Mechanistically, hsa_circ_0000231 acted as a sponge of miR-502-5p and miR-502-5p bound to MYO6. Furthermore, hsa_circ_0000231 knockdown decreased tumor volume and weight of CRC in vivo. CONCLUSION: Hsa_circ_0000231 knockdown inhibited CRC progression and glycolysis by downregulating MYO6 expression through sponging miR-502-5p, which might provide a theoretical basis in further studying circ_0000231-directed therapy in CRC.

Melatonin rescues the aneuploidy in mice vitrified oocytes by regulating mitochondrial heat product.

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.

Toxic effects of 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal on the reproduction of female mice.

Cigarette smoke can affect female reproductive health by causing follicle destruction and oocyte dysfunction. Third-hand smoke has received increasing attention as a public health issue. However, the effects of third-hand smoke on the female reproductive system, particularly the ovaries, remain unclear. 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA) can be used as a biomarker of third-hand smoke. We studied the in vivo toxic effects of NNA on mice ovaries and offspring development. Three-week-old premature female mice were exposed to NNA at two different concentrations (0.075 µg/kg and 0.15 µg/kg body weight) and tap water (blank control) and diluted dimethylsulfoxide (solvent control) for 30 days. We found that oral administration of NNA (0.075 µg/kg and 0.15 µg/kg) significantly reduced ovary weight (the 0.15 µg/kg group was reduced to 18.69% ±â€¯0.89%) and ovarian follicle number (reduced by about 30%) (p < 0.05). Consumption of 0.15 µg/kg NNA reduced the survival rate of superovulated oocytes from 91.36% to 60.55% (p < 0.05). In addition, treated female mice in each group were mated with normal male mice to observe the effects of NNA on the F1 offspring, and during mating and lactation, all groups were given tap water. Two different concentrations of NNA exposure also significantly reduced body weight and impaired ear opening, tooth eruption and eye opening in F1 offspring, especially those exposed to 0.15 µg/kg NNA (p < 0.05). Our study suggested that NNA exposure had toxic effects on the reproductive health of female mice and their offspring. The results obtained may help evaluate the risks of third-hand smoke to women's reproductive health and to the health of their offspring.

Toxic effects of 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal on the maturation and subsequent development of murine oocyte.

Cigarette smoke can cause follicle destruction and oocyte dysfunction and increase the risks of spontaneous abortion, stillbirth, and tubal ectopic pregnancy, affecting female reproductive health. Third-hand smoke (THS) is residual tobacco smoke existing in the environment long after cigarettes are extinguished, which can react with other compounds in the environment to produce secondary pollutants. However, the effects of THS on the female reproductive system, particularly the maturation of the oocyte, remain unclear. 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA), a component of THS, is a logical biomarker of THS exposure. Thus, this study aims to investigate the toxic effects of NNA on the maturation of murine oocytes and subsequent developmental competence. Herein, murine oocytes were exposed to 0 (control group), 0.1, 1.0, 10, and 50 µM NNA for 24 h. Our results showed that NNA exposure reduced the polar body extrusion rate by causing 8-oxo-deoxyguanosine (8-OHdG) to increase and disrupting the meiotic spindle morphology by inhibiting ERK1/2 activation during in vitro maturation. Additionally, NNA exposure resulted in cleavage and blastocyst rate reduction by altering DNA and histone methylations by reducing 5 mC and H3K4me2 levels and by inducing apoptosis caused by mitochondrial dysfunction and reactive oxygen species accumulation, as shown by the increased superoxide dismutase mRNA level and by the decreased Bcl-x mRNA level. Collectively, our results demonstrate that NNA exposure reduces the maturation and developmental capability of murine oocytes by increasing the risk of DNA damage and abnormal spindle morphology, altering epigenetic modifications, and inducing apoptosis, suggesting the toxic effect of NNA on mammalian productive health.

The impacts of laser zona thinning on hatching and implantation of vitrified-warmed mouse embryos.

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.

Toxicity and related mechanisms of dihydroartemisinin on porcine oocyte maturation in vitro.

Dihydroartemisinin (DHA), the main active metabolite of artemisinin, has been used to treat malaria and has anticancer activities. Previous work has shown that DHA has negative impacts on embryos in rodents and primates. However, whether DHA has adverse effects on oocyte maturation is unknown. In the present study, we evaluated the toxic effects and possible mechanisms of DHA on porcine oocyte maturation. The results showed that exposure to DHA inhibited porcine oocyte polar body extrusion, and blocked cell cycle progression. Meanwhile, early embryo development after parthenogenetic activation was also impaired. DHA disturbed spindle morphology and actin assembly in porcine oocytes by reducing phosphorylation levels of MAPK. Moreover, the ROS content was increased and the mitochondrial membrane potential decreased in oocytes treated with DHA. DHA also increased the levels of intracellular and mitochondrial calcium. Furthermore, Annexin V-FITC staining showed that early apoptosis occurred in DHA-treated oocytes. The mRNA levels of apoptosis-related genes BAX and CASP3 were increased, and the anti-apoptotic gene BCL2 was decreased in oocytes exposed to DHA. Taken together, these results indicate that DHA exposure impairs porcine oocyte maturation in vitro via mechanisms involved in cytoskeleton dynamics, oxidative stress, calcium homeostasis, and apoptosis.

BAPTA-AM dramatically improves maturation and development of bovine oocytes from grade-3 cumulus-oocyte complexes.

Intracellular free calcium ([Ca2+ ]i ) is essential for oocyte maturation and early embryonic development. Here, we investigated the role of [Ca2+ ]i in oocytes from cumulus-oocyte complexes (COCs) with respect to maturation and early embryonic development, using the calcium-buffering agent BAPTA-AM (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid tetrakis [acetoxymethyl ester]). COCs were graded based on compactness of the cumulus mass and appearance of the cytoplasm, with Grade 1 indicating higher quality and developmental potential than Grade 3. Results showed that: (i) [Ca2+ ]i in metaphase-II (MII) oocytes from Grade-3 COCs was significantly higher than those from Grade-1 COCs, and was significantly reduced by BAPTA-AM; (ii) nuclear maturation of oocytes from Grade-3 COCs treated with BAPTA-AM was enhanced compared to untreated COCs; (iii) protein abundance of Cyclin B and oocyte-specific Histone 1 (H1FOO) was improved in MII oocytes from Grade-3 COCs treated with BAPTA-AM; (iv) Ca2+ transients were triggered in each group upon fertilization, and the amplitude of [Ca2+ ]i oscillations increased in the Grade-3 group upon treatment with BAPTA-AM, with the magnitude approaching that of the Grade-1 group; and (v) cleavage rates and blastocyst-formation rates were improved in the Grade-3 group treated with BAPTA-AM compared to untreated controls following in vitro fertilization and parthenogenetic activation. Therefore, BAPTA-AM dramatically improved oocyte maturation, oocyte quality, and embryonic development of oocytes from Grade-3 COCs.

The Extracellular Calcium-Sensing Receptor (CASR) Regulates Gonadotropins-Induced Meiotic Maturation of Porcine Oocytes.

Gonadotropins and epidermal growth factor (EGF) play crucial roles in promoting oocyte maturation. The regulatory network downstream of these key factors is not well understood. The present study was designed to investigate the role of the calcium-sensing receptor (CASR) in porcine oocyte in vitro maturation. CASR expression was up-regulated in oocytes matured in gonadotropin-containing medium. Cortical distribution of CASR was enhanced with gonadotropins but not EGF. Supplementation of a CASR agonist (NPS R-568) in the gonadotropin (FSH and/or LH)-containing maturation medium significantly enhanced oocyte nuclear maturation. Addition of NPS2390, a CASR antagonist, compromised oocyte nuclear maturation. Furthermore, increased cortical distribution and decreased expression of CASR was observed after the NPS R-568 treatment. Oocytes treated with NPS R-568 had higher concentration of CYCLIN B1, decreased reactive oxygen species, and increased glutathione levels, indicative of advanced cytoplasmic maturation. In contrast, NPS2390 treatment compromised oocyte cytoplasmic maturation. A higher blastocyst formation rate after parthenogenetic activation was observed when oocytes were matured in the presence of the CASR agonist, NPS R-568. MAPK3/1 phosphorylation was increased during in vitro maturation and after NPS R-568 treatment, and decreased following CASR antagonist supplementation. Taken together, our data showed that the CASR is a gonadotropin-regulated factor that promotes porcine oocyte maturation in a MAPK-dependent manner.

A SIMPLE AND EFFICIENT VITRIFICATION METHOD FOR IN-STRAW DILUTION AND DIRECT TRANSFER OF BOVINE EMBRYOS.

BACKGROUND: An easy and user friendly protocol that produces consistent results will facilitate the commercial application of embryo vitrification technology in the field. OBJECTIVE: This study was designed to develop a simple and efficient vitrification, in-straw dilution and direct transfer method for bovine embryos. METHODS: After being vitrified and in-straw thawed, in vivo-derived and in vitro-produced bovine embryos were subjected to in vitro culture or embryo transplantation. RESULTS: There were no significant differences (P > 0.05) in survival rates (100.0% vs. 93.9%) and expansion rates (93.8% vs. 87.5%) between in vivo-derived and in vitro-produced blastocysts after vitrification and in-straw dilution. And there was also no significant difference (P > 0.05) in conception rates (56.5% vs. 58.8%) after ET between cryopreserved and fresh in vivo-derived blastocysts. CONCLUSION: Vitrification using EG-based vitrification solution and in-straw dilution with PBS-based diluent is a simple and efficient method for cryopreservation and direct transfer of bovine embryos.

trans-10, cis-12 conjugated linoleic acid enhances in vitro maturation of porcine oocytes.

The aim of this study was to determine the effects of trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) supplementation on oocyte maturation and embryo development in pigs. Compared with the control, supplementation of 50 µM t10c12 CLA to in vitro maturation (IVM) medium significantly increased the proportion of oocytes at the metaphase-II (MII) stage and subsequent parthenogenetic embryo development in terms of cleavage rate, blastocyst formation rate, and cell numbers in blastocysts. The t10c12 CLA-treated oocytes resumed meiotic maturation and progressed to the MII stage significantly faster than those of control. The expression of phosphorylated mitogen-activated protein kinase 3/1 (p-MAPK3/1) and cyclooxygenase-2 (COX2) in cumulus oocyte complexes (COCs) at 5, 10, and 22 hr of IVM were significantly increased in the t10c12 CLA-treatment group. The level of p-MAPK3/1 in t10c12 CLA-treated MII oocytes was also higher (P < 0.05) than that of control. Moreover, t10c12 CLA supplementation partially overcame the negative effects of U0126 on cumulus expansion and nuclear maturation, and completely recovered COX2 protein levels in the presence of U0126. Treatment of COCs with NS398 also significantly suppressed cumulus expansion and nuclear maturation, which was overcome by t10c12 CLA. Yet, this simulatory effect of t10c12 CLA was blocked in the presence of both U0126 and NS398. The t10c12 CLA treatment significantly reduced reactive oxygen species level and increased glutathione concentrations in MII oocyte. In conclusion, supplementation of t10c12 CLA during porcine oocyte maturation exerts its beneficial effects on nuclear and cytoplasmic maturation, which contributes to enhancing subsequent embryo development.

DNA methylation pattern in mouse oocytes and their in vitro fertilized early embryos: effect of oocyte vitrification.

This study was conducted to investigate the pattern of DNA methylation in vitrified-thawed mouse oocytes and their in vitro fertilized early embryos. Firstly, mouse oocytes at metaphase II (MII) stage of meiosis were allocated randomly into three groups: (1) untreated (control); (2) exposed to vitrification solution without being plunged into liquid nitrogen (toxicity); or (3) vitrified by open-pulled straw (OPS) method (vitrification). Oocytes from all three groups were fertilized subsequently in vitro. The level of DNA methylation in the MII oocytes and their early embryos was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Developmental rates to 2-cell embryos (62.28%) and blastocysts (43.68%) of the vitrified-thawed oocytes were lower (P < 0.01) than those of fresh oocytes (81.47%, 61.99%) and vitrification solution treated (79.20%, 60.04%) oocytes. DNA methylation (as reflected by 5-MeC fluorescence intensity) in the vitrification group was less (P < 0.01) for MII oocyte and 2- to 8-cell stages compared with that in the control and toxicity groups. Accordingly, a reduction in global genomic methylation due to vitrification of MII oocytes may result in compromised in vitro developmental potential in early mouse embryos.

The association between the oocyte pool and aneuploidy: a comparative study of the reproductive potential of young and aged mice.

PURPOSE: The present study examined the effect of aging on female reproductive potential. METHODS: Six-week-old and 9-month-old CD1 mice were referred to as the 'young' and 'aged' groups, respectively. Oocytes were collected after superovulation, and their viability were compared using parthenogenetic activation. The aneuploidy of the oocytes (MII) was assessed using chromosome spread, and the whole ovarian follicle number was counted using an unbiased stereological method. Serum hormone levels were measured using the radio-immunity method, and the expression of the Cohesin subunit genes in the oocytes (GV) were assessed using RT-PCR. RESULTS: The mean number of recovered (25.8 vs. 16.2; P < 0.05) and live oocytes (24.0 vs. 11.73; P <0.01) per head in the young-mice group (6-week-old) was significantly higher than that of the aged group (9-month-old). The aneuploidy rate of the ovulated oocytes in the aged group was significantly higher than that of the young group (36.8% vs. 10%; P < 0.01), and the rate of blastocyst formation in the young group (85.23%) was significantly higher than that of the aged group (81.2%; P <0.05). The number of primordial follicles (the oocyte pool) per ovary in the aged group was significantly decreased compared with the young group (330 ± 33.51 vs. 2079.6 ± 420.70; P < 0.01), and the level of AMH in the aged group was significantly higher than that of the young group (4.66 ± 0.11 ng/ml vs. 4.07 ± 0.18 ng/ml; P < 0.01). CONCLUSIONS: We propose that maternal aging significantly reduces the oocyte pool, superovulation efficiency and developmental potential and increases the oocyte aneuploidy rate.

Vitrification of Mammalian Oocytes: Recent Studies on Mitochondrial Dysfunction.

Vitrification of reproductive cells is definitely essential and integral in animal breeding, as well as in assisted reproduction. However, issues accompanied with this technology such as decreased oocyte competency and relatively low embryo survival rates appear to be a tough conundrum that has long perplexed us. As significant organelles in cell metabolism, mitochondria play pivotal roles in numerous pathways. Nonetheless, extensive evidence has demonstrated that vitrification can seriously impair mitochondrial function in mammalian oocytes. Thus, in this article, we summarize the current progress in oocyte vitrification and particularly outline the common mitochondrial abnormalities alongside subsequent injury cascades seen in mammalian oocytes following vitrification. Based on existing literature, we tentatively come up with the potential mechanisms related to mitochondrial dysfunction and generalize efficacious ways which have been recommended to restore mitochondrial function.

Superior performance of biocomposite nanoparticles PLGA-RES in protecting oocytes against vitrification stimuli.

Irreversible cryogenic damage caused by oocyte vitrification limits its widespread use in female fertility preservation. In recent years, nanoparticles (NPs) have gained great attention as potential alternatives in protecting oocytes against cryoinjuries. In this paper, a novel composite nanoparticle, poly (lactic-co-glycolic acid)-resveratrol (PLGA-RES) was designed to improve the biocompatibility and sustained release properties by encapsulating natural antioxidant RES into PLGA NPs. Firstly, biotoxicity and oxidation resistance of PLGA-RES were determined, and the results showed that PLGA-RES had nontoxic effect on oocyte survival during in vitro maturation (IVM) (97.08% ± 0.24% vs. 98.89% ± 1.11%, p > 0.05). Notably, PLGA-RES even increased maturation (65.10% ± 4.11% vs. 52.85% ± 2.87%, p < 0.05) and blastocyst rate (56.13% ± 1.36% vs. 40.91% ± 5.85%, p < 0.05). Moreover, the reduced reactive oxygen species (ROS) level (13.49 ± 2.30 vs. 34.07 ± 3.30, p < 0.01), increased glutathione (GSH) (44.13 ± 1.57 vs. 37.62 ± 1.79, p < 0.01) and elevated mitochondrial membrane potential (MMP) levels (43.10 ± 1.81 vs. 28.52 ± 1.25, p < 0.01) were observed in oocytes treated with PLGA-RES when compared with that of the control group. Subsequently, the role of PLGA-RES played in oocytes during vitrification was systematically evaluated. The results showed that the addition of PLGA-RES during vitrification and thawing significantly improved the survival rate (80.42% ± 1.97% vs. 75.37% ± 1.3%, p < 0.05). Meanwhile, increased GSH (15.09 ± 0.86 vs. 14.51 ± 0.78, p < 0.01) and mitochondrial membrane potential (22.56 ± 3.15 vs. 6.79 ± 0.60, p < 0.01), decreased reactive oxygen species levels (52.11 ± 2.95 vs. 75.41 ± 7.23, p < 0.05) and reduced mitochondrial abnormality distribution rate (25.00% ± 0.29% vs. 33.33% ± 1.15%, p < 0.01) were assessed in vitrified MII oocytes treated with PLGA-RES. Furthermore, transcriptomic analyses demonstrated that PLGA-RES participated in endocytosis and PI3K/AKT/mTOR pathway regulation, which was verified by the rescued expression of ARRB2 and ULK3 protein after PLGA-RES treatment. In conclusion, PLGA-RES exhibited potent antioxidant activity, and could be used as an efficacious strategy to improve the quality of vitrified oocytes.