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1.
Wingless‐int (WNT) signaling pathway is vital to modulate life processes, including cell fate determination, cell differentiation, cell proliferation, cell apoptosis and embryogenesis. To demonstrate the uncertain effect of the canonical WNT signaling pathway on oocyte maturation, immature porcine oocytes were collected and cultured in vitro with the WNT/β‐catenin inhibitor FH535. The concentrations of FH535 were selected as 0.00, 0.01, 0.10, 1.00 and 10.00 μmol/L. The results showed that the optimum concentration of FH535 on oocyte maturation was 1.00 μmol/L. In this concentration, the proportion of MII oocytes increased (P < 0.05). The rate of cleavage was the same with the control (P > 0.05), while the rate of blastocysts in the 1.00 μmol/L FH535 treated group was higher than that of control (P < 0.01). Additionally, the average number of nuclei in blastocysts raised significantly (P < 0.05). The inhibition of WNT could regulate expression of maturation‐related genes, including Cdc‐2, Bmp‐15, Gdf‐9 and Mos. In the 1.00 μmol/L FH535 treated group, the messenger RNA level of β‐catenin showed no significant change compared to the control (P > 0.05), but the protein abundance was decreased (P < 0.05). This study revealed that the inhibition of FH535 on the WNT signaling pathway could promote the maturation of porcine oocytes and altered gene expressions in vitro.  相似文献   

2.
Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.  相似文献   

3.
This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus‐oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA‐fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.  相似文献   

4.
P38α mitogen‐activated protein kinase (MAPK), which is a member of the canonical MAPK family, is activated in response to various extracellular stresses and plays a role in multiple cellular processes. In this study, we investigated the expression, subcellular localization and functional roles of p38α MAPK during the meiotic maturation of rat oocytes. We found that p38α MAPK phosphorylation (p‐p38α MAPK, indicative of p38α MAPK activation) was low at the germinal vesicle (GV) stage, increased 3 hr after germinal vesicle breakdown (GVBD) and maintained its maximum at metaphase I (MI) or metaphase II (MII). The p‐p38α MAPK mainly accumulated in the GV and had no obvious expression in the nucleus. From GVBD to MII, p‐p38α MAPK was distributed in the cytoplasm around either the chromosomes or the spindle. We used SB203580, an inhibitor of p38α MAPK, to investigate the possible functional role of p38α MAPK during rat oocyte meiotic maturation. Treatment of GV stage oocytes with 20 μM SB203580 blocked p‐p38α MAPK activity, and the spindles appeared abnormal. Additionally, the rate of GVBD after 3 hr of culture with 20 μM SB203580 (58.8%) was significantly inhibited compared with the control (82.5%, p < .05), and the polar body extrusion rate after 12 hr of culture with SB203580 was also significantly decreased compared with the control (40.1% vs 73.3%, p < .05). Taken together, these data indicate that p38α MAPK may play a vital role in rat oocyte meiotic maturation.  相似文献   

5.
This study examined the effects of treatment with U0126, which inhibits MAPK by inhibiting MAPK kinase, during the first 2 hr of in vitro maturation on bovine developmental competence and on gap junction (GAPJ) communication between the oocyte and cumulus cells. The percentage of oocytes developing to the blastocyst stage in the group treated with 5 μM U0126 (28%) was significantly higher than that in controls (15%, p < .05), while that in the group treated with 10 μM U0126 (18%) was not. Breakdown of the GAPJs was delayed in the group treated with 5 μM U0126 when compared to controls, as estimated by immunohistochemical examination of connexin 43, which is a primary constituent of the GAPJs. These results indicate that treatment with 5 μM U0126 during in vitro maturation delays GAPJ breakdown and improves bovine oocyte developmental competence.  相似文献   

6.
We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre‐culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L‐carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non‐presence of oxidant stress induced by H2O2 supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H2O2. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.  相似文献   

7.
Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti‐inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging‐related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1 μM KAE was significantly higher than in untreated aging oocytes (36.78 ± 0.86% vs. 27.55 ± 2.60%, respectively, p < .05). The KAE‐treated aging oocytes had significantly reduced levels of reactive oxygen species (p < .05). Furthermore, the mRNA levels of the embryonic pluripotency‐related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE‐treated oocytes (p < .05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.  相似文献   

8.
Oxidative stress inevitably occurs during oocyte maturation in vitro. α-lipoic acid (α-LA) has a strong antioxidant capacity, but the effect of α-LA on parthenogenetic activation of oocytes was rarely reported. This study aims to investigate the effect of supplementing α-LA to in vitro maturation medium on the subsequent developmental ability of goat parthenogenetic embryos during oocytes maturation. In the study, the goat cumulus-oocyte complex was divided into the experimental (with 25 μmol/L α-LA) and the control (without α-LA) groups. Oxidase expression was measured using RT-qPCR. After 18–22 hr of maturation, the oocytes were then parthenogenetic activated. The total antioxidant capacity of embryos was measured after 0, 24, 48, 72 and 96 hr of culture. Rates of oocyte maturation and the rates of development for parthenogenetic embryos in the α-LA group were significantly improved by 7.88% (p < .05) and 5.41% (p < .05) compared with those in the control group, respectively. After 24 hr, the difference in total antioxidant capacity was extremely significant in both groups. An evident decrease in the control group and a minor decrease in the α-LA group were observed (p < .01). The ratio of inner cell mass cells to the total cell number of blastocysts in the α-LA group increased compared with that in the control group (p < .05) on day 8. α-LA significantly promoted the expression of SOD and GPX4 of parthenogenetic blastocysts and maturated oocytes. α-LA (25 μmol/L) improved the maturation rate and the developmental competence of the parthenogenetic activation of oocytes, which might be mediated by maintaining the total antioxidant ability of oocytes during the culture period.  相似文献   

9.
SUMOylation is a dynamic post-translational modification process. However, the function of small ubiquitin-like modifiers (SUMOs) in the maturation of porcine oocytes and embryo growth is not well known. Therefore, the aim of this study was to investigate the effect of E2 binding enzyme UBC9 on the expression of SUMO-1 protein during the in vitro maturation of porcine oocytes and embryo development after in vitro fertilization. Four groups were used: 0 (Control), 5, 10 and 15 µg/ml UBC9. Western blotting, flow cytometry and RT-qPCR were used to detect the in vitro maturation of porcine oocytes, SUMO-1 content, viability and the expression of apoptotic genes. Compared to those in the control treatment, the maturation rate (p < .05) and viability (p < .01) of oocytes in the 5 μg/ml treatment group decreased significantly. SUMO-1 protein markers appeared at 59 and 71 kDa and the content of SUMO-1 protein in the 10 µg/ml treatment group decreased significantly (p < .05). In the expression of apoptosis-related genes, Bcl-2 gene expression was significantly downregulated in the 10 μg/ml treatment group (p < .05). However, Bax and Caspase-3 were significantly upregulated in the 5 μg/ml treatment group (p < .05). During embryonic development, the cleavage rate of oocytes in the 10 µg/ml treatment group was significantly reduced (p < .05), whereas blastocyst formation rate in the 5 µg/ml treatment group was significantly reduced. UBC9 regulates SUMO-1 content in mature pig oocytes in vitro, which affects oocyte maturation rate, viability, apoptotic genes expression and embryo development after fertilization.  相似文献   

10.
Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 μM CGA were significantly (p < .05) higher than those of the control oocytes. Hydrogen peroxide (H2O2) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H2O2 to assess the protective effect of CGA, 50 μM CGA supplementation improved the maturation rate and the proportion of DNA‐fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 μM CGA (control) or caffeic acid (10, 50 and 100 μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 μM CGA were similar to those of oocytes matured with 10 and 50 μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.  相似文献   

11.
The objectives of present study were to evaluate the effect of casein kinase 1 (CK1) inhibition D4476 on in vitro maturation (IVM) and developmental competence of bovine oocytes. The cumulus oocyte complexes (COCs) were cultured in maturation medium with D4476 (0, 2, 5, 10, 20 μM) for 24 hr. After IVM and in vitro fertilization, through expansion average scores of cumulus cells (CCs), oocyte maturation efficiency, cleavage rate and blastocyst rate of zygote, we found 5 μM D4476 could increase the development potential of oocytes. After the COCs were treated with 5 μM D4476, the results of quantitative real‐time PCR analysis, Lichen red staining and PI staining showed that under without affecting germinal vesicle breakdown and nuclear morphology, D4476 could significantly decrease CK1 and upregulate TCF‐4 in oocytes. Furthermore, without influencing the level of Bad and CTSB, D4476 could significantly increase the expression of β‐catenin, TCF‐4, Cx43, MAPK, PTGS‐2, PTX‐3, TGS‐6, Bax and Bcl‐2 in CCs. Western blot analysis revealed that the addition of 5 μM D4476 during the maturation of COCs resulted in a lower level of Cx43 protein at 12 hr and a higher expression of Cx43 protein at 24 hr compared to the group without D4476. These results indicate that adding optimum D4476 (5 μM) to maturation medium is beneficial to maturity efficiency and development competence of bovine oocytes.  相似文献   

12.

High blood urea nitrogen (BUN) concentration decreases fertility in ruminants. Nguni cattle are reported to maintain BUN concentration more efficiently than other beef breeds. Our objectives were to determine if BUN concentration differed between Nguni and Hereford cows exposed to a high protein ration, and if breed or BUN and serum protein concentrations at the time of oocyte pick-up affected oocyte quantity, quality, and viability. Twelve Nguni and 10 Hereford cows were randomized into high or normal BUN-inducing diets in a crossover design. Ultrasound-guided oocyte pick-up was performed twice weekly; oocytes were counted, visually graded and the viable oocytes were pooled by treatment and breed for in vitro maturation, fertilization, and culture. Nguni cows on the highest protein ration achieved lower mean BUN concentration than Herefords (P < 0.05), and Nguni cows reached BUN concentrations above 20 mg/dL less frequently than Herefords (P = 0.03). Donor BUN concentration above 20 mg/dL at the time of oocyte pick-up, but not breed, independently decreased the number of good quality oocytes harvested. Increasing weighted mean serum albumin of donor cows was independently associated with the number of oocytes that cleaved by day 2 and that reached morula stage by day 7 (P = 0.01). In conclusion, Nguni cows reached the critical threshold of 20 mg/dL BUN less frequently than Herefords; BUN of donor cows above 20 mg/dL negatively affected visual oocyte quality independent of breed, and increasing serum albumin of donor cows improved viability of bovine oocytes.

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13.
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14.
The aim of this study was to investigate the influence of zinc (Zn) on the health of cumulus–oocyte complex (COC) during in vitro maturation (IVM). Experiments were designed to evaluate the effect of Zn added to IVM medium on: DNA integrity, apoptosis, cumulus expansion and superoxide dismutase (SOD) activity of cumulus cells (CC). Also, role of CC on Zn transport during IVM was evaluated on oocyte developmental capacity. DNA damage and early apoptosis were higher in CC matured with 0 μg/ml Zn compared with 0.7, 1.1 and 1.5 μg/ml Zn (p < 0.05). Cumulus expansion did not show differences in COC matured with or without Zn supplementation (p > 0.05). Superoxide dismutase activity was higher in COC matured with 1.5 μg/ml Zn than with 0 μg/ml Zn (p < 0.05). Cleavage and blastocyst rates were recorded after IVM in three maturation systems: intact COCs, denuded oocytes with cumulus cells monolayer (DO + CC) and denuded oocytes (DO). Cleavage rates were similar when COC, DO + CC or DO were matured with 1.5 μg/ml Zn compared with control group (p > 0.05). Blastocyst rates were significantly higher in COC than in DO + CC and DO with the addition of 1.5 μg/ml Zn during IVM (p < 0.01). Blastocyst quality was enhanced in COC and DO + CC compared with DO when Zn was added to IVM medium (p < 0.001). The results of this study indicate that Zn supplementation to IVM medium (i) decreased DNA damage and apoptosis in CC; (ii) increased SOD activity in CC; (iii) did not modify cumulus expansion and cleavage rates after in vitro fertilization; (iv) improved subsequent embryo development up to blastocyst stage; and (v) enhanced blastocyst quality when CC were present either in intact COC or in coculture during IVM.  相似文献   

15.
Sirtuins have been widely reported to be involved in multiple biological processes. However, their function during pig oocyte aging has not been reported yet. Here, we first identify that sirt1 expression is dramatically reduced in pig in vitro‐aged oocytes. Furthermore, by confocal scanning and quantitative analysis, we find the increased frequency of spindle defects and chromosome misalignment, disturbed redistribution of cortical granules and mitochondria during oocyte in vitro‐aging. Importantly, these aging‐associated defective phenotypes can be ameliorated through resveratrol (sirt1 activator) treatment during pig oocyte maturation, providing the evidence for the hypothesis that decreased sirt1 is one of a number of factors contributing to oocyte in vitro‐aging. In summary, our data indicate a role for sirt1 in pig oocytes and uncover a striking beneficial effect of sirt1 expression on aged oocytes.  相似文献   

16.
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17.
The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p < 0.05). When cumulus cells were removed from cumulus‐oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7 h of in vitro maturation (IVM) (p < 0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0 h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p < 0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.  相似文献   

18.
This study evaluated the effect of three reversible meiotic inhibitors (MINs) and their interaction with gonadotrophins (Gns) on the meiotic maturation and developmental competence of porcine oocytes. In experiment 1, the oocytes were matured for 22 hr in the presence or absence of dbcAMP (1 mM), cycloheximide (7 μM) or cilostamide (20 μM) with or without Gns, and for an additional 22 hr in the absence of MINs and Gns. At 22 hr of maturation, regardless of the presence of Gns, a higher proportion (p < .001) of oocytes cultured in the presence of MINs were effectively arrested at the germinal vesicle stage compared with the oocytes cultured without MINs. At 44 hr of maturation, the proportion of oocytes that reached MII was higher (p < .05) in groups with Gns compared with groups without Gns. In experiment 2, oocytes that were matured as in experiment 1 were inseminated and cultured for 7 days to evaluate fertilization parameters and blastocyst formation. Only oocytes from the dbcAMP + Gns group had higher (p < .05) efficiency of fertilization compared with the other treatment groups. The presence of dbcAMP during maturation also increased (p < .05) blastocyst formation and efficiency of blastocyst formation in both the presence and absence of Gns. These results indicate that the interaction of Gns with the tested MINs improved meiotic progression. In addition, regardless of supplementation with Gns, the presence of dbcAMP during the first maturation period increased and even doubled the capacity of oocytes to develop to the blastocyst stage.  相似文献   

19.
Spontaneous nuclear maturation of mammalian oocytes can occur when physically removed from the ovarian follicle during in vitro oocyte maturation (IVM), largely because of a decrease in cyclic adenosine monophosphate (cAMP) concentration. Modulation of oocyte cAMP during IVM by using phosphodiesterase inhibitors has been shown to maintain elevated oocyte cAMP concentrations and control meiotic resumption of bovine and ovine oocytes. This study determined the effect of inclusion of isobutyl-1-methylxanthine (IBMX) during collection and the first 12 hours of incubation of equine oocytes on cAMP concentration and glucose metabolism of cumulus–oocyte complexes (COCs). Abattoir-derived COCs were collected in aspiration medium with (Asp-IBMX) or without (Asp) IBMX. Cumulus–oocyte complexes were then incubated for 12 hours in IVM medium with (Mat-IBMX) or without (Mat) IBMX, followed by additional 24 hours in Mat medium. The cAMP concentration, glucose consumption, lactate production, and metaphase II rates of the COCs were assessed. Cumulus–oocyte complexes aspirated into Asp-IBMX (62.2 ± 2.6 fmol per COC) medium had higher cAMP concentration than Asp (31.8 ± 2.8 fmol per COC) control group (P < .05). Likewise, at 12 hours of IVM, Mat-IBMX group (33.2 ± 2.1 fmol per COC) had higher cAMP concentration than the Mat group (7.68 ± 0.5 fmol per COC; P < .05). Glucose consumption and lactate production were lower during the first 12 hours of incubation in COCs cultured in Mat-IBMX (P < .05). Isobutyl-1-methylxanthine prevented the rapid drop in cAMP concentration and altered metabolism of glucose by the COC. Preventing the sudden drop in cAMP prevents the premature nuclear maturation of in vitro–matured oocytes causing poor developmental competence.  相似文献   

20.
As a natural plant‐derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 μM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti‐apoptotic B‐cell lymphoma 2 (BCL‐2) gene and significant downregulation of the pro‐apoptotic BCL2‐associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 μM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 μM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT.  相似文献   

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