Effects of the E1 activating enzyme UBA2 on porcine oocyte maturation,apoptosis, and embryonic development in vitro

The purpose of this study was to investigate the effect of the E1 activating enzyme UBA2 on the expression of the SUMO-1 protein during in vitro maturation (IVM) of pig oocytes and embryonic development. In the 5 μg/ml UBA2 treatment group, the expression of the anti-apoptotic gene Bcl-2 and the embryo cleavage rate was significantly increased, while the proapoptotic gene Bax was significantly reduced. When 10 μg/ml UBA2 was added, the in vitro maturation rate, blastocyst rate, and SUMO-1 protein content of oocytes increased significantly (p < .05), and the expression of proapoptotic gene Caspase3 was significantly decreased (p < .05), while the viability of cumulus cells was extremely significantly reduced (p < .01). In summary, UBA2 can regulate the content of the SUMO-1 protein in mature pig oocytes in vitro, which in turn affects the maturation rate of oocytes, expression of apoptosis genes, cumulus cell viability, and the development of embryos after fertilization.     

Effect of follistatin on pre‐implantational development of pig parthenogenetic embryos

The present study was designed to explore effects of follistatin (FST) on pre‐implantational development of parthenogenetically activated embryos (PAEs) in pigs. First, we investigated the FST messenger RNA expression level and dynamic FST protein expression patterns in porcine oocytes and PAEs. Then, PAEs were placed in embryo culture medium supplemented with 10 ng/mL of FST‐288, FST‐300, and FST‐315. Next, PAEs were cultured with 0, 1, 10 and 100 ng/mL of FST‐315 protein throughout the in vitro culture (IVC) duration. Further, 10 ng/mL of FST‐300 was added from the start of IVC in which PAEs were treated for 30, 48 and 60 h. The results showed that 1 ng/mL FST‐315 could significantly increase the total cell numbers of blastocyst and trophectoderm cell number in PAEs. Exogenous FST‐300 supplementation could significantly promote the early cleavage divisions and improve the blastocyst formation rate of porcine embryos. FST‐300 appeared to affect early embryonic development before activation of the embryonic genome. In all, the study confirmed for the first time that FST plays a role in promoting early embryonic development in pigs, which differed with different FST subtypes. FST‐300 could facilitate the initial cleavage time and improve the blastocyst formation rate, and FST‐315 could improve the blastocyst quality.     

Effects of melatonin on maturation,histone acetylation,autophagy of porcine oocytes and subsequent embryonic development

Melatonin (MLT) is an endogenous hormone with roles in animal germ cell development. However, the effect of MLT on porcine oocyte maturation and its underlying mechanisms remain largely unknown. Here, we investigated the effects of exogenous MLT on oocyte maturation, histone acetylation, autophagy and subsequent embryonic development. We found that 1 nmol/L MLT supplemented in maturation medium was the optimal concentration to promote porcine oocyte maturation and subsequent developmental competence and quality of parthenogenetic embryos. Interestingly, the beneficial effects of 1 nmol/L MLT treatment on porcine oocyte maturation and embryo development were mainly attributed to the first half period of in vitro maturation. Simultaneously, MLT treatment could also improve maturation of small follicle‐derived oocytes, morphologically poor (cumulus cell layer ≤1) and even artificially denuded oocytes and their subsequent embryo development. Furthermore, MLT treatment not only could decrease the levels of H3K27ac and H4K16ac in metaphase II (MII) oocytes, but also could increase the expression abundances of genes associated with cumulus cell expansion, meiotic maturation, histone acetylation and autophagy in cumulus cells or MII oocytes. These results indicate that MLT treatment can facilitate porcine oocyte maturation and subsequent embryonic development probably, through improvements in histone acetylation and autophagy in oocytes.     

Effects of FGF10 on Bovine Oocyte Meiosis Progression,Apoptosis, Embryo Development and Relative Abundance of Developmentally Important Genes In Vitro

Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion‐related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22 h in TCM‐199 supplemented with 0, 2.5, 10 or 50 ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5 ng/ml FGF10 increased (p < 0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50 ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real‐time RT‐PCR showed a tendency of 50 ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10 ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.     

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.     

Effect of Different Manganese Concentrations during in vitro Maturation of Bovine Oocytes on DNA Integrity of Cumulus Cells and Subsequent Embryo Development

Manganese (Mn) is a trace element present in forages and cereals, and its concentration depends on soil status. Manganese deficiency in cattle, goats and ewes not only impairs oestrous cycle but reduces calf birth weight. The achievement of the first oestrus is delayed, and more attempts are necessary to obtain a successful conception. This study was conducted to investigate the effect of the availability of supplemental Mn during IVM on DNA damage of cumulus cells and total glutathione (GSH) content in oocytes and cumulus cells. The effect of supplementary Mn during IVM on subsequent embryo development was also studied. The results reported here indicate (i) DNA damage in cumulus cells decreased with 0, 2, 5 and 6 ng/ml Mn supplementation during IVM (p < 0.05). (ii) Intracellular GSH‐GSSG content increased (p < 0.01) with different Mn concentrations in oocytes and cumulus cells. Also, cumulus cell number per cumulus oocyte‐complexes (COC) did not differ either before or after IVM. (iii) Addition of Mn to maturation medium resulted in similar cleavage rates (p > 0.05) at 0, 2, 5 and 6 ng/ml Mn. However, subsequent embryo development to blastocyst stage was significantly higher (p < 0.01) in oocytes matured with 5 and 6 ng/ml Mn. (iv) There was also an increase (p < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 5 and 6 ng/ml respect to zero Mn (IVM alone) and 2 ng/ml Mn. This study provides evidence that optimal embryo development to the blastocyst stage was partially dependent on the presence of Mn during IVM. Moreover, the availability of Mn during oocyte maturation ensures ‘normal’ intracellular GSH content in COCs and protects DNA integrity of cumulus cells.     

Ghrelin and Leptin Did Not Improve Meiotic Maturation of Porcine Oocytes Cultured In Vitro

To improve culture system for in vitro maturation (IVM) of porcine oocytes, ghrelin, leptin or growth hormone (GH), at concentration of 0, 0.5, 5, 50 and 500 ng/ml were added to the porcine follicular fluid (pFF)‐supplemented medium NCSU23, and their effects on the maturation and cytoskeletal distribution of the oocytes with or without cumulus cells were compared. In the cumulus‐denuded oocytes, no significant changes were noted in the maturation rate by different hormone treatments due to a marked decline in the controls. Maturation of the cumulus intact oocytes was moderately interfered by ghrelin (0.5–50 ng/ml, p < 0.01), but not significantly affected by leptin and GH. Distribution density of the cytoplasmic microtubules was decreased significantly by addition of ghrelin (by approximately 30% in 50–500 ng/ml, p < 0.01), whereas no remarkable effect was noted by leptin supplementation. High concentration (500 ng/ml) of ghrelin or leptin decreased significantly the cytoplasmic microfilaments in density (by 43% and 38%, p < 0.01, respectively). GH did not affect cytoskeletal distribution. The results suggest, in the culture system using pFF‐supplemented medium that (i) ghrelin may have some inhibitory effect on the organization of microtubules and microfilaments, probably being a factor in lowered maturation rate and (ii) the addition of higher concentration of leptin may decrease microfilaments in density with no effect on meiotic maturation of the porcine oocytes.     

Growth differentiation factor‐9 improves development,mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles

This study analysed the effect of growth differentiation factor‐9 (GDF‐9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α‐MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α‐MEM⁺—control medium) or α‐MEM⁺ supplemented with 1, 10, 50 or 100 ng/ml GDF‐9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF‐9 than other treatments, except for 10 ng/ml of GDF‐9 (p > 0.05). Treatment containing 100 ng/ml GDF‐9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF‐9 showed more oocytes in MI than α‐MEM⁺, 1 or 50 ng/ml GDF‐9 (p < 0.05). In conclusion, 100 ng/ml GDF‐9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.     

Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte–cumulus cell complexes in vitro

During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. We examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes collected from early antral follicles (1.2–1.5 mm). First, we confirmed that GDF9 and BMP15 mRNAs were expressed almost exclusively in the oocytes. Oocyte–cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with 0–100 ng/ml of GDF9 or BMP15 for 5 days. GDF9 dose-dependently increased the OCC diameter, while BMP15 did not. GDF9 and BMP15 had no significant effects on oocyte growth (P > 0.05). When OCCs that had been cultured with 50 and 100 ng/ml BMP15 were subjected to a subsequent maturation culture, they expanded fully by gonadotropic stimulation and 49% and 61% of oocytes matured to metaphase II (MII), respectively. In contrast, GDF9 did not promote cumulus expansion, and < 10% of oocytes matured to MII. Based on the difference in cumulus expansion, we compared the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) mRNAs in cumulus cells. The level of LHCGR mRNA was increased in cumulus cells of the BMP15 group, although there were no significant differences in FSHR mRNA levels among the groups. These results suggest that GDF9 promotes the growth of OCCs and that BMP15 promotes LHCGR mRNA expression in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.     

卵丘细胞对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响

为探讨表皮生长因子(epidermal growth factor,EGF)的添加浓度及脱卵丘细胞时间对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响.试验通过在体外成熟液中添加不同浓度(0、10、15、20、30、40 ng/mL)的EGF来研究其对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响;在培养开始后的不同时间(18、24、38、44 h)进行脱卵丘细胞处理来研究不同时间脱卵丘处理对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响.结果表明,成熟培养基中添加10 ng/mL EGF能显著提高卵母细胞的卵裂率和囊胚率(P <0.05).共培组和独培组卵母细胞培养18 h后脱卵丘细胞成熟率均低于44 h,但差异不显著(P >0.05);共培组卵母细胞培养18 h后脱卵丘细胞的卵裂率和囊胚率显著高于培养44 h(P <0.05);独培组卵母细胞培养18 h后脱卵丘细胞的卵裂率与44 h无显著差异(P >0.05),但囊胚率显著高于培养44 h后脱卵丘细胞(P <0.05).添加10 ng/mL EGF对猪卵母细胞体外成熟及孤雌胚胎体外发育较好;卵母细胞培养18 h后脱卵丘细胞可提高孤雌胚胎早期发育能力.     

All‐trans retinoic acid improves goat oocyte nuclear maturation and reduces apoptotic cumulus cells during in vitro maturation

All‐trans retinoic acid (t‐RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t‐RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus‐oocyte complexes (COCs) were matured in vitro in the absence or presence of t‐RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real‐time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t‐RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t‐RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t‐RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t‐RA inhibits the apoptosis of cumulus cells (P < 0.01). t‐RA treatment up‐regulated the expression of B‐cell lymphoma 2 (BCL‐2), catalase (CAT) (P < 0.05) and down‐regulated the expression of Caspase‐8 (P < 0.05). In conclusion, t‐RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.     

Ghrelin Accelerates In Vitro Maturation of Bovine Oocytes

Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap‐frozen cumulus cells, oocytes and day‐7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin‐treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin‐treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes’ expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over‐maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.     

Ascorbic acid–cyclodextrin complex alters the expression of genes associated with lipid metabolism in bovine in vitro produced embryos

Ascorbic acid (AC) used as antioxidant in embryo culture is very sensitive and degrades unavoidably in aqueous solution. Methyl‐β‐cyclodextrin (CD) improved the stability of AC in solution to elevated temperature, light, humidity and oxidation. The aim of this study was to evaluate the effect of the complex AC‐CD during in vitro maturation (IVM) or in vitro culture (IVC) on oocyte developmental competence and subsequent embryo development and quality. AC‐CD (100 µM) was added to IVM media, and maturation level and embryo development were examined. Matured oocytes, their cumulus cells and produced blastocysts were snap‐frozen for gene expression analysis by RT‐qPCR. Besides, in vitro‐produced zygotes were cultured with 100 µM of AC‐CD and blastocysts were as well snap‐frozen for gene expression analysis. A group without AC‐CD (control^?) and other with CD (control⁺) were included. No differences were found on maturation, cleavage or blastocyst rates. However, in matured oocytes, AC‐CD downregulated BAX, GPX1 and BMP15. In cumulus cells, AC‐CD downregulated BAX/BCL2 and GSTA4 while upregulated BCL2 and CYP51A1. The expression of SL2A1, FADS1, PNPLA and MTORC1 was downregulated in blastocysts derived from oocytes matured with AC‐CD, while in blastocysts derived from zygote cultured with AC‐CD, CYP51A1 and IGF2R were downregulated and PNPLA2 was upregulated. In conclusion, AC‐CD in both IVM and IVC media may reduce accumulated fat by increasing lipolysis and suppressing lipogenesis in blastocysts derived from both oocytes and zygotes cultured with AC‐CD, suggesting that CD improves the quality of embryos and bioavailability of AC during IVM and IVC.     

Maternal Liver Damage Delays Meiotic Resumption in Bovine Oocytes through Impairment of Signalling Cascades Originated from Low p38MAPK Activity in Cumulus Cells

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.     

Effect of estradiol‐17β during in vitro growth culture on the growth,maturation, cumulus expansion and development of porcine oocytes from early antral follicles

Growing porcine oocytes from early antral follicles can acquire meiotic and developmental competence under suitable culture conditions, but at lower rates compared to full‐grown oocytes. We postulated that estradiol‐17β (E₂) supported the acquisition of meiotic and developmental competence as well as cumulus‐expansion ability during growth culture. Growing oocytes from early antral follicles (1.2 to 1.5 mm in diameter) were grown in vitro for 5 days in a medium containing 0, 10^?7, 10^?6, 10^?5 or 10^?4 mol/L E₂; after in vitro maturation, 35, 58, 47, 74 and 49% of oocytes matured to metaphase II, 25, 79, 77, 90 and 97% acquired cumulus‐expansion ability, and 23, 54, 63, 89 and 64% were fully surrounded by cumulus cells, respectively. Following maturation, electro‐stimulation was applied to the oocytes grown with 10^?5 mol/L E₂. After 6 days of culture, in vitro‐grown oocytes developed to the blastocyst stage at a rate similar to that for full‐grown oocytes (31% and 40%, respectively). Therefore, we suggest that the use of E₂ during growth culture improves the meiotic and developmental competence of oocytes, cumulus‐expansion ability, and cumulus cell attachment to the oocytes.     

Study on the expression patterns and function of JUNO and CD9 in bovine oocytes during in vitro maturation

Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 μg/ml) or (ii) CD9 antibody (1, 5 and 25 μg/ml) or (iii) CD9 antibody (5 μg/ml) + JUNO antibody (5 μg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 μg/ml) + JUNO antibody (5 μg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.     

A new three-dimensional glass scaffold increases the in vitro maturation efficiency of buffalo (Bubalus bubalis) oocyte via remodelling the extracellular matrix and cell connection of cumulus cells

At present, many three-dimensional (3D) culture systems have been reported, improving the oocyte quality of in vitro maturation (IVM), yet the mechanism still needs to be further explored. Here we examined the effects of a new self-made 3D glass scaffold on buffalo oocyte maturation; meanwhile, the underlying mechanism on buffalo oocyte maturation was also detected. Compared to the two-dimensional (2D) glass dish culture, results revealed that the 3D culture can improve the first polar body rate of oocytes, subsequent cleavage and blastocysts rate of parthenogenetic activation embryos (p < .05). The extracellular matrix-related proteins COL1A1, COL2A1, COL3A1, FN and cell connection-related proteins N-cadherin, E-cadherin, GJA1 were found higher in cumulus cells of 3D culture. Moreover, in cumulus cells, proteins of the PI3K/AKT pathway reported being regulated by FN and E-cadherin including PI3K P85 and p-AKT were also higher in 3D culture. Furthermore, proapoptosis proteins P53, BAX, caspase-3 were lower in both cumulus cells and oocytes in 3D culture, while proteins PCNA and BCL2 showed the opposite result. Results also showed that the apoptosis was inhibited, and the proliferation was enhanced in cumulus cells of 3D culture. Finally, the cumulus expansion-related genes HAS2, CD44, HMMR, PTX3, PTGS2 were found higher in cumulus cells of 3D culture. Taken together, the 3D culture could promote oocyte maturation by regulating proteins correlated with the ECM, cell connection and PI3K/AKT pathway, inhibiting the apoptosis of cumulus cells and oocytes, enhancing the proliferation of cumulus cells and the cumulus expansion.     

Effect of permeable cryoprotectant‐free vitrification on DNA fragmentation of equine oocyte–cumulus cells

DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post‐mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus–oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO₂) before (control group) or after a permeable cryoprotectant‐free vitrification method using 1 M sucrose (vitrification group). After in vitro maturation, COCs were denuded, and cumulus cells were washed and stored at ?80°C until thawing. Cumulus cell samples were processed with the chromatin dispersion test (Ovoselect, Halotech DNA, Spain). Low, high and total DNA fragmentation percentages of cumulus cells were recorded and compared between the two groups by Student's t test. Results were expressed as mean ± SEM. The vitrified group resulted in significantly higher (p < 0.05) percentages for low (16.81 ± 1.62 vs. 6.63 ± 0.77) and total (21.14 ± 1.84 vs. 12.76 ± 1.48) DNA fragmentation of cumulus cells. There were no significant differences between groups for high DNA fragmentation of cumulus cells. In conclusion, permeable cryoprotectant‐free vitrification of equine oocytes increased the total DNA fragmentation rate of cumulus cells but protected them against high DNA fragmentation rates. Further studies are needed to examine the relationship between DNA fragmentation of cumulus cells and the developmental competence of equine oocytes.