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.     

NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse

Nlrp9a, Nlrp9b and Nlrp9c are preferentially expressed in oocytes and early embryos in the mouse. Simultaneous genetic ablation of Nlrp9a and Nlrp9c does not affect early embryonic development, but the function of Nlrp9b in the process of oocyte maturation and embryonic development has not been elucidated. Here we show that both Nlrp9b mRNA and its protein are expressed in ovaries and the small intestine. Moreover, the NLRP9B protein was restricted to oocytes in the ovary and declined with oocyte aging. After ovulation and fertilization, NLRP9B protein was found in preimplantation embryos. Confocal microscopy demonstrated that it was mainly localized in the cytoplasm in the oocytes and blastomeres. Thus, this protein might play a role in oocyte maturation and early embryonic development. However, knockdown of Nlrp9b expression in GV-stage oocytes using RNA interference did not affect oocyte maturation or subsequent parthenogenetic development after Nlrp9b-deficient oocytes were activated. Furthermore, Nlrp9b knockdown zygotes could reach the blastocyst stage after being cultured for 3.5 days in vitro. These results provide the first evidence that the NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse.     

Oocyte and Embryo Quality: Effect of Origin, Culture Conditions and Gene Expression Patterns

In general, the majority of immature bovine oocytes fail to develop to the blastocyst stage following maturation, fertilization and culture in vitro. The evidence suggests that while culture conditions during in vitro embryo production can impact on the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. In addition, evidence suggests that the period of post‐fertilization embryo culture is the most critical in determining blastocyst quality. This paper reviews the current literature, with emphasis on the bovine model, demonstrating evidence for an effect of oocyte origin and/or in vitro maturation conditions on the developmental capacity and gene expression patterns in the oocyte. Furthermore, the well‐documented effects of post‐fertilization culture environment on embryo gene expression and quality are highlighted.     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响.结果显示,添加颗粒细胞传代接种第2天收集的20％颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P＜0.05);然...     

Developmental competence and glutathione content of maternally heat-stressed mouse oocytes and zygotes

The loss of developmental competence and the glutathione (GSH) content of maternally heat‐stressed mouse oocytes and zygotes were determined. In experiment 1, zygotes were collected from female mice that were heat‐stressed at 35°C for 10 h after hCG injection (oocyte maturation stage), or for 12 h on Day 1 of pregnancy (zygote stage), followed by in vitro culture. To minimize the effects of heat stress on the fertilization process, heat‐stressed oocytes that were fertilized in vitro were also included in this experiment. In experiment 2, heat‐stressed oocytes and zygotes were assayed for GSH content. The application of heat stress to the oocytes resulted in a significant decrease in the percentage of zygotes that developed to morulae or blastocysts, both for naturally fertilized oocytes (56.9% for heat‐stressed vs 85.4% for control) or in vitro‐fertilized oocytes (54.5%vs 73.6%). In the heat‐stressed zygotes, the disruption of embryonic development was more drastic (24.3%vs 90.3%), with the majority of zygotes being arrested at the two‐cell stage. In contrast, the GSH content decreased significantly in heat‐stressed zygotes, but not in heat‐stressed oocytes. These results demonstrate that the loss of developmental competence of early embryos is associated with a decrease in the GSH content of maternally heat‐stressed zygotes, but not of maternally heat‐stressed oocytes.     

Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system

Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.     

Beta‐mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes

In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low‐molecular thiol compounds can be added to culture media. Beta‐mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recovered from slaughterhouse ovaries were used. Meiotic configuration after in vitro maturation (IVM) and early embryo production after intracytoplasmic sperm injection (ICSI) were considered as criteria for assessing nuclear and cytoplasmic maturation, respectively. A total of 1,076 oocytes were analysed over two experiments: 848 (control n = 293, BME 0.1 n = 270, BME 0.7 n = 285) were stained with Hoechst 33342 and examined for nuclear stage after 26 hr of IVM, and 228 MII oocytes were fertilized by ICSI (control n = 83, BME 0.1 n = 65, BME 0.7 n = 80). Cleavage rates were determined after 60 hr of culture. Unlike results obtained in other species, the addition of BME did not influence maturation rates (51.9% control vs 55.6% BME 0.1 mM and 55.1% BME 0.7 mM), nor cleavage rates after ICSI (38.6% vs 38.5% and 41.3%, respectively). In conclusion, the addition of BME at 0.1 and 0.7 mM to the maturation medium, in our culture conditions, has no effect on nuclear and cytoplasmic maturation of equine oocytes.     

Effect of L‐carnitine on maturation,cryo‐tolerance and embryo developmental competence of bovine oocytes

In this study, the effects of the addition of L‐carnitine in in vitro maturation (IVM) medium for bovine oocytes on their nuclear maturation and cryopreservation were investigated; they were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of L‐carnitine (control, 0.3, 0.6 and 1.2 groups, respectively) and some of them were vitrified by Cryotop. Moreover, the effects of L‐carnitine during in vitro fertilization (IVF) and in vitro culture (IVC) on the developmental potential and quality of IVF embryos were also examined. A significantly higher maturation rate of oocytes was obtained for 0.3 and 0.6 mg/mL groups compared with the control (P < 0.05). The blastocyst formation rate in the 0.6 group was significantly improved, whereas the rate in the 1.2 group was significantly decreased when compared with the control group (P < 0.05). No significant difference was found in embryo development between the control and the L‐carnitine group after oocyte vitrification. Supplementation of IVF and IVC media with L‐carnitine had no effect on development to the blastocyst stage of IVM oocytes treated with 0.6 mg/mL L‐carnitine. In conclusion, the supplementation of L‐carnitine during IVM of bovine oocytes improved their nuclear maturation and subsequent embryo development after IVF, but when they were vitrified the improving effects were neutralized.     

Naloxone increases maturation rate and ratio of inner cell mass to total cells in blastocysts in pigs

The purposes of the present study were to examine the effect of naloxone, a mu‐opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M‐II) porcine oocytes, one‐, four‐cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10^?8 mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10^?4 mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10^?3 mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10^?8 mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10^?6 mol/L and 10^?8 mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.     

Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress

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.     

The effects of ceftiofur sodium (Naxcel) on bovine oocyte and preimplantation embryonic development assessed by in vitro embryo production techniques

Ceftiofur sodium is a third-generation cephalosporin antibiotic with broad spectrum bactericidal activity against Gram-positive and Gram-negative bacteria including the beta-lactamase producing strains. In this study, we use in vitro techniques to examine the effects of low and high levels of ceftiofur sodium on the development of bovine oocytes/embryos during oocyte maturation, oocyte fertilization and embryo culture. A total of 8590 oocytes was used in six independent experiments, each in a randomized complete block design. Each replication within each experiment consisted of oocytes from the same abattoir collection of ovaries. There was no difference in embryo development when oocytes were exposed to ceftiofur sodium during oocyte maturation or fertilization at low (10 and 50 μg/mL) or high (100 and 200 μg/mL) concentrations. However, when fertilized oocytes were exposed to concentrations 50 μg/mL during culture, ceftiofur sodium significantly retarded embryo development (e.g. the numbers of ova developing to the morula and blastocyst stages were reduced, and a large proportion of embryos were blocked at the 8-cell stage). We conclude that ceftiofur sodium does not appear to have detrimental effects on oocyte maturation and fertilization. However, long term exposure to high dosages of ceftiofur sodium during post-fertilization culture adversely affects embryo development in vitro.     

In Vitro Maturation and Fertilization of Buffalo Oocytes: Effects of Storage of Ovaries, IVM Temperatures, Storage of Processed Sperm and Fertilization Media

Studies were conducted to examine the possibility of preserving slaughterhouse‐derived buffalo ovaries at 4°C for 0 (control), 12 and 24 h to maintain the developmental competence of the oocytes (experiment 1), to assess the effect of incubation temperature during oocyte maturation on rates of in vitro maturation (IVM) and in vitro fertilization (IVF) of buffalo oocytes and embryo development (experiment 2), and to examine the effect of storage at 25°C for 0 (control), 4 and 8 h of frozen–thawed buffalo sperm and BO and H‐TALP as sperm processing and fertilization media on cleavage and embryo development in vitro of buffalo oocytes (experiment 3) in order to optimize the IVF technology in buffalo. Results suggested that storage of ovaries at 4°C for 12 or 24 h significantly (p < 0.05) reduced the developmental potential of oocytes. Incubation temperatures during the IVM influenced the fertilization rate but had no significant effect on maturation and subsequent embryo development. The incubation temperature of 38.5°C during IVM was found to be optimum for embryo production in vitro. Storage of frozen–thawed sperm at 25°C for 8 h significantly (p < 0.05) decreased its ability to cleave the oocytes. Sperm processed in BO medium had significantly (p < 0.05) higher ability to cleave the oocytes than the H‐TALP medium.     

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.     

Effect of FH535 on in vitro maturation of porcine oocytes by inhibiting WNT signaling pathway

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.     

Supplementing media with NAD+ precursors enhances the in vitro maturation of porcine oocytes

In vitro maturation (IVM) is an important reproductive technology used to produce embryos in vitro. However, the developmental potential of oocytes sourced for IVM is markedly lower than those matured in vivo. Previously, NAD⁺-elevating treatments have improved oocyte quality and embryo development in cattle and mice, suggesting that NAD⁺ is important during oocyte maturation. The aim of this study was to examine the effects of nicotinic acid (NA), nicotinamide (NAM) and nicotinamide mononucleotide (NMN) on oocyte maturation and subsequent embryo development. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation medium supplemented with NA, NAM and resveratrol or NMN. Mature oocytes were artificially activated and presumptive zygotes cultured for 7 days. Additionally, oocytes were matured without treatment then cultured for 7 days with NMN. Supplementing the IVM medium with NA improved maturation and blastocyst formation while NAM supplementation improved cleavage rates compared with untreated controls. Supplementing the IVM or embryo culture media with NMN had no effect on maturation or embryo development. The results show that supplementing the maturation medium with NA and NAM improved maturation and developmental potential of porcine oocytes.     

Adiponectin enhances in vitro development of swine embryos

Recent studies have shown that factors from adipose tissue influence and regulate the reproductive system. Hormones such as leptin and resistin are now known to regulate several reproductive processes. Adiponectin is the most abundant protein secreted by adipose tissue, and its circulating concentration is inversely related to adiposity and body mass index. Little is known about the involvement of adiponectin in reproduction. In the present study, the effect of recombinant adiponectin on the meiotic maturation and early embryo development in vitro was investigated, using porcine oocytes. Adiponectin receptors, AdipoR1 and AdipoR2, were found to be expressed in porcine oocytes and cumulus cells of both small and large follicles. Both AdipoR1 and AdipoR2 were immunolocalized to cumulus-oocyte complexes (COCs), oocytes, and early developing embryos. When included in oocyte maturation medium for 46 h, adiponectin significantly decreased the frequency of meiotic immature oocytes derived from large follicles (3-6 mm) but not from small follicles (<3mm). From studies of oocytes matured in the presence of adiponectin and mitogen-activated protein kinase (MAPK) pathway inhibitors MEK1 (PD98059), MEK1/2 (U0126), and p38MAPK (SB203580) it was concluded that adiponectin enhances oocyte maturation thought the p38MAPK pathway. Finally, a superior rate of embryo development to the blastocyst stage was achieved by embryos cultured in the presence of adiponectin. These results indicate that adiponectin has a positive effect on the meiotic maturation and in vitro embryo development of porcine oocytes and suggests a physiological role for this adipokine in early development in mammals.     

Effect of Preserving Condition of Porcine Ovaries on the Development of In Vitro Matured Oocytes

The effect of preservation condition of ovaries on the in vitro maturation of the porcine oocytes was studied. Cumulus‐oocyte complexes (COCs) were obtained from the ovaries preserved in Dulbecco’s phosphate buffered saline (PBS) solution at various temperatures for different time intervals, and cultured in M199 maturation medium. Matured oocytes were obtained from the ovaries preserved in PBS for 8 h and electrically activated. The activated oocytes were then cultured in NCSU23 embryo culture medium for 16 h to observe activation or 144 h to observe embryo development. It was found that the preservation temperature affected the maturation of porcine oocytes greatly. The effect was described as a compromise of the suppressions of autolysis at physiological temperature and frostbite because of low temperature. A preservation temperature of approximately 25°C showed the maximum maturation rate for a preservation time of 8 h. Preservation temperature also affected the activation and embryo development of porcine oocytes greatly, following a trend similar to the effect of preservation temperature on the maturation. Based on maturation rate, activation rate and cleavage rate, a preservation temperature of approximately 25°C would be optimum for a preservation time of 8 h.