Earticle

Cumulus-oocyte complexes (COCs), which contain immature oocytes, are matured in vitro for in vitro embryo production. Oocyte and cumulus cells are then separated using hyaluronidase. To date, there have only been a few reported cases of the toxic effects of hyaluronidase on porcine oocytes. The aim of this study was to compare the effects of bovine testis-derived hyaluronidase and recombinant human hyaluronidase on oocyte denudation and quality. Porcine COCs were matured for 44 h and denuded using different hyaluronidase concentrations and exposure times. Then, oocytes were activated by electrical parthenogenesis. In experiment 1, COCs were denuded using bovine-derived, ovine-derived (Hirax), and human recombinant (ALT-BC4) hyaluronidases for 10 and 20 min. In experiment 2, bovine-derived and human recombinant (ALT-BC4 and ICSI Cumulase®) hyaluronidases were used to denude the COCs for 2 and 20 min. In both experiments the oocytes were all completely denuded, and there was no degeneration. Rate of embryo development was significantly increased in group treated ALT-BC4 for 2 min and not significantly different in other treatment groups. In general it slightly decreased with longer exposure times. These results have confirmed that different sources of hyaluronidase do not have detrimental effects on the quality of porcine oocytes and suggest that the human recombinant hyaluronidase ALT-BC4 is suitable for oocyte denudation with an increased blastocyst rate.

Mycotoxin contamination of animal feed is a major threat to livestock reproduction. Deoxynivalenol (DON), a trichothecene mycotoxin, disrupts cellular function by binding to ribosomes and inducing ribotoxic stress, oxidative imbalance, and apoptosis. Its primary microbial metabolite, deepoxydeoxynivalenol (DOM-1), is thought to be less toxic. However, its reproductive effects remain unclear. DON contamination is widely reported in cereal grains used in animal feed, and pigs exposed to contaminated diets have been shown to exhibit detectable DON and DOM-1 levels in blood, urine, and fetal tissues (approximately 8?9 ng/g), highlighting the biological relevance of DON exposure during reproduction. Here, we evaluated the effects of DON (250, 500, and 1,000 ng/mL) and DOM-1 (250, 500, and 1,000 ng/mL) on porcine oocyte maturation and embryonic development in vitro, and examined whether resveratrol (Res, 2 μM) could mitigate DON-induced toxicity. Exposure to DON inhibited cumulus cell expansion, maturation rates and impaired developmental competence, at a concentration of 1,000 ng/mL, developmental arrest was complete. DON-treated oocytes showed increased ROS, decreased GSH, upregulated ER stress and apoptosis-related gene levels (ATF4, XBP1, CHOP, BAX), with downregulated antiapoptosis gene levels (BCL2). In contrast, DOM-1 had no significant effects compared with those of the controls, except for a modest reduction in the blastocyst rate at the highest concentration. Although Res did not show restorative effects on cumulus cell expansion and nuclear maturation, Res supplementation reduced DON-induced ER stress markers, increased BCL2 levels, and induced blastocyst development in DON-exposed embryos. These findings demonstrate that DON exerts dose-dependent toxicity in porcine oocytes via oxidative stress and ER stress? mediated apoptosis, whereas DOM-1 is largely nontoxic. Res partially protected against DONinduced oocyte damage and confirms its potential as a supplemen tto combat mycotoxins.

The Conical 9 well dish (C9 well dish) is characterized by a decreasing cross-sectional area towards the base. This design was hypothesized to enhance embryonic development by emulating the in vivo physical environment through density modulation. Comparative analyses revealed no significant difference in nuclear maturation rates between the C9 well dish and the 5-well dish. Reactive oxygen species (ROS) generation was lower in the C9 well dish compared to the 5-well dish; however, this difference was not statistically significant. On the second day of in vitro culture, the cleavage rate in the C9 well dish was 4.66% higher, although not statistically significant, and the rates of blastocyst development were similar across both dishes. No significant differences were observed in the intracellular levels of glutathione (GSH) and ROS, as well as in the total cell number within the blastocysts between the dish types. The expression of mitogen-related factors, TGFα and IGF-1, in the blastocysts was consistent between the dishes. However, PDGFβ expression was significantly lower in the C9 well dish compared to the 35 mm petri dish. Similarly, the expression of the apoptosis factor Bax/Bcl2l2 showed no significant differences between the two dishes. Despite the marked difference in PDGFβ expression, its impact on blastocyst formation appeared negligible. The study also confirmed the feasibility of culturing a small number of oocytes per donor, collected via Ovum Pick-Up (OPU), with reduced volumes of culture medium and mineral oil, thus offering economic advantages. In conclusion, the present study indicates that the C9 well dish is effective for in vitro development of a small quantity of oocytes and embryos, presenting it as a viable alternative to traditional cell culture dishes.

Alanine known as non-essential amino acid was detected at high concentration in reproductive tracts and follicular fluid in developing porcine antral follicle. The purpose of this study was to determine the effect of alanine supplementation during in vitro maturation (IVM) of porcine oocyte. We investigated nuclear maturation, intraoocyte glutathione (GSH) contents in metaphase II (MII) oocytes, and subsequent embryonic development. And also, We detected the gene expression pattern in MII oocytes, early embryo and blastocyst derived parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). The base medium for IVM was North Carolina State University-23 (NCSU-23) medium, modified by supplementing 10 ng/mL EGF, 0.5 ug/mL FSH, and 0.5 ug/ LH, replacing BSA with 0.1% (w/v) PVA, and deleting glutamine. Alanine of various concentrations (0, 0.363, 1, 5, and 10 mM) were added to base IVM medium. The proportion of mature oocyte after IVM did not increase by alanine treatment at various concentrations. However, The intraoocyte GSH content was higher (p<0.05) in oocytes treated with 0.363 mM alanine (1.17±0.01 pixels per oocyte) than non-treated oocytes (1.00±0.02 pixels per oocyte). Blastocyst formation of PA (31.2±2.5% vs. 19.8±2.5%) and SCNT (20.5±1.9% vs. 10.1±2.0%) embryos was significantly (p<0.05) improved by treatment with 0.363 mM alanine during IVM compared with embryos derived from the non-treated oocytes. Supplementation of oocytes IVM medium with 0.363 mM alanine significantly (p<0.05) increased the gene expression of POU5F1 and FGFR2 levels in early embryo and blastocyst derived PA and SCNT embryos. In MII oocytes, transcript levels of POU5F1 and FGFR2 as well as CDK1 gene were significantly (p<0.05) increased in 0.363 mM alanine-treated oocytes. Our results demonstrate that treatment with 0.363 mM alanine during pig oocyte maturation improves developmental competence after PA and SCNT by influencing cytoplasmic maturation, such as improved GSH content in IVM oocyte and increasing gene expression associated with embryonic development in oocyte and embryo.

Background: Cadmium (Cd) is toxic heavy metal that accumulates in organisms after passing through their respiratory and digestive tracts. Although several studies have reported the toxic effects of Cd exposure on human health, its role in embryonic development during preimplantation stage remains unclear. We investigated the effects of Cd on porcine embryonic development and elucidated the mechanism. Methods: We cultured parthenogenetic embryos in media treated with 0, 20, 40, or 60 μM Cd for 6 days and evaluated the rates of cleavage and blastocyst formation. To investigate the mechanism of Cd toxicity, we examined intracellular reactive oxygen species (ROS) and glutathione (GSH) levels. Moreover, we examined mitochondrial content, membrane potential, and ROS. Results: Cleavage and blastocyst formation rates began to decrease significantly in the 40 μM Cd group compared with the control. During post-blastulation, development was significantly delayed in the Cd group. Cd exposure significantly decreased cell number and increased apoptosis rate compared with the control. Embryos exposed to Cd had significantly higher ROS and lower GSH levels, as well as lower expression of antioxidant enzymes, compared with the control. Moreover, embryos exposed to Cd exhibited a significant decrease in mitochondrial content, mitochondrial membrane potential, and expression of mitochondrial genes and an increase in mitochondrial ROS compared to the control. Conclusions: We demonstrated that Cd exposure impairs porcine embryonic development by inducing oxidative stress and mitochondrial dysfunction. Our findings provide insights into the toxicity of Cd exposure on mammalian embryonic development and highlight the importance of preventing Cd pollution.

The acetyltransferase TIP60 (also known as Kat5) is a member of the MYST family of histone acetyltransferases and was initially identified as a cellular protein. TIP60 acetylates histone and nonhistone proteins and is involved in diverse biological processes, including apoptosis, cell cycle, and DNA damage responses. In this study, a specific inhibitor of TIP60 was used to detect the function of TIP60 in porcine parthenogenetic embryos. The results showed that TIP60 inhibition impaired porcine parthenogenetic embryonic development. The mechanism of TIP60 was also determined. We found that the TIP60 inhibition impaired embryonic development by ROS induced DNA damage, as demonstrated by the number of γH2A in the nuclei. TIP60 inhibition triggered DNA damage through the regualetion of p53-p21 pathway and TIP60 played a role in DNA repair. TIP60 inhibition decreased the efficiency of DNA repair by regulating 53BP1-dependent repair after DNA damage. Inhibition of TIP60 also increased the adaptive response, autophagy, by modulating LC3. Therefore, TIP60 plays a role in early porcine parthenogenetic embryonic development by regulating DNA damage and repair.

Sphingosine-1-phosphate (S1P) has a many function involved proliferation, differentiation and survival of many cells. In this study, to investigate whether S1P improve the developmental competence of porcine embryos, 50 nM of S1P were supplemented during in vitro maturation (with EGF or without EGF) medium and/or in vitro culture (IVC) medium. Addition of S1P was significantly increased the rate of oocytes reaching metaphase II (MII) compared to the control (83.5 vs. 64.1%) in without EGF medium, but not with EGF medium (89.5 vs. 84.6%). When treated with 1 μM of N1N-dimethylsphingosine (DMS), a sphingosine kinase inhibitor which is blocked endogenous generation of S1P, the meiotic progression rates to MII stage (without EGF: 45.2 and with EGF: 66.7%) were significantly decreased and degeneration rates (without EGF: 51.2 and with EGF: 30.1%) were increased in both medium compared to control group during IVM periods. Also, the rates of blastocyst formation was significantly increased in the S1P treated group compared to control group (29.0 vs. 19.2%) of EGF supplemented medium, whereas there were no effect in the EGF free medium (9.0 vs. 10.5%). After 12 h IVM, the phosphorylation of ERK1 and ERK2, which is major signaling pathway of MAP kinase, were increased in the S1P group than that of control or DMS group. When supplemented of S1P during IVC, the rates of blastocyst formation and total cell number (30.2% and 40.6) were significantly increased in S1P-treated group compared with control (20.1% and 32.5), DMS (12.3% and 25.1), and S1P plus DMS group (24.7% and 33.6). The percentage of apoptosis nuclei in the S1P group was significantly decreased than other groups. Also, the rates of blastocyst formation (26.7 vs. 14%) and total cell number (42.8 vs. 32.5) were significantly increased in the S1P group than those of control group when S1P added during the entire IVM and IVC periods. Taken together, our results indicate that S1P supplementation in IVM and/or IVC medium affects beneficial effect of meiotic maturation and subsequent developmental competence of porcine embryos.

Cell adhesion plays an important role in the differentiation of the morphogenesis and the trophectoderm epithelium of the blastocyst. In the porcine embryo, CDH1 mediated adhesion initiates at compaction before blastocyst formation, regulated post-translationally via protein kinase C and other signaling molecules. Here we focus on muscle RAS oncogene homolog (M-RAS), which is the closest relative to the RAS related proteins and shares most regulatory and effector interactions. To characterize the effects of M-RAS on embryo compaction, we used gain- and loss-of-function strategies in porcine embryos, in which M-RAS gene structure and protein sequence are conserved. We showed that knockdown of M-RAS in zygotes reduced embryo development abilities and CDH1 expression. Moreover, the phosphorylation of ERK was also decreased in M-RAS KD embryos. Overexpression of M-RAS allows M-RAS KD embryos to rescue the embryo compaction and blastocyst formation. Collectively, these results highlight novel conserved and multiple effects of M-RAS during porcine embryo development.

Objective: To analyze the potential beneficial effects and mechanisms of action of resveratrol on the maturation of bovine oocytes that were incubated in different concentrations of resveratrol (0.1, 1.0 or 10μM) as germinal vesicle (GV) oocytes. Design: In vitro prospective study. Setting: University research laboratory Patient(s): Animal models for human studies. Intervention(s): In vitro culture in the presence of various concentrations of the antioxidant resveratrol. Main Outcome Measures: The parameters of hormone levels, oocyte nuclear maturation, cumulus expansion, levels of intracellular glutathione and reactive oxygen species, embryonic cleavage, blastocyst formation, gene expression associated with mature bovine oocytes and cumulus cells and the level of sirtuin 1 gene expression were detected. Results: Resveratrol significantly increased progesterone secretion and decreased estradiol- 17β secretion by cumulus cells. The elevated levels of progesterone activated the Mos/MEK/p42 MAPK cascade in the oocytes. At a concentration of 1.0 μM, resveratrol significantly improved cumulus expansion, polar body formation, the (hatched)blastocyst rate and the mean number of cells/blastocyst. Meanwhile, resveratrol significantly reduced the level of ROS, increased the level of GSH. For the first time, the expression of the sirtuin 1 gene was identified in granulosa cells, cumulus cells, oocytes and blastocysts. Further studies revealed that resveratrol promoted sirtuin 1 gene expression.Conclusion(s): Resveratrol promoted bovine oocyte maturation and subsequent post- IVF embryonic development by inducing progesterone secretion and antioxidant effect, probably in a manner dependent on Sirtuin 1.