Earticle

Bisphenol A (BPA) is an environmental contaminant widely used in the plastic industry. BPA has been demonstrated to be an endocrine disruptor and has an adverse effect on the embryonic development of mammals. However, the mechanism of action of BPA is limited. In this study, we investigated the role and mechanism of BPA in porcine embryonic development. First, the parthenotes were treated with different concentrations of BPA. We found that blastocyst formation was impaired and the parthenotes were arrested at the 4-cell stage after treatment with 100 μm BPA. Second, ROS increased following the addition of BPA, which further caused mitochondrial damage, and cytochrome c was released from the mitochondria to induce apoptosis. The adaptive response was demonstrated through LC3 immunofluorescence staining and by assessing autophagy-related gene expression. In addition, BPA caused DNA damage through the p53-p21 signaling pathway. Thus, our results indicate that BPA displays an adverse effect on porcine early embryonic development through mitochondrial and DNA damage.

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, gene expressions in matured oocytes, cumulus cells, and IVF-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). In the nuclear maturation after 44 h IVM, the groups of 0.1, 0.5, and 2.0 μM (83.0%, 84.1%, and 88.3%, respectively) had no significant difference compared to the control (84.1%), but the group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (p<0.05). The groups of 0.5 and 2.0 μM showed a significant (p<0.05) increase in intracellular GSH levels compared to the control and 10.0 μM groups. Intracellular ROS level of oocytes matured with 2.0 μM resveratrol was significantly (p<0.05) decreased compared to the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rate, and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) compared to the control group. Cumulus-oocytes complex (COCs) treated with 2.0 μM resveratrol were showed lower (p<0.05) expressions of apoptosis-related genes in both matured oocytes (Bax, Bak, and Caspase-3) and cumulus cells (Bax). In IVF-derived blastocysts derived from 2.0 μM resveratrol treated oocytes had also decreased (p<0.05) expression of Bak compared to the control. In conclusion, the 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating apoptosis-related genes expression during oocyte maturation.

Alpha lipoic acid (ALA) is a biological membranes compound. as the antioxidant, it decreases the oxidized forms of other antioxidant substances such as vitamin C, vitamin E and glutathione (GSH). To examine the effect of ALA on the in vitro maturation (IVM) of porcine oocytes, we investigated intracellular GSH and reactive oxygen species (ROS) levels, and subsequent embryonic development after parthenogenetic activation (PA). Intracellular GSH levels in oocytes treated with 50uM of ALA increased significantly (p<0.05) and exhibited a significant (p<0.05) decrease in intracellular ROS levels compared with the control group. Oocytes matured with 50uM during IVM displayed significantly higher cleavage rates (67.8% vs. 83.4%, respectively), and higher blastocyst formation rates and total cell number of blastocysts after PA (31.6%, 58.49 vs. 46.8%, 68.58, respectively) than the control group. In conclusion, these results suggest that treatment with ALA during IVM improves the nuclear maturation of porcine oocytes by increasing the intracellular GSH levels, thereby decreasing the intracellular ROS levels and subsequent embryonic developmental potential of PA.

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in human and animals. However, in our preliminary experiment, it was observed that SSC was fragile when used as nuclei donor for SCNT. This study investigated the potential of porcine SSC as donor nuclei for somatic cell nuclear transfer (SCNT) and developmental competence of SSC-derived cloned embryos. In addition, it was examined whether demecolcine could prevent rupture of SSC during SCNT. SCNT embryos were produced using a standard protocol of our laboratory. After electric activation, SCNT embryos were treated with demecolcine combined with 6-DMAP for 4 h, washed, and then cultured in a porcine zygote medium-3 at 39℃ in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 7 days. When SSC was compared with porcine fetal fibroblast (PFF) in the potential to support embryonic development after SCNT, SSC-derived SCNT embryos showed higher (p< 0.05) developmental competence to the blastocyst stage (53.7%) than PFF-derived embryos (28.6%). Treatment of SSC with demecolcine significantly (p<0.01) inhibited the rupture of SSC during SCNT (7.8% vs. 16.4%) and increased fusion of cell-ooplasm couplets compared to no treatment (71.9% vs. 62.8%). In addition, even after demecolcine treatment, SSC-derived SCNT embryos showed a higher blastocyst formation (46.4%) than PFF-derived embryos (27.8%). Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.

C-phycocyanin (C-PC) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, antiapoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. But the protective effect of C-PC on porcine embryo developmental competence in in vitro is little known. In the present study, we investigated the effect of C-PC on the development of porcine early embryos as well as the underlying its mechanisms. Different concentrations of C-CP (1, 2, 5, 8, 10 μg/mL) was added to the porcine zygote medium 5 (PZM-5) during in vitro culture. The results showed that 5 μg/mL C-PC significantly increased blastocyst formation. Blastocyst formation and its quality were significantly increased in 50 μM H2O2 treatment group following 5μg/mL C-PC addition. C-PC prevented H2O2-induced compromise of mitochondrial membrane potential, release of cytochrome C from the mitochondria and reactive oxygen species generation. Furthermore, apoptosis, DNA damage level and autophagy in the blastocysts were attenuated by supplement of C-PC in H2O2-induce doxidativ injury group compared with control.Taken together, these results suggest that C-PC has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.

Cyclin E1 (CCNE1) is a core component of cell cycle regulation that drives the transition into the S phase. CCNE1 plays critical roles in cell cycle, cell proliferation, and cellular functions. However, the function of CCNE1 in early embryonic development is limited. In the present study, the function and expression of Ccne1 in porcine early parthenotes were examined. Immunostaining experiments showed that CCNE1 localized in the nucleus, starting at the four-cell stage. Knockdown of Ccne1 by double-stranded RNA resulted in the failure of blastocyst formation and induced blastocyst apoptosis. Ccne1 depletion increased expression of the pro-apoptotic gene Bax, and decreased the expression of Oct4 and the rate of inner cell mass (ICM)/trophectoderm formation. The results indicated that CCNE1 affects blastocyst formation by inducing cell apoptosis and ICM formation during porcine embryonic development.

C-phycocyanin (C-PC) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, antiapoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. But the protective effect of C-PC on porcine embryo developmental competence in in vitro is little known. In the present study, we investigated the effect of C-PC on the development of porcine early embryos as well as the underlying its mechanisms. Different concentrations of C-CP (1, 2, 5, 8, 10 μg/mL) was added to the porcine zygote medium 5 (PZM-5) during in vitro culture. The results showed that 5 μg /mL C-PC significantly increased blastocyst formation. Blastocyst formation and its quality were significantly increased in 50 μM H2O2 treatment group following 5 μg/mL C-PC addition. C-PC prevented H2O2-induced compromise of mitochondrial membrane potential, release of cytochrome c from the mitochondria and reactive oxygen species generation. Furthermore, apoptosis, DNA damage level and autophagy in the blastocysts were attenuated by supplement of C-PC in H2O2-induced oxidative injury group compared with control. Taken together, these results suggest that C-PC has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.

In a conventional sense, dried-spermatozoa are all dead and motionless due to the lost of their natural ability to penetrate oocytes both in vivo and in vitro. However, their nuclei are completely able to contribute to normal embryonic development even after long-term preservation in a dried state when the dried-spermatozoa are microinjected into the oocytes. In this sense, dried spermatozoa must still be alive. Thus, defining spermatozoa as alive or dead seems rather arbitrary. Several drying method of sperm including freeze-drying, evaporative/convective-drying and heat-drying were represented in this review. Although the drying protocol reported here will need further improvement, the results suggest that it may be possible to store the male genetic resources.

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.

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.