Embryonic Development In Pigs Research Articles

MCT1-mediated transport of valeric acid promotes porcine preimplantation embryo development by improving mitochondrial function and inhibiting the autophagic AMPK-ULK1 pathway

Oocytes and embryos are highly sensitive to environmental stress in vivo and in vitro. During in vitro culture, many stressful conditions can affect embryo quality and viability, leading to adverse clinical outcomes such as abortion and congenital abnormalities. In this study, we found that valeric acid (VA) increased the mitochondrial membrane potential and ATP content, decreased the level of reactive oxygen species that the mitochondria generate, and thus improved mitochondrial function during early embryonic development in pigs. VA decreased expression of the autophagy-related factors LC3B and BECLIN1. Interestingly, VA inhibited expression of autophagy-associated phosphorylation-adenosine monophosphate-activated protein kinase (p-AMPK), phosphorylation-UNC-51-like autophagy-activated kinase 1 (p-ULK1, Ser555), and ATG13, which reduced apoptosis. Short-chain fatty acids (SCFAs) can signal through G-protein-coupled receptors on the cell membrane or enter the cell directly through transporters. We further show that the monocarboxylate transporter 1 (MCT1) was necessary for the effects of VA on embryo quality, which provides a new molecular perspective of the pathway by which SCFAs affect embryos. Importantly, VA significantly inhibited the AMPK-ULK1 autophagic signaling pathway through MCT1, decreased apoptosis, increased expression of embryonic pluripotency genes, and improved embryo quality.

Expression pattern of ATG4C and its effect on early embryonic development of porcine oocytes

Autophagy is essential for oocyte maturation and preimplantation embryo development. ATG4C, a member of the ATG4 family, plays a crucial role in the autophagy process. The effect of ATG4C on the early embryonic development in pig has not been studied. In this study, the expression patterns of ATG4C were explored using qRT-PCR and immunofluorescence staining. Different concentrations of serum were added to in vitro maturation (IVM) medium to investigate its effects on oocyte maturation and embryonic development. Finally, the developmental potential of parthenogenetic embryos was detected by downregulating ATG4C in MII stage oocytes under 0% serum condition. The results revealed that ATG4C was highly expressed in porcine oocytes matured in vitro and in parthenogenetic embryos. Compared with the 10% serum group, the cumulus cell expansion, first polar body (PB1) extrusion rate, and subsequent developmental competence of embryos were reduced in the 0% and 5% serum groups. The mRNA levels of LC3, ATG5, BECLIN1, TFAM, PGC1α, and PINK1 were significantly increased (P ˂ 0.05) in the 0% serum group. ATG4C was significantly upregulated in the embryos at the 1-cell, 2-cell, 8-cell, and 16-cell stages in the 0% serum group (P ˂ 0.05). Compared with the negative control group, downregulation of ATG4C significantly decreased the 4-cell, 8-cell, and blastocyst rates (P < 0.05), and the expression of genes related to autophagy, mitochondria, and zygotic genome activation (ZGA) was significantly decreased (P ˂ 0.05). The relative fluorescence intensity of LC3 and mitochondrial content in the ATG4C siRNA group was significantly reduced (P ˂ 0.05). Collectively, the results indicate that ATG4C is highly expressed in porcine oocytes matured in vitro and in early embryos, and inhibition of ATG4C effects embryonic developmental competence by decreasing autophagy, mitochondrial content, and ZGA under serum-free condition.

Deoxynivalenol leads to endoplasmic reticulum stress-mediated apoptosis via the IRE1/JNK/CHOP pathways in porcine embryos

The cytotoxic mycotoxin deoxynivalenol (DON) reportedly has adverse effects on oocyte maturation and embryonic development in pigs. Recently, the interplay between cell apoptosis and endoplasmic reticulum (ER) stress has garnered increasing attention in embryogenesis. However, the involvement of the inositol-requiring enzyme 1 (IRE1)/c-jun N-terminal kinase (JNK)/C/EBP homologous protein (CHOP) pathways of unfolded protein response (UPR) signaling in DON-induced apoptosis in porcine embryos remains unknown. In this study, we revealed that exposure to DON (0.25 μM) substantially decreased cell viability until the blastocyst stage in porcine embryos, concomitant with initiation of cell apoptosis through the IRE1/JNK/CHOP pathways in response to ER stress. Quantitative PCR confirmed that UPR signaling-related transcription factors were upregulated in DON-treated porcine blastocysts. Western blot analysis showed that IRE1/JNK/CHOP signaling was activated in DON-exposed porcine embryos, indicating that ER stress-associated apoptosis was instigated. The ER stress inhibitor tauroursodeoxycholic acid protected against DON-induced ER stress in porcine embryos, indicating that the toxic effects of DON on early developmental competence of porcine embryos can be prevented. In conclusion, DON exposure impairs the developmental ability of porcine embryos by inducing ER stress-mediated apoptosis via IRE1/JNK/CHOP signaling.

Methoxychlor induces oxidative stress and impairs early embryonic development in pigs.

Introduction: Methoxychlor (MXC) is an organochlorine pesticide (OCP) that was formerly used worldwide as an insecticide against pests and mosquitoes. However, MXC is not biodegradable and has lipophilic characteristics; thus, it accumulates in organisms and affects reproductive function. MXC, as an estrogenic compound, promotes oxidative stress, induces oxidative stress damage to ovarian follicles, and causes miscarriages and stillbirths in females. In this research endeavor, our primary objective was to explore the ramifications of MXC regarding the developmental processes occurring during the initial stages of embryogenesis in pigs. Methods: In this study, we counted the blastocyst rate of early embryos cultured in vitro. We also examined the reactive oxygen species level, glutathione level, mitochondrial membrane potential, mitochondrial copy number and ATP level in four-cell stage embryos. Finally, apoptosis and DNA damage in blastocyst cells, as well as pluripotency-related and apoptosis-related genes in blastocyst cells were detected. The above experiments were used to evaluate the changes of MXC damage on early parthenogenetic embryo development. Results and Discussion: The results showed that early embryos exposed to MXC had a significantly lower cleavage rate, blastocyst rate, hatching rate, and total cell count compared with the control group. It was also of note that MXC not only increased the levels of reactive oxygen species (ROS), but also decreased the mitochondrial membrane potential (ΔΨm) and mitochondrial copy number during the development of early embryos. In addition, after MXC treatment, blastocyst apoptosis and DNA damage were increased, decreased cell proliferation, and the expression of pluripotency-related genes SOX2, NANOG, and OCT4 was down-regulated, while the expression of apoptosis-related genes BAX/BCL-2 and Caspase9 was up-regulated. Our results clearly show that MXC can have deleterious effects on the developmental processes of early porcine embryos, establishing the toxicity of MXC to the reproductive system. In addition, the study of this toxic effect may lead to greater concern about pesticide residues in humans and the use of safer pesticides, thus potentially preventing physiological diseases caused by chemical exposure.

KLF4 regulates skeletal muscle development and regeneration by directly targeting P57 and Myomixer

Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell proliferation, apoptosis, and differentiation. Our previous study showed that KLF4 expression is upregulated in skeletal muscle ontogeny during embryonic development in pigs, suggesting its importance for skeletal muscle development and muscle function. We revealed here that KLF4 plays a critical role in skeletal muscle development and regeneration. Specific knockout of KLF4 in skeletal muscle impaired muscle formation further affecting physical activity and also defected skeletal muscle regeneration. In vitro, KLF4 was highly expressed in proliferating myoblasts and early differentiated cells. KLF4 knockdown promoted myoblast proliferation and inhibited myoblast fusion, while its overexpression showed opposite results. Mechanically, in proliferating myoblasts, KLF4 inhibits myoblast proliferation through regulating cell cycle arrest protein P57 by directly targeting its promoter; while in differentiated myoblasts, KLF4 promotes myoblast fusion by transcriptionally activating Myomixer. Our study provides mechanistic information for skeletal muscle development, reduced muscle strength and impaired regeneration after injury and unveiling the mechanism of KLF4 in myogenic regulation.

CircKDM5B sponges miR-128 to regulate porcine blastocyst development by modulating trophectoderm barrier function.

Circular RNAs (circRNAs), which exert critical functions in the regulation of transcriptional and posttranscriptional gene expression, are found in mammalian cells but their functions in mammalian preimplantation embryo development remain poorly understood. Here, we showed that circKDM5B mediated miRNA128 to regulate porcine early embryo development. We screened circRNAs potentially expressed in porcine embryos by integrated analysis of sequencing data from mouse and human embryos as well as porcine oocytes. An authentic circRNA originating from histone demethylase KDM5B (referred to as circKDM5B) was abundantly expressed in porcine embryos. Functional studies revealed that circKDM5B knockdown not only significantly reduced blastocyst formation but also decreased the number of total cells and trophectoderm (TE) cells. Moreover, the knockdown of circKDM5B resulted in the disturbance of tight junction assembly and impaired paracellular sealing within the TE epithelium. Mechanistically, miR-128 inhibitor injection could rescue the early development of circKDM5B knockdown embryos. Taken together, the findings revealed that circKDM5B functions as a miR-128 sponge, thereby facilitating early embryonic development in pigs through the modulation of gene expression linked to tight junction assembly.

Proteomics reveals the underlying mechanism by which the first uneven division affects embryonic development in pig

One of the most typical abnormal cleavage patterns during early embryonic development is uneven division, but the first uneven division of pig zygote is common. Uneven division results in different daughter cell sizes and an uneven distribution of organelles such as lipid droplet, mitochondria, but the developmental capacity of daughter cells and proteomic changes of daughter cells are still unclear. Therefore, the developmental ability and proteomic quantification were investigated on blastomeres from even division (ED) or uneven division (UD) embryos at 2-cell stage in the present study. Firstly, the developmental ability was affected by the blastomeric size, when compared with medium blastomeres (MBs), the large blastomeres (LBs) with the higher cleavage rate but the small blastomeres (SBs) with the lower rate was observed. Subsequently, proteomic analysis was performed on blastomeres of LBs, MBs and SBs, a total of 109 DEPs were detected, which were involved in protein metabolism and processing, energy metabolism and ribosome. In particular, DEPs in LBs vs. SBs were focused on RNA binding and actin cytoskeletal tissue. Two protein-dense networks associated with RNA binding and cytoskeleton were revealed by further protein-protein interaction (PPI) analysis of DEPs in LBs vs. SBs, that DDX1 related to RNA binding and ACTB related to cytoskeleton were confirmed in UD embryos. Therefore, a briefly information of DEPs in blastomeres of 2-cell stage pig embryos was described in the present study, and it further confirmed that the formation of uneven division of the first cell cycle of pig embryos might be controlled by the cytoskeleton; the developmental capacity of daughter cells might be affected by the energy metabolism, RNA binding and ribosome, and further account for the developmental potential of the whole embryo.

Profiling the transcriptomic signatures and identifying the patterns of zygotic genome activation – a comparative analysis between early porcine embryos and their counterparts in other three mammalian species

BackgroundThe transcriptional changes around zygotic genome activation (ZGA) in preimplantation embryos are critical for studying mechanisms of embryonic developmental arrest and searching for key transcription factors. However, studies on the transcription profile of porcine ZGA are limited.ResultsIn this study, we performed RNA sequencing in porcine in vivo developed (IVV) and somatic cell nuclear transfer (SCNT) embryo at different stages and compared the transcriptional activity of porcine embryos with mouse, bovine and human embryos. The results showed that the transcriptome map of the early porcine embryos was significantly changed at the 4-cell stage, and 5821 differentially expressed genes (DEGs) in SCNT embryos failed to be reprogrammed or activated during ZGA, which mainly enrichment to metabolic pathways. c-MYC was identified as the highest expressed transcription factor during ZGA. By treating with 10,058-F4, an inhibitor of c-MYC, the cleavage rate (38.33 ± 3.4%) and blastocyst rate (23.33 ± 4.3%) of porcine embryos were significantly lower than those of the control group (50.82 ± 2.7% and 34.43 ± 1.9%). Cross-species analysis of transcriptome during ZGA showed that pigs and bovines had the highest similarity coefficient in biological processes. KEGG pathway analysis indicated that there were 10 co-shared pathways in the four species.ConclusionsOur results reveal that embryos with impaired developmental competence may be arrested at an early stage of development. c-MYC helps promote ZGA and preimplantation embryonic development in pigs. Pigs and bovines have the highest coefficient of similarity in biological processes during ZGA. This study provides an important reference for further studying the reprogramming regulatory mechanism of porcine embryos during ZGA.

E2F4 regulates cell cycle to mediate embryonic development in pigs

In mammals, E2 factor (E2F) acts as a cell cycle regulator. E2F transcription factor 4 (E2F4) is a member of the E2F family of transcription factors and usually represents predominant E2F activity in cells. The E2F4 gene has been extensively studied in animals and is associated with multiple functions, such as cell cycle regulation and apoptosis; however, little is known about its role during embryonic development. In this study, we investigated the function of E2F4 and its mechanism of action in porcine embryo development. For this purpose, we knocked down E2F4 by microinjecting double-stranded RNA of E2F4 at the 1-cell stage. The results showed that E2F4 knockdown in porcine embryos led to a significant decrease in the blastocyst rate and total cell number. Defective E2F4 expression reduced the level of G1/S checkpoints (cyclin E-cyclin-dependent kinase 2) and cell cycle-related gene expression at the 4-cell embryo stage and blastocyst. Moreover, a decrease in E2F4 expression increased phosphorylated H2A.X variant histones and activated ataxia telangiectasia mutated (ATM) and p53-p21 pathway. In addition, E2F4 depletion caused a significant decrease in histone acetylation. Taken together, E2F4 plays a critical role as a transcriptional activator in the development of porcine embryos, an observation that contradicts its well-established role as a transcription repressor.

Stabilization of F-Actin Cytoskeleton by Paclitaxel Improves the Blastocyst Developmental Competence through P38 MAPK Activity in Porcine Embryos.

Changes in F-actin distribution and cortical F-actin morphology are important for blastocyst developmental competence during embryogenesis. However, the effect of paclitaxel as a microtubule stabilizer on embryonic development in pigs remains unclear. We investigated the role of F-actin cytoskeleton stabilization via P38 MAPK activation using paclitaxel to improve the developmental potential of blastocysts in pigs. In this study, F-actin enrichment and adducin expression based on blastomere fragment rate and cytokinesis defects were investigated in cleaved embryos after in vitro fertilization (IVF). Adducin and adhesive junction F-actin fluorescence intensity were significantly reduced with increasing blastomere fragment rate in porcine embryos. In addition, porcine embryos were cultured with 10 and 100 nM paclitaxel for two days after IVF. Adhesive junction F-actin stabilization and p-P38 MAPK activity in embryos exposed to 10 nM paclitaxel increased significantly with blastocyst development competence. However, increased F-actin aggregation, cytokinesis defects, and over-expression of p-P38 MAPK protein by 100 nM paclitaxel exposure disrupted blastocyst development in porcine embryos. In addition, exposure to 100 nM paclitaxel increased the misaligned α-tubulin of spindle assembly and adhesive junction F-actin aggregation at the blastocyst stage, which might be caused by p-P38 protein over-expression-derived apoptosis in porcine embryos.

ELF4 is critical to zygotic gene activation and epigenetic reprogramming during early embryonic development in pigs.

Zygotic gene activation (ZGA) and epigenetic reprogramming are critical in early embryonic development in mammals, and transcription factors are involved in regulating these events. However, the effects of ELF4 on porcine embryonic development remain unclear. In this study, the expression of ELF4 was detected in early porcine embryos and different tissues. By knocking down ELF4, the changes of H3K9me3 modification, DNA methylation and ZGA-related genes were analyzed. Our results showed that ELF4 was expressed at all stages of early porcine embryos fertilized in vitro (IVF), with the highest expression level at the 8-cell stage. The embryonic developmental competency and blastocyst quality decreased after ELF4 knockdown (20.70% control vs. 17.49% si-scramble vs. 2.40% si-ELF4; p < 0.001). Knockdown of ELF4 induced DNA damage at the 4-cell stage. Interfering with ELF4 resulted in abnormal increases in H3K9me3 and DNA methylation levels at the 4-cell stage and inhibited the expression of genes related to ZGA. These results suggest that ELF4 affects ZGA and embryonic development competency in porcine embryos by maintaining genome integrity and regulating dynamic changes of H3K9me3 and DNA methylation, and correctly activating ZGA-related genes to promote epigenetic reprogramming. These results provide a theoretical basis for further studies on the regulatory mechanisms of ELF4 in porcine embryos.

Effects of pyruvate on early embryonic development and zygotic genome activation in pigs

Pyruvate is an important energy substance during early embryonic development of mammals. However, the underlying mechanisms of pyruvate during early embryonic development in pigs and its role in zygotic genome activation (ZGA) are not fully understood. Here, based on a previous RNA-seq dataset of porcine early embryos, we found that pyruvate metabolism-related genes started to be expressed at the 4-cell stage and that pyruvate metabolism-related genes were correlated with porcine ZGA marker genes. To determine the function of pyruvate in porcine embryos, in vitro fertilization (IVF) embryos were cultured in PZM-3 medium (control group); modified PZM-3 medium that only contains pyruvate and lactate plus salts (+P group); or modified PZM-3 medium lacking pyruvate (-P group). The 4-cell arrest rate at 72 h was significantly increased in the -P group compared to the +P group (P < 0.05). In addition, we observed that the reactive oxygen species (ROS) level was significantly increased and that the adenosine triphosphate (ATP) level was significantly (P < 0.05) decreased in the -P group compared to the +P group. Moreover, the expression of ZGA marker genes and SIRT1 protein in embryos was significantly decreased in the -P group compared to the +P group (P < 0.05). Furthermore, the acetylation level of H3K9 was significantly decreased (P < 0.05) and the methylation level of H3K9 was significantly increased (P < 0.05) in the -P group compared to the +P group. In summary, our findings demonstrate that pyruvate affects early embryonic development in pigs by promoting ZGA and reducing oxidative stress levels.

Cycloleucine negatively regulates porcine oocyte maturation and embryo development by modulating N6-methyladenosine and histone modifications

Maturation of oocytes and early embryo development are regulated precisely by numerous factors at transcriptional and posttranslational levels through precise mechanisms. N6-methyladenosine (m6A) is the most common modification in mRNA which regulates RNA metabolism and gene expression. However, the role of RNA m6A on porcine oocyte maturation and early embryogenesis is largely unknown. Here, we found that oocytes treated with cycloleucine (CL), an RNA m6A inhibitor, express impaired cumulus expansion, increased production of reactive oxygen species (ROS) in the mitochondria, and delayed maturation of oocytes by disrupting spindle organization and chromosome alignment. Also, CL halted the development of embryos at the 4-cell stage and resulted in low-quality blastocysts. Furthermore, CL treatment decreased the RNA m6A, H3K4me3, and H3K9me3 levels, but increased the acetylation level of H4K16 during parthenogenetic embryonic development in pigs. Single-cell RNA-seq (scRNA-seq) analysis further revealed that CL treatment dramatically up-regulated the expression of metabolism-related genes (SLC16A1, and MAIG3 etc.) and maternal related genes, including BTG4, WEE2, and BMP15 among others, at the blastocyst stage. Taken together, inhibition of RNA m6A by CL impaired meiosis of oocytes and early embryonic development of porcine via RNA m6A methylation, histone modifications, and altering the expression of metabolism-related genes in blastocysts.

Tannin Supplementation Improves Oocyte Cytoplasmic Maturation and Subsequent Embryo Development in Pigs.

To investigate the effects of tannins (TA) on porcine oocyte in vitro maturation (IVM), different concentrations of TA (0, 1, 10 and 100 μg/mL) were supplemented with a maturation medium and the COCs and subsequent embryonic development were examined. The results showed that 10 µg/mL TA significantly improved the cumulus expansion index (CEI), cumulus-expansion-related genes (PTGS1, PTGS2, PTX-3, TNFAIP6 and HAS2) expression and blastocyst formation rates after parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) compared to the control groups, but not oocyte nuclear maturation. Nevertheless, 10 µg/mL TA dramatically enhanced the mRNA expression of oocyte-development-related genes (BMP15, GDF9, CDC2 and CYCLIN B1), GSH, ATP, SOD1, PGC1α, BMP15, GDF9 and CDC2 levels and reduced intracellular ROS level in porcine oocytes. These results indicated that porcine oocyte cytoplasmic maturation was improved by 10 µg/mL TA treatment during IVM. In contrast, a high concentration of TA (100 μg/mL) significantly decreased the CEI and PTGS1, PTGS2, PTX-3 and HAS2 mRNA expressions in cumulus cells, and reduced oocyte nuclear maturation and the total cell numbers/blastocyst. In general, these data showed that 10 μg/mL TA supplementation has beneficial effects on oocyte cytoplasmic maturation and subsequent embryonic development in pigs.

Transcriptome Analysis of In Vitro Fertilization and Parthenogenesis Activation during Early Embryonic Development in Pigs.

Parthenogenesis activation (PA), as an important artificial breeding method, can stably preserve the dominant genotype of a species. However, the delayed development of PA embryos is still overly severe and largely leads to pre-implantation failure in pigs. The mechanisms underlying the deficiencies of PA embryos have not been completely understood. For further understanding of the molecular mechanism behind PA embryo failure, we performed transcriptome analysis among pig oocytes (meiosis II, MII) and early embryos at three developmental stages (zygote, morula, and blastocyst) in vitro fertilization (IVF) and PA group. Totally, 11,110 differentially expressed genes (DEGs), 4694 differentially expressed lincRNAs (DELs) were identified, and most DEGs enriched the regulation of apoptotic processes. Through cis- and trans-manner functional prediction, we found that hub lincRNAs were mostly involved in abnormal parthenogenesis embryonic development. In addition, twenty DE imprinted genes showed that some paternally imprinted genes in IVF displayed higher expression than that in PA. Notably, we identified that three DELs of imprinted genes (MEST, PLAGL1, and DIRAS3) were up regulated in IVF, and there was no significant change in PA group. Disordered expression of key genes for embryonic development might play key roles in abnormal parthenogenesis embryonic development. Our study indicates that embryos derived from different production techniques have varied in vitro development to the blastocyst stage, and they also affect the transcription level of corresponding genes, such as imprinted genes. This work will help future research on these genes and molecular-assisted breeding for pig parthenotes.

The landscape of chromatin accessibility in skeletal muscle during embryonic development in pigs

BackgroundThe development of skeletal muscle in pigs during the embryonic stage is precisely regulated by transcriptional mechanisms, which depend on chromatin accessibility. However, how chromatin accessibility plays a regulatory role during embryonic skeletal muscle development in pigs has not been reported. To gain insight into the landscape of chromatin accessibility and the associated genome-wide transcriptome during embryonic muscle development, we performed ATAC-seq and RNA-seq analyses of skeletal muscle from pig embryos at 45, 70 and 100 days post coitus (dpc).ResultsIn total, 21,638, 35,447 and 60,181 unique regions (or peaks) were found across the embryos at 45 dpc (LW45), 70 dpc (LW70) and 100 dpc (LW100), respectively. More than 91% of the peaks were annotated within − 1 kb to 100 bp of transcription start sites (TSSs). First, widespread increases in specific accessible chromatin regions (ACRs) from embryos at 45 to 100 dpc suggested that the regulatory mechanisms became increasingly complicated during embryonic development. Second, the findings from integrated ATAC-seq and RNA-seq analyses showed that not only the numbers but also the intensities of ACRs could control the expression of associated genes. Moreover, the motif screening of stage-specific ACRs revealed some transcription factors that regulate muscle development-related genes, such as MyoG, Mef2c, and Mef2d. Several potential transcriptional repressors, including E2F6, OTX2 and CTCF, were identified among the genes that exhibited different regulation trends between the ATAC-seq and RNA-seq data.ConclusionsThis work indicates that chromatin accessibility plays an important regulatory role in the embryonic muscle development of pigs and regulates the temporal and spatial expression patterns of key genes in muscle development by influencing the binding of transcription factors. Our results contribute to a better understanding of the regulatory dynamics of genes involved in pig embryonic skeletal muscle development.

Glucose in a maturation medium with reduced NaCl improves oocyte maturation and embryonic development after somatic cell nuclear transfer and in vitro fertilization in pigs.

This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.

Lycopene Improves In Vitro Development of Porcine Embryos by Reducing Oxidative Stress and Apoptosis

In vitro culture (IVC) for porcine embryo development is inferior compared to in vivo development because oxidative stress can be induced by the production of excessive reactive oxygen species (ROS) under high oxygen tension in the in vitro environment. To overcome this problem, we investigated the effect of lycopene, an antioxidant carotenoid, on developmental competence and the mechanisms involved in mitochondria-dependent apoptosis pathways in porcine embryos. In vitro fertilized (IVF) embryos were cultured in IVC medium supplemented with 0, 0.02, 0.05, 0.1, or 0.2 μM lycopene. The results indicate that 0.1 μM lycopene significantly increased the rate of blastocyst formation and the total cell numbers, including trophectoderm cell numbers, on Day In terms of mitochondria-dependent apoptosis, IVF embryos treated with 0.1 μM lycopene exhibited significantly decreased levels of ROS, increased mitochondrial membrane potential, and decreased expression of cytochrome c on Days 2 and Furthermore, 0.1 μM lycopene significantly decreased the number and percentage of caspase 3-positive and apoptotic cells in Day-6 blastocysts. In addition, Day-2 embryos and Day-6 blastocysts treated with 0.1 μM lycopene showed significantly reduced mRNA expression related to antioxidant enzymes (SOD1, SOD2, CATALASE) and apoptosis (BAX/BCL2L1 ratio). These results indicate that lycopene supplementation during the entire period of IVC enhanced embryonic development in pigs by regulating oxidative stress and mitochondria-dependent apoptosis.

Effects of electromagnetic waves on oocyte maturation and embryonic development in pigs.

Our living environment has been full of electromagnetic radiation (EMR) due to the prevailing electronic devices and equipment. Intermediate frequency electromagnetic field (IF-EMF) or waves constitute a significant part of EMR; therefore, an increasing number of household electrical appliances have become a source of IF-EMF, and concerns about IF-EMF on health are gaining more attention. However, little information is available about its impact on female reproductive traits, such as germ cell viability and early embryonic development, particularly at the cellular and molecular levels. In this study, we used porcine oocytes as a model system to explore the effect of IF-EMF at various intensities on the in vitro maturation (IVM) of oocytes and their subsequent embryonic development. Our results showed that no difference in oocyte maturation rates was detected among groups, but the cleavage and blastocyst rates of parthenotes derived from EMF-treated oocytes decreased with the weaker IF-EMF intensity (25 and 50 Gauss) groups compared to the control group (P < 0.05). For cytoplasmic maturation, the weaker IF-EMF intensity groups also showed a peripheral pattern of mitochondrial distribution resembling that of immature oocytes and increased autophagy activity. No obvious differences in cytoskeletal distribution and total cell numbers of blastocysts were investigated in the four IF-EMF treatments compared to those in the control group. Although the underlying mechanism associated with EMF effects on oocytes and embryos is still elusive, we have demonstrated that low intensity IF-EMF exerts harmful effects on porcine oocytes during the maturation stage, carrying over such effects to their subsequent embryonic development.

Asiatic acid protects oocytes against in vitro aging-induced deterioration and improves subsequent embryonic development in pigs.

As a pentacyclic triterpene in Centella asiatica, asiatic acid (AA) is a powerful antioxidant with many bioactivities. In the present research, we investigated whether AA has the potential to rescue the decrease in porcine oocyte quality that occurs during in vitro aging (IVA). Mature porcine oocytes were collected and then continuously cultured for an additional 24 h or 48 h with or without AA in maturation medium as an IVA model. The results revealed that AA supplementation reduced the percentage of abnormal aged porcine oocytes during IVA. Furthermore, AA supplementation effectively maintained aged porcine oocyte developmental competence, both parthenogenetic activation and in vitro fertilization. The number of sperm that bound to the zona pellucida on aged porcine oocytes was higher in the AA-supplemented group than in the non-supplemented group. Moreover, AA supplementation not only blocked IVA-induced oxidative stress but also maintained intracellular GSH levels and reduced the percentage of early apoptosis aged porcine oocytes. Mitochondrial functions were disordered during the IVA process. The intracellular ATP levels and mitochondrial membrane potential in aged porcine oocytes were dramatically increased by AA supplementation. Therefore, AA has beneficial effects on porcine oocyte quality and developmental potential maintenance during IVA.