Bovine Preimplantation Embryo Development Research Articles

173 Effects of aggregated protein content in sperm head on pre-implantation development of bovine embryos

173 Effects of aggregated protein content in sperm head on pre-implantation development of bovine embryos

Sperm-borne lncRNA loc100847420 improves development of early bovine embryos

Long non-coding RNAs (lncRNAs) act as competing endogenous RNAs (ceRNAs) that play a significant role in bovine embryo development; but the influence of sperm-borne lncRNA on the preimplantation development of bovine embryos has not been reported in detail. In this study, we aimed to clarify how sperm-borne lncRNAs can act to regulate early development of bovine embryos. Utilizing high-throughput sequencing technology and quantitative real-time PCR (qPCR), we found that the lncRNA, loc100847420, was highly enriched in bovine sperm and was carried into the oocyte during fertilization. Introduction of wild-type loc100847420 had no effect on cleavage rate of parthenogenetic embryos compared with injection of mutant loc100847420 (70.58 ± 2.85% vs 70.46 ± 1.98%, p > 0.05), but significantly improved the blastocyst rate (33.67 ± 2.40% vs 28.35 ± 3.06%, p < 0.05), total numbers of cells (p < 0.05), numbers of inner cell mass (ICM) cells (p < 0.05) and numbers of trophoblast (TE) cells (p < 0.05). In summary, the sperm-borne lncRNA, loc100847420, can improve the developmental potential of early bovine embryos.

Molecular and cellular programs underlying the development of bovine pre-implantation embryos.

Early embryonic mortality is a major cause of infertility in cattle, yet the underlying molecular causes remain a mystery. Over the past half century, assisted reproductive technologies such as in vitro fertilisation and somatic cell nuclear transfer have been used to improve cattle reproductive efficiency; however, reduced embryo developmental potential is seen compared to their in vivo counterparts. Recent years have seen exciting progress across bovine embryo research, including genomic profiling of embryogenesis, new methods for improving embryo competence, and experimenting on building bovine embryos from stem cell cultures. These advances are beginning to define bovine embryo molecular and cellular programs and could potentially lead to improved embryo health. Here, I highlight the current status of molecular determinants and cellular programs of bovine embryo development and new opportunities to improve the bovine embryo health.

Embryonic microRNAs are essential for bovine preimplantation embryo development

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression after transcription. miRNAs are present in transcriptionally quiescent full-grown oocytes and preimplantation embryos that display a low level of transcription prior to embryonic genome activation. The role of miRNAs, if any, in preimplantation development is not known. The temporal pattern of expression of miRNAs during bovine preimplantation development was determined by small RNA-sequencing using eggs and preimplantation embryos (1-cell, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst). Embryos cultured in the presence of α-amanitin, which permitted the distinguishing of maternal miRNAs from embryonic miRNAs, indicated that embryonic miRNA expression was first detected at the two-cell stage but dramatically increased during the morula and blastocyst stages. Targeting DGCR8 by a small-interfering RNA/morpholino approach revealed a role for miRNAs in the morula-to-blastocyst transition. Knockdown of DGCR8 not only inhibited expression of embryonically expressed miRNAs but also inhibited the morula-to-blastocyst transition. In addition, RNA-sequencing identified an increased relative abundance of messenger RNAs potentially targeted by embryonic miRNAs in DGCR8-knockdown embryos when compared with controls. Results from these experiments implicate an essential role for miRNAs in bovine preimplantation embryo development.

Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development

We report a novel method to profile intrcellular oxygen concentration (icO2) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO2 and decreasing oxygen consumption to increase icO2. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9–4 × 106 copies and did not correlate with icO2. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.

Sperm-borne miR-202 targets SEPT7 and regulates first cleavage of bovine embryos via cytoskeletal remodeling.

In mammals, sperm-borne regulators can be transferred to oocytes during fertilization and have different effects on the formation of pronuclei, the first cleavage of zygotes, the development of preimplantation embryos and even the metabolism of individuals after birth. The regulatory role of sperm microRNAs (miRNAs) in the development of bovine preimplantation embryos has not been reported in detail. By constructing and screening miRNA expression libraries, we found that miR-202 was highly enriched in bovine sperm. As a target gene of miR-202, co-injection of SEPT7 siRNA can partially reverse the accelerated first cleavage of bovine embryos caused by miR-202 inhibitor. In addition, both a miR-202 mimic and SEPT7 siRNA delayed the first cleavage of somatic cell nuclear transfer (SCNT) embryos, suggesting that miR-202-SEPT7 mediates the delay of first cleavage of bovine embryos. By further exploring the relationship between miR-202/SEPT7, HDAC6 and acetylated α-tubulin during embryonic development, we investigated how sperm-borne miR-202 regulates the first cleavage process of bovine embryos by SEPT7 and demonstrate the potential of sperm-borne miRNAs to improve the efficiency of SCNT.

Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1.

In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18–20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P < 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P < 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P < 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.

Quercetin supports bovine preimplantation embryo development under oxidative stress condition via activation of the Nrf2 signalling pathway.

Nrf2 is a master regulator for antioxidant machinery against oxidative stress in bovine preimplantation embryos. The endogenous or exogenous modulation of Nrf2-KEAP1 system in bovine embryos may contribute to the understanding of the mechanisms behind the response of embryos to stress conditions. Therefore, here we aimed to investigate the protective effect of quercetin on bovine preimplantation embryos exposed to higher atmospheric oxygen concentration. For that, blastocysts, which were developed from zygotes cultured in media supplemented with or without quercetin under high oxygen level (20%), were subjected intracellular ROS level and mitochondrial analysis, and determining blastocyst formation rate and total cell number. Moreover, mRNA and protein expression level of Nrf2 and selected downstream antioxidant genes were investigated in the resulting blastocysts. Quercetin supplementation in vitro culture did not affect cleavage and blastocyst rate until day 7. However, quercetin supplementation resulted in higher blastocyst total cell number and reduction of intracellular ROS level accompanied by increasing mitochondrial activity compared with control group in both day 7 and day 8 blastocysts. Moreover, quercetin supplementation induced mRNA and protein of Nrf2 with subsequent increase in the expression of downstream antioxidants namely: NQO1, PRDX1, CAT and SOD1 antioxidants. In conclusion, quercetin protects preimplantation embryos against oxidative stress and improves embryo viability through modulation of the Nrf2 signalling pathway.

The landscape of accessible chromatin in bovine oocytes and early embryos

ABSTRACT Chromatin reorganization governs the regulation of gene expression during preimplantation development. However, the landscape of chromatin dynamics in this period has not been explored in bovine. In this study, we constructed a genome-wide map of accessible chromatin in bovine oocytes and early embryos using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) which revealed unique features of the accessible chromatin during bovine early embryo development. We found that chromatin accessibility is low in oocytes and 2-/4-cell embryos, followed by a significant increase in embryos during major embryonic genome activation (EGA), and peaked in elongating day 14 embryos. Genome-wide characteristics of open chromatin showed that ATAC-seq signals in both transcription start sites (TSS) and transcription end sites (TES) were strong. Additionally, the distal ATAC-seq peaks were enriched in repeat elements in a type-specific and stage-specific manner. We further unveiled a series of transcription factor (TF) motifs with distinct variation of enrichment from distal ATAC-seq peaks. By integrated analysis of chromatin accessibility with transcriptomes and DNA methylomes in bovine early embryos, we showed that promoter accessibility was positively correlated with gene expression, especially during major EGA, and was strongly correlated to DNA methylation and CpG density. Finally, we identified the critical chromatin signatures and TFs that differ between in vivo and in vitro derived blastocysts, which provides insights to the potential mechanisms leading to low quality of embryos produced in vitro. Together, this comprehensive analysis revealed critical features of chromatin landscape and epigenetic reprogramming during bovine preimplantation embryo development.

Low-dose Roundup induces developmental toxicity in bovine preimplantation embryos in vitro.

Roundup is a widely used glyphosate-based herbicide worldwide. Roundup residues can be detected in the organs and urine of animals. However, its toxicity on mammalian preimplantation embryos has not been well investigated. Here, we show Roundup impairs the development and quality of bovine preimplantation embryos in a dose-dependent manner. Exposure to the agricultural recommended doses of Roundup caused in vitro developmental arrest and quick death of bovine embryos. Furthermore, even a very low concentration (0.9ppm) of Roundup was harmful to bovine preimplantation development. In addition, Roundup increases intracellular calcium levels and induces oxidative stress and apoptosis in bovine embryos. Even if the embryos developed to morphologically normal blastocysts when cultured with low concentrations of Roundup, abnormal intracellular calcium and oxidative stress could be detected inside the embryos and led to an increased incidence of apoptosis in the blastocysts. These data suggest Roundup residues from the agricultural application are potentially dangerous to mammalian preimplantation embryos.

85 Regulation of mammalian target of rapamycin signaling post-fertilisation is essential for efficient development of bovine pre-implantation embryos

The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is considered a key regulator of various cellular responses related to growth, proliferation, survival, apoptosis, and autophagy. The expression of mTOR in cumulus cells and all oocyte stages highlights a role in embryonic development, but its function in the context of embryo development is not completely clarified. In the current study, we investigated the effect of mTOR activation on the total cleavage and blastocyst development rates in bovine. Oocytes collected from bovine ovaries were invitro matured (around 50 oocytes per group) and fertilised before incubation with the mTOR activator MHY1485 (1 µM) for 72h at 38.5°C and 5% CO2. The total cleavage rate at Day 4 post-fertilisation and the blastocyst development rate at Day 8 post-fertilisation were recorded. The expression levels of the mTOR and PI3K in Day 8 blastocysts were investigated using immunofluorescence and imaged using confocal laser-scanning microscope, whereas the fluorescence intensity was analysed using ImageJ software. The difference between groups was analysed using Student's t-test (GraphPad Prism). Results of microscopic investigations showed higher rates of Day 4 cleavage (83.7±1.3 vs. 72±0.8% for control) and Day 8 blastocyst development (40.4±1.7 vs. 33±1.8% for control) upon addition of MHY1485 (P&amp;lt;0.05). The immunofluorescence showed higher mTOR and PI3K expression levels in MHY1485-treated group than the untreated control (P&amp;lt;0.05). The total number of cells per blastocyst, using 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei, was higher in the mTOR-activated group (194.9±4.8 vs. 171.3±3.5 for control). In conclusion, our data reflect the essential role of the mTOR signaling for bovine pre-implantation embryonic development.

188 Improvement of bovine oocyte maturation invitro through cytokine supplementation

Oocyte competence is one of the key factors determining the proportion of embryos that develop to the blastocyst stage. There is vast evidence that IVM oocytes exhibit less developmental potential than their invivo counterparts. Here, we tested whether supplementation of three cytokines [FGF2 (40 ngmL−1), LIF (20ngmL−1), and IGF1 (20 ngmL−1), termed FLI] improved oocyte maturation, and as a consequence, preimplantation development of bovine embryos invitro. In the first experiment, cumulus-oocyte complexes (COCs) were collected from abattoir-derived ovaries and placed in maturation medium,±FLI, for 18 to 22h. At the end of maturation, COCs were fertilized with sperm from a single Holstein bull known to have high fertility. After an 18- to 20-h fertilization period, putative zygotes were cultured in synthetic oviductal fluid for 8 days. The number of embryos that underwent at least one cellular division (cleavage) and the number of embryos that developed to the blastocyst stage was recorded on Days 3 and 8 after insemination, respectively. The COCs supplemented with FLI (n=554) and controls (n=534) were evaluated across 5 replicates. There was no difference in the cleavage rate (P&amp;gt;0.05) between the two treatments. Development to the blastocyst stage was higher (P=0.05) for FLI-treated COCs (34.9%±1.96) than for the control group (23.9%±1.96). In a second experiment, COCs (n=204) supplemented±FLI were collected and fixed at 6, 12, 18, and 24h after placement in maturation medium. The number of transzonal projections in the COCs was determined by localization of actin filaments by using confocal microscopy. Data were analysed by ANOVA using the GLM procedure of SAS software (version 9.4; SAS Institute Inc.). The model included treatment, time, and the interaction of treatment×time as fixed effects. There was no difference (P&amp;gt;0.05) in the number of transzonal projections at 6h (166.3±8.6 vs. 143.9±8.8) and 12h (107.8±8.4 vs. 128.3±7.7) between FLI-treated and control COCs. However, FLI-treated COCs had fewer (P&amp;lt;0.05) transzonal projections at 18h (67.9±7.8 vs. 100.1±7.7) and 24h (56.4±7.2 vs. 80.6±7.4) compared with the controls. There was a significant treatment×time interaction (P=0.006). In a third experiment, we tested whether the timing of transzonal projection disassociation affected lipid accumulation in the embryo. Blastocysts (n=59) on Day 8 produced from COCs matured±FLI were collected and lipid content was determined by using Nile Red staining. There was no difference (P&amp;gt;0.05) in lipid content between treatments. Thus, supplementation of maturation medium with FLI accelerates the disassociation of transzonal projections in COCs and improves subsequent embryonic development to the blastocyst stage while having no detectable effect on lipid content. Further research is necessary to understand how these cytokines modulate IVM of bovine oocytes. This project was supported by Food for the 21st Century and the Clifton Murphy scholarship fund.

The PPARδ Agonist GW501516 Improves Lipolytic/Lipogenic Balance through CPT1 and PEPCK during the Development of Pre-Implantation Bovine Embryos.

The PPARs (peroxisome proliferator-activated receptors) play critical roles in the regulation of lipid and glucose metabolism. PPARδ, a member of the PPARs family, is associated with decreased susceptibility to ectopic lipid deposition and is implicated in the regulation of mitochondrial processes. The current study aimed to determine the role of PPARδ in fatty acid β-oxidation and its influence on PEPCK for the lipogenic/lipolytic balance during in vitro bovine oocyte maturation and embryo development. Activation of PPARδ by GW501516, but not 2-BP, was indicated by intact embryonic PEPCK (cytosolic) and CPT1 expression and the balance between free fatty acids and mitochondrial β-oxidation that reduced ROS and inhibited p-NF-κB nuclear localization. Genes involved in lipolysis, fatty acid oxidation, and apoptosis showed significant differences after the GW501516 treatment relative to the control- and 2-BP-treated embryos. GSK3787 reversed the PPARδ-induced effects by reducing PEPCK and CPT1 expression and the mitochondrial membrane potential, revealing the importance of PPARδ/PEPCK and PPARδ/CPT1 for controlling lipolysis during embryo development. In conclusion, GW501516-activated PPARδ maintained the correlation between lipolysis and lipogenesis by enhancing PEPCK and CPT1 to improve bovine embryo quality.

Characterisation of the Hippo signalling pathway during bovine preimplantation embryo development.

Blastocyst formation is an important milestone during preimplantation embryo development. During murine preimplantation embryogenesis, the Hippo signalling pathway is known to play a significant role in lineage segregation and henceforth the formation of blastocysts. However, the role of this cell signalling pathway during bovine embryogenesis remains unknown. Thus, the aim of the present study was to characterise the Hippo signalling pathway during bovine preimplantation embryo development. mRNA transcripts of Hippo signalling pathway constituents (i.e. crumbs cell polarity complex component 3 (CRB3), mammalian sterile 20-like 1 (MST1), mammalian sterile 20-like 2 (MST2), Yes associated protein 1 (YAP1), transcriptional coactivator with PDZ-binding motif (TAZ)) were observed during all stages of bovine preimplantation embryo development. To evaluate the localisation of Hippo pathway components, bovine embryos at timed stages of development were stained using specific antibodies and observed under a laser confocal microscope. Although MST1/2 proteins were in the cytoplasm during various stages of bovine embryonic development, TAZ and phosphorylated (p-) YAP were detected in the nucleus during the blastocyst stages. Localisation of TAZ and p-YAP proteins was distinct in the bovine compared with mouse model, suggesting that the Hippo signalling pathway is regulated differently in early bovine embryos.

60 Quercetin protects bovine pre-implantation embryos against oxidative stress via activation of Nrf2 signaling pathway

High atmospheric oxygen level during in vitro embryo culture system is reported to induce oxidative stress, leading to an increased intracellular reactive oxygen species (ROS) accumulation and lower the quality and development of the embryos. The Nrf2 is a redox sensitive transcription factor, which regulates the antioxidant machinery against oxidative stress in bovine pre-implantation embryos (Amin et al. 2014Mol. Reprod. Dev. 81, 497-513). Quercetin is a plant-derived flavonoid found in fruits and vegetables with antioxidant, anti-inflammatory, and anti-apoptotic properties. The effect of these antioxidants on the development and quality of bovine pre-implantation embryos with respect to the activation of the Nrf2 signalling pathways is not yet discovered. Here, we aimed to investigate the effect of quercetin supplementation on the activation of the Nrf2 signalling pathway and the subsequent impact on the quality of bovine embryos. For this, presumptive bovine zygotes were cultured in SOFaa embryo culture media supplemented with or without 10 µM quercetin (482 and 485 zygotes, respectively) and incubated under high oxygen level (20%) conditions. Day 7 and Day 8 blastocysts were analysed for the accumulation intracellular ROS and mitochondrial activity using H2DCFDA and MitoTracker® Red, respectively. Thereafter, blastocysts from both groups were subjected to total RNA isolation using Norgen total RNA purification plus kit (BioTek, Winooski, VT, USA). The relative abundance of Nrf2 and its downstream antioxidant genes (NQO1, PRDX1, SOD1, and CAT) was quantified using quantitative PCR. Moreover, the protein level of Nrf2 was detected using immunofluorescence staining. Data from 4 independent biological replicates were statistically analysed using a 2-tailed Student’s t-test. A significant difference in the mean value among treatments was determined at P=0.05. Results showed that quercetin supplementation in embryo culture medium significantly increased the blastocyst total cell number both at Day 7 (125±7.98 v. 100±3.74; P=0.007) and Day 8 (128±4.49 v. 95±3.15; P&amp;lt;0.0001). However, no difference was observed in the quercetin-treated group compared with control with respect to the cleavage rate (85.89±1.11v. 83.86±1.27; P=0.2) as well as the blastocyst rate at Day 7 (24.57±4.94 v. 27.23±1.68; P=0.64) and Day 8 (41.17±3.70 v. 38.87±0.85; P=0.58). Interestingly, quercetin-supplemented blastocysts showed a significant reduction in intracellular ROS level and increment in the mitochondrial activity compared with untreated counterparts. Supplementation of quercetin activated the Nrf2 transcriptional factor both at the mRNA and protein level, which subsequently activated the transcription of the aforementioned downstream antioxidant genes. In conclusion, supplementation of quercetin to embryo culture media protects embryos against oxidative stress by activating the Nrf2-mediated oxidative stress response mechanism, which leads to reduced accumulation of ROS and elevated mitochondrial activity.

The necessity of ZSCAN4 for preimplantation development and gene expression of bovine embryos.

Zinc finger and SCAN domain containing 4 (Zscan4) is a gene that is specifically expressed during zygotic genome activation (ZGA) in mouse preimplantation embryos, and a reduction of Zscan4 transcripts leads to developmental failure. In mouse embryonic stem cells (ESCs), Zscan4 is expressed transiently in as little as 1–5% of the cell population. Zscan4 has also been shown to enhance the efficiency of mouse induced pluripotent stem cells (iPSCs) generation and their quality. Although ZSCAN4 plays important roles in murine embryos and stem cells, its expression and role in bovine embryos is unknown. This study examines ZSCAN4 transcripts in bovine embryos at various developmental stages and attempts to elucidate the functions of ZSCAN4 during bovine preimplantation development. ZSCAN4 transcripts were found to be upregulated at the 8- and 16-cell stages. We next attempted ZSCAN4 downregulation in bovine early embryos by RNA interference and evaluated developmental competency and transcripts levels of genes involved in ZGA and iPSCs generation. Although the bovine embryos injected with ZSCAN4-siRNA could develop to the 8-cell stage, very few were developing beyond the 16-cell stage. PIWIL2 expression was reduced in ZSCAN4 downregulated embryos. It is possible that ZSCAN4 downregulated embryos fail to regulate gene expression during ZGA. Our results indicate that ZSCAN4 is an important factor for the preimplantation development of bovine embryos.

Active H3K27me3 demethylation by KDM6B is required for normal development of bovine preimplantation embryos

ABSTRACTThe substantial epigenetic remodeling that occurs during early stages of mammalian embryonic development likely contributes to reprogramming the parental genomes from a differentiated to a totipotent state and activation of the embryonic genome. Trimethylation of lysine 27 of histone 3 (H3K27me3) is a repressive mark that undergoes global dynamic changes during preimplantation development of several species. To ascertain the role of H3K27me3 in bovine preimplantation development we perturbed the activity of KDM6B, which demethylates H3K27me3. Knockdown of maternal KDM6B mRNA inhibited the reduction in global levels of H3K27me3 from 2-cell to 8-cell embryo stages and compromised development to the blastocyst stage; embryos that developed to the blastocyst stage had fewer inner cell mass (ICM) and trophectoderm (TE) cells. In addition, the transcriptome of KDM6B knockdown embryos was altered at the 8-cell stage and characterized by downregulation of transcripts related to transcriptional regulation, chromatin remodeling, and protein catabolism. Inhibiting the catalytic activity of KDM6B with a specific small molecule inhibitor also prevented the global decrease in H3K27me3 and compromised development to the blastocyst stage. These results indicate that histone demethylation activity, mediated by KDM6B, is required for the global decrease in H3K27me3, correct activation of the embryonic genome, and development to the blastocyst stage in bovine embryos.

Native plants (Phellodendron amurense and Humulus japonicus) extracts act as antioxidants to support developmental competence of bovine blastocysts.

ObjectivePhellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos.MethodsAfter in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense (0.01 μg/mL) and H. japonicus (0.01 μg/mL). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated.ResultsWe observed that the blastocysts rate was significantly increased (p<0.05) in P. amurense (28.9%±2.9%), H. japonicus (30.9%±1.5%), and a mixture of P. amurense and H. japonicus (34.8%± 2.1%) treated groups compared with the control group (25.4%±1.6%). We next confirmed that the intracellular levels of reactive oxygen species (ROS) were significantly decreased (p<0.01) in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05) in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos.ConclusionThese results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

Pyridoxine supplementation during oocyte maturation improves the development and quality of bovine preimplantation embryos

Recently, inhibition of cathepsin B (CTSB) activity during in vitro maturation (IVM) and culture (IVC) improved the developmental competence and quality of bovine oocytes and embryos. E-64 is a widely used inhibitor to inhibit CTSB activity, however, E-64 inhibits not only CTSB activity but also the activities of other proteases including cathepsin L (CTSL), papain, calpain, and trypsin. Pyridoxine, the catalytically active form of vitamin B6, plays a crucial role in several cellular processes and has the ability to inhibit CTSB activity. However, whether pyridoxine has an improving effect during IVM of bovine oocytes is still unknown. In this study, we investigated the effect of pyridoxine supplementation during IVM on the developmental competence of bovine oocytes and the quality of the produced blastocysts. Supplementation of pyridoxine to the maturation medium significantly decreased the activity of CTSB in both bovine cumulus cells and oocytes. Moreover, pyridoxine improved both the blastocyst and hatched blastocyst rates. In addition, the presence of pyridoxine during IVM also significantly improved the quality of the produced embryos by increasing the total cell number as well as decreasing the CTSB mRNA expression and apoptotic rate. These results indicate that pyridoxine is a promising tool to improve the developmental competence of bovine oocytes and subsequent embryo quality.

Exploring timing activation of functional pathway based on differential co-expression analysis in preimplantation embryogenesis.

Recent genome-wide omics studies have confirmed the early embryogenesis strictly dependent on the rigorous spatiotemporal activation and multilevel regulation. However, the full effect of functional pathway was not considered. To obtain complete understanding of the gene activation during early development, we performed systematic comparisons based on differential co-expression analysis for bovine preimplantation embryo development (PED). The results confirmed that the functional pathways actively transcribes as early as the 2-cell and 4-cell waves, which Basal transcription factor, Endocytosis and Spliceosome pathway can represent first signs of embryonic activity. Endocytosis act as one of master activators for uncovering a series of successive waves of maternal pioneer signal regulator with the help of Spliceosome complex. Furthermore, the results showed that pattern recognition receptors began to perform its essential function at 4-cell stage, which might be needed to coordinate the later major activation. And finally, our work presented a probable dynamic landscape of key functional pathways for embryogenesis. A clearer understanding of early embryo development will be helpful for Assisted Reproductive Technology (ART) and Regenerative Medicine (RM).