Cultured Mouse Embryos Research Articles

Comparison of the developmental competence of in vitro-produced mouse embryos cultured under 5 versus 2% O2 with in vivo-derived blastocysts.

The prevalence of infertility in Canada has substantially increased over 30 years, and plateaued success rates of culture systems warrant further optimization for transfer outcomes. In clinical programs, embryos commonly undergo extended culture under 5% O2 until the blastocyst stage. The aim of this study is to characterize the developmental competence and stress-related responses of embryos cultured under 5 versus 2% O2 in comparison to in vivo-derived blastocysts. We hypothesized 2% O2 compromises developmental competence through altered embryonic stress responses and induction of apoptosis-related genes relative to those cultured under 5% O2 and in vivo-derived blastocysts. Quantitative measures of development and relative expressions of a cohort of stress-related genes in CD1 mouse zygotes cultured to blastocysts under 5 or 2% O2 were compared to in vivo-derived embryos. Apoptotic responses were evaluated using an immunofluorescence assay for Caspase-3. The mean percentage of blastocysts developed, and total cell number of embryos derived in vivo or cultured under 5% O2 was significantly higher than those cultured under 2% O2. Blastocyst expansion was greatest in embryos cultured under 5% O2. Stress response genes were significantly upregulated in embryos cultured under 2% O2, and expression of antioxidant-related genes was significantly lower in cultured versus in vivo-derived embryos. Caspase-3 immunofluorescence was significantly higher in cultured embryos versus in vivo-derived embryos. We inferred that 5% O2 systems better approximate physiologic oxygen availability for culture of mouse embryos, warranting re-evaluation of culturing embryos under threshold or sub-physiologic oxygen concentrations during clinical IVF programs.

Enhancement of preimplantation mouse embryo development with optimized in vitro culture dish via stabilization of medium osmolarity.

We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

Caudal Fgfr1 disruption produces localised spinal mis-patterning and a terminal myelocystocele-like phenotype in mice.

Closed spinal dysraphisms are poorly understood malformations classified as neural tube (NT) defects. Several, including terminal myelocystocele, affect the distal spine. We have previously identified a NT closure-initiating point, Closure 5, in the distal spine of mice. Here, we document equivalent morphology of the caudal-most closing posterior neuropore (PNP) in mice and humans. Closure 5 forms in a region of active FGF signalling, and pharmacological FGF receptor blockade impairs its formation in cultured mouse embryos. Conditional genetic deletion of Fgfr1 in caudal embryonic tissues with Cdx2Cre diminishes neuroepithelial proliferation, impairs Closure 5 formation and delays PNP closure. After closure, the distal NT of Fgfr1-disrupted embryos dilates to form a fluid-filled sac overlying ventrally flattened spinal cord. This phenotype resembles terminal myelocystocele. Histological analysis reveals regional and progressive loss of SHH- and FOXA2-positive ventral NT domains, resulting in OLIG2 labelling of the ventral-most NT. The OLIG2 domain is also subsequently lost, eventually producing a NT that is entirely positive for the dorsal marker PAX3. Thus, a terminal myelocystocele-like phenotype can arise after completion of NT closure with localised spinal mis-patterning caused by disruption of FGFR1 signalling.

O-235 Antioxidants in culture media improve ICSI fertilisation rate: results from a randomised controlled trial

Abstract Study question Does addition of three antioxidants to G-Series media during gamete collection, insemination, and embryo culture increase the clinical pregnancy rate from fresh blastocyst transfers? Summary answer Antioxidants increased the ICSI fertilization rate, and consequently the number of blastocysts utilised, but the clinical pregnancy rate from fresh blastocyst transfers was not affected. What is known already Supplementation of IVF media with a combination of three antioxidants known as A3 (10 μmol/l acetyl-L-Carnitine, 5 μmol/l α-Lipoic acid and 10 μmol/l N-acetyl-L-cysteine) is beneficial in mouse IVF, embryo culture and cryopreservation, and improves post-transfer outcomes. Further, in a prospective randomised trial on sibling oocytes, addition of A3 to G-1/G-2 media (Vitrolife, Sweden) resulted in more good quality embryos on day 3 (Gardner et al., 2020). The study was not powered to determine the effect of A3 on pregnancy rate, but an increase in ongoing pregnancy rate was observed in women aged 35-40 years following frozen blastocyst transfer. Study design, size, duration Single-centre, prospective randomised controlled trial, superiority study comparing G-series media (G-MOPS, G-IVF and G-TL (Vitrolife)) with or without the addition of A3 from January 2019 to November 2021. 1482 patients were randomized before egg collection. Patients and their doctors were blinded to the treatment group. The primary endpoint was clinical pregnancy per randomised couple from the fresh transfer of a single blastocyst. Participants/materials, setting, methods Patients undergoing IVF/ICSI cycles and intending to undergo a fresh transfer of a single blastocyst were recruited. Exclusion criteria were previous participation in the study, freeze-all cycle, or extraction of sperm from testicular biopsy. 743 patients were randomised to the A3 media, 739 to the control. Main results and the role of chance Patients randomised to A3 media had significantly higher fertilisation rate per inseminated oocyte (64.4 ± 25.6%) compared to the control (59.2% ± 26.3%, P < 0.001). This was more pronounced in patients undergoing ICSI (68.1% ± 24.9% vs 57.9% ± 27.2%, P < 0.0001), where the number of cycles with failed fertilisation decreased from 8.0% to 3.9% with A3 media (P < 0.05). There was no effect of A3 on fertilisation rate following IVF. Blastocyst development rate was unaffected by A3, but the higher fertilisation rate resulted in more blastocysts available for transfer or cryopreservation per patient in the A3 group (3.06 ± 2.97 vs 2.67 ± 2.60, P < 0.01). Clinical pregnancy rate from fresh cycles was not different between the control (26.1%) and A3 media (23.0%; P > 0.05; RR 0.88, 95% CI 0.73-1.05). When patients who did not have a fresh transfer were excluded (due to freeze-all or no embryo available), there was also no difference between the control (35.8%) and A3 media (32.6%; P > 0.05; RR 0.91; 95% CI 0.77-1.08). Limitations, reasons for caution This was a single-centre study, so the effects of A3 supplementation in clinics with different media and protocols are unknown. Detection of fetal heart by ultrasound was the primary endpoint, birth outcomes are currently being investigated. Outcomes of frozen embryo transfers and cumulative pregnancy rates are yet to be determined. Wider implications of the findings Addition of antioxidants to media during gamete collection, incubation and ICSI can increase fertilisation rate and reduce the frequency of failed fertilization cycles. This results in more blastocysts available for transfer and cryopreservation per egg collection, potentially leading to higher cumulative pregnancy rates. Trial registration number ACTRN12618001479291

The surface ectoderm exhibits spatially heterogenous tension that correlates with YAP localisation during spinal neural tube closure in mouse embryos

The single cell layer of surface ectoderm (SE) which overlies the closing neural tube (NT) plays a crucial biomechanical role during mammalian NT closure (NTC), challenging previous assumptions that it is only passive to the force-generating neuroepithelium (NE). Failure of NTC leads to congenital malformations known as NT defects (NTDs), including spina bifida (SB) and anencephaly in the spine and brain respectively. In several mouse NTD models, SB is caused by misexpression of SE-specific genes and is associated with disrupted SE mechanics, including loss of rostrocaudal cell elongation believed to be important for successful closure. In this study, we asked how SE mechanics affect NT morphology, and whether the characteristic rostrocaudal cell elongation at the progressing closure site is a response to tension anisotropy in the SE. We show that blocking SE-specific E-cadherin in ex utero mouse embryo culture influences NT morphology, as well as the F-actin cable. Cell border ablation shows that cell shape is not due to tension anisotropy, but that there are regional differences in SE tension. We also find that YAP nuclear translocation reflects regional tension heterogeneity, and that its expression is sensitive to pharmacological reduction of tension. In conclusion, our results confirm that the SE is a biomechanically important tissue for spinal NT morphogenesis and suggest a possible role of spatial regulation of cellular tension which could regulate downstream gene expression via mechanically-sensitive YAP activity.

Effects of crocin supplementation during in vitro culture of intact and half-destroyed four-cell mouse embryos.

We evaluated the effects of crocin supplementation during culture of intact and half-destroyed four-cell mouse embryos. Outcomes measured included rate of cleavage arrest, blastocyst formation, and blastocyst cell number. We used laser to create two zonal holes without blastomere destruction in Groups 1 (n=100) and 2 (n=100), and to destroy two of the four blastomeres in Groups 3 (n=150) and 4 (n=150). Embryos were cultured in groups of ten in drops of medium without (Groups 1 and 3) or with 20 μg/ml of crocin supplementation (Groups 2 and 4). Embryos in Groups 1 and 2 had no difference in the rate of cleavage arrest (6.0% vs. 7.0%, respectively; p=0.774) or blastocyst formation (89.0% vs. 86.0%, respectively; p=0.521). Neither was there a difference in the number of cells in the blastocysts (99.6±23.5 vs. 95.6± 8.2, respectively, p=0.83). Half-destroyed embryos cultured in crocin-supplemented medium (Group 4) had a lower rate of cleavage arrest (14.7% vs. 30.0%, p=0.001), and a higher rate of blastocyst formation (51.3% vs. 37.3%, p=0.015), than those in non-supplemented medium (Group 3). In blastocysts derived from half-destroyed embryos, there was no difference in the number of cells in ICM (14.5±3.9 vs. 13.7±2.9, p=0.285), TE (45.2±12.3 vs. 46.0±13.3, p=0.764), or total cells (59.7±12.2 vs. 59.7±14.8, respectively, p=0.990) among the two groups. Crocin supplementation during in vitro development of impaired embryos improved their development, but had no effect on intact embryos.

KSOM-R supports both mouse and rat preimplantation embryo development in vitro

A modified KSOM for rat embryo culture (KSOM-R), which has enriched taurine, glycine, glutamic acid, and alanine, promoted rat embryo development in vitro. Since mice and rats share similar amino acid profiles in their female reproductive tracts, this study explored whether KSOM-R would also have a positive effect on mouse embryo development and if KSOM-R modifications could extend its shelf time at 2–8 °C for consistency. We first examined the effects of newly made (≤1 month at 2–8 °C) antibiotics-free KSOM-R (mKSOM-R), antibiotics-free KSOM (mKSOM) and KSOM on the development of in vivo or in vitro derived C57BL/6NJ zygotes. We then investigated the effect of extended shelf life (6 months at 2–8 °C) of mKSOM-R and mKOSM on the development of C57BL/6NJ mouse and Sprague Dawley (SD) rat embryos. The results showed that there were no significant differences in cleavage, blastocyst, and hatching rates of C57BL/6NJ embryos among the three freshly made media. After 6 months of storage at 2–8 °C, mKSOM-R and mKSOM were still able to support the development of in vivo C57BL/6NJ zygotes at comparable rates seen with newly made (≤1 month at 2–8 °C) KSOM (control) in terms of cleavage, blastocyst formation and hatching. There were also no significant differences in total cell numbers in day 4 blastocysts among the three groups. After surgical embryo transfers, C57BL/6NJ blastocysts cultured in mKSOM-R (6 months at 2–8 °C) and newly made (≤1 month at 2–8 °C) KSOM culture developed into live pups. These pups had no gross abnormalities in animal morphology and growth. SD zygotes cultured in mKSOM-R stored at 2–8 °C for 6 months developed at comparable rates in cleavage, blastocyst and hatching rates when compared to those cultured in newly made mKSOM-R (≤1 month at 2–8 °C). The data showed that, although no significant beneficial effects were observed on mouse embryo development, mKSOM-R was able to support both mouse and rat embryo development in vitro. Additionally, mKSOM-R and mKSOM can be stored at 2–8 °C for at least 6 months without significantly compromising quality. This study suggests that it is possible to reduce the media inventory by using only mKSOM-R to culture both mouse and rat embryos, and quality media with extended shelf life can be made through modifications.

Author Correction: Mitochondrial function and intracellular distribution is severely affected in in vitro cultured mouse embryos

Author Correction: Mitochondrial function and intracellular distribution is severely affected in in vitro cultured mouse embryos

TOP2A deficiency leads to human recurrent spontaneous abortion and growth retardation of mouse pre-implantation embryos

BackgroundRecurrent spontaneous abortion (RSA), is a dangerous pregnancy-related condition and is a subject of debate in the gynaecology and obstetrics communities. The objective of this study was to determine the function of DNA Topoisomerase II Alpha (TOP2A) in RSA and elucidate the underlying molecular mechanisms.MethodsIn vitro models of TOP2A-knockdown and -overexpression were generated by transfecting specific sh-RNA lentivirus and overexpression plasmid, respectively. An in vitro TOP2A inhibition model was established by culturing mouse embryos at the two-cell stage in a medium containing PluriSIn2, a TOP2A inhibitor. Immunohistochemical staining was used to analyse expression of TOP2A in villi tissues of patients with RSA. Western blotting and qRT-PCR were used to analyse the expression of TOP2A and proteins involved in trophoblast functions, the FOXO signalling pathway, and the development of pre-implantation embryos. 5-Ethynyl-2′-deoxyuridine staining, TUNEL assay and flow cytometry were used to further evaluate the effect of TOP2A on cell proliferation and apoptosis. Transwell and wound healing assays were used to evaluate migration and invasion. Moreover, the effect of TOP2A inhibitor on embryos was determined by immunofluorescence and mitochondrial-related dyes.ResultsEvaluation of clinical samples revealed that the villi tissues of patients that have experienced RSA had lower TOP2A expression compared with that from women who have experienced normal pregnancy (P < 0.01). In vitro, TOP2A knockdown decreased the proliferation, migration, and invasion of trophoblast cell lines, and increased apoptosis and activation of the FOXO signalling pathway (P < 0.05). Conversely, TOP2A overexpression reversed these effects. Moreover, in vivo experiments confirmed that inhibition of TOP2A impairs trophectoderm differentiation, embryonic mitochondrial function as well as the developmental rate; however, no differences were noted in the expression of zygotic genome activation-related genes.ConclusionsCollectively, our data suggest that lower TOP2A expression is related to RSA as it inhibits trophoblast cell proliferation, migration, and invasion by activation of the FOXO signalling pathway. Additionally, TOP2A inhibition resulted in impaired development of pre-implantation embryos in mice, which could be attributed to excessive oxidative stress.Graphical

Expression and localization of NRF2/Keap1 signalling pathway genes in mouse preimplantation embryos exposed to free fatty acids

Obese women experience greater incidence of infertility, with reproductive tracts exposing preimplantation embryos to elevated free fatty acids (FFA) such as palmitic acid (PA) and oleic acid (OA). PA treatment impairs mouse preimplantation development in vitro, while OA co-treatment rescues blastocyst development of PA treated embryos. In the present study, we investigated the effects of PA and OA treatment on NRF2/Keap1 localization, and relative antioxidant enzyme (Glutathione peroxidase; Gpx1, Catalase; Cat, Superoxide dismutase; Sod1 and γ-Glutamylcysteine ligase catalytic unit; Gclc) mRNA levels, during in vitro mouse preimplantation embryo development. Female mice were superovulated, mated, and embryos cultured in the presence of bovine Serum albumin (BSA) control or PA, or OA, alone (each at 100 μM) or PA + OA combined (each at 100 μM) treatment. NRF2 displayed nuclear localization at all developmental stages, whereas Keap1 primarily displayed cytoplasmic localization throughout control mouse preimplantation development in vitro. Relative transcript levels of Nrf2, Keap1, and downstream antioxidants significantly increased throughout control mouse preimplantation development in vitro. PA treatment significantly decreased blastocyst development and the levels of nuclear NRF2, while OA and PA + OA treatments did not. PA and OA treatments did not impact relative mRNA levels of Nrf2, Keap1, Gpx1, Cat, Sod1 or Gclc. Our outcomes demonstrate that cultured mouse embryos display nuclear NRF2, but that PA treatment reduces nuclear NRF2 and thus likely impacts NRF2/KEAP1 stress response mechanisms. Further studies should investigate whether free fatty acid effects on NRF2/KEAP1 contribute to the reduced fertility displayed by obese patients.

Mitochondrial function and intracellular distribution is severely affected in in vitro cultured mouse embryos

Studies of mitochondrial dynamics have identified an intriguing link between energy supply balance and mitochondrial architecture. This suggests that inappropriate culture conditions might inhibit mitochondrial functions, and affect embryonic development. Therefore, this study was conducted to determine whether in vitro culture (IVC) might affect mitochondrial function, distribution, organization (by Mitotracker Green), gene expression on RNA level (by qPCR), and protein expression and localization (by western blot and immunostaining) involved in regulation of mitochondrial functions. Mitochondria in 2-cell IVC embryos were less numerous compare to IN VIVO while the localization and distribution do not differ between the groups. Mitochondria of in vivo blastocysts formed elongated network along the cells, while in IVC were fragmented, rounded, and aggregated mainly in the perinuclear region. Additionally, mitochondria of IN VIVO embryos moved back and forth along their long axis on radial tracks, while in IVC blastocysts were much less active. mtDNA copy number in IVC blastocysts (92,336.65 ± 5860.04) was significantly lower than that of IN VIVO (169,103.92 ± 16,322.41; P < 0.02) as well as lower protein expressions responsible for mitochondrial fusion was observed in IVC blastocysts. Results indicate that in vitro culture affect on perturbations in mitochondrial number and function, which is associated with decreased developmental competence of in vitro produced mouse embryos.

Antioxidants in Mouse Embryo Culture and Vitrification Media Maintain an In Vivo Like Gene Expression Profile

Background: Embryo culture and cryopreservation causes detrimental effects on blastocyst gene expression. While combined antioxidants (acetyl-L-carnitine/N-acetyl-L-cysteine/[Formula: see text]-lipoic acid (A3)) in culture and vitrification media have beneficial effects on embryo and fetal development, their impact on fetal gene expression has not been assessed. Therefore, the effects of antioxidants on global gene expression were examined from fetal and placental tissue of transferred blastocysts and compared to in vivo derived embryos. Aim: To determine the effect of A3 in culture and vitrification media on mouse blastocyst, fetal and placental gene expression. Method: Embryo transfers were conducted on in vivo flushed blastocysts or blastocysts cultured or vitrified with and without A3. Transcriptional profiles of E14 fetal liver and placental tissue in all groups were quantified using RNAseq and functional analyses (KEGG). Results: Compared to in vivo derived embryos, in vitro culture altered the expression of 3601 fetal liver and 408 placental genes. Functional analysis showed upregulation/enrichment of oxidative phosphorylation and mitochondrial function and activity. Similarly, vitrification led to 2018 liver and 216 placental differentially expressed genes (DEGs). Upregulated KEGG pathways were enriched for cell and tissue development and cell cycle regulation. Down regulated pathways were associated with metabolism and immune response. Interestingly, exposure to A3 during development significantly reduced the number of DEGs with 1855 and 4 identified in liver and placentae respectively with no KEGG pathways identified. Correspondingly, exposure to A3 during vitrification reduced the number of DEGs to 1017 in liver and 206 in placentae. Functional pathway enrichment was similar to embryos vitrified without A3, although with decreased expression. Conclusion: Supplementation with antioxidants reduce the number of DEGs and biological processes altered by in vitro culture and vitrification, in fetal liver and placentae, establishing a more in vivo like gene expression profile which may help maintain the viability of vitrified human embryos.

P-412 3D live-imaging reconstruction of the human embryo implantation ex vivo

Abstract Study question How do human embryos implant in physiological conditions and develop beyond blastocyst stage? Summary answer Human embryos apply forces during invasion of the matrix, the mural trophectoderm undergoes a quick compaction leading to a fast radial expansion of polar side What is known already Human embryos can be cultured beyond blastocyst stage using supplements of animal origin, such as fetal bovine serum. However, two main issues limit our understanding of the implantation of the human embryo. First, human embryos do not express fluorescent proteins, severely limiting the access to advanced live-imaging tools. Second, the animal serums may not represent the physiological peri-implantation conditions of the human embryo, thus limiting the reproducibility of the in vitro experiments. Here we combine label-free multiphoton imaging with media formulations including clinical-grade protein supplements from human plasma unlocking the observation of the human embryo in 3D and physiological conditions Study design, size, duration We have cultured 150 human embryos from D3 until blastocyst stage and also more than 600 mouse embryos from zygote until blastocyst stage and then transferred them into a 3D implantation platform. All embryos were supplemented with clinical-grade human-derived serum, which is rich in globulins and growth factors, and commercial basal media. The implantation was monitored for 3-to-5 days up until day 11. Traction-force microscopy and molecular imaging was employed to quantify implantation markers. Participants/materials, setting, methods Implantation was measured using our novel version of traction-force microscopy, which reveals the force applied by entire embryos during live-imaging experiments. Multiphoton illumination of autofluorescent molecules allows to reconstruct images of live human embryos on 3D at unprecedented resolution. Key molecular markers for the formation of the epiblast rosette (OCT4), extra-embryonic visceral endoderm (GATA4), extra-embryonic ectoderm and trophoblast (CDX2) and primary yolk sac and pre-amniotic cavity were reconstructed in 3D using high resolution confocal imaging. Main results and the role of chance The implantation of human embryos was visualized in 3D movies at high resolution, during time-resolved experiments and compared to implantation in mouse embryos. Human embryos undergo a remarkable compaction upon attachment of the polar trophectoderm and thereafter they implant by engaging the matrix through many different points (&amp;gt;8). The post-implantation compaction leads to a strong invasion of the polar trophectoderm, digging large holes in the matrix and expanding radially. Some trophoblast cells escape the embryo colonizing the matrix. Human embryos expressed the correct pattern of molecular markers by day 11 of culture. Actin staining and OCT4 expression revealed the formation of a central rosette defining the pre-amniotic cavity. GATA6 expression defined the visceral endoderm and the formation of a primordial yolk sac. Mouse embryos conversely, compact upon implantation of the mural trophectoderm and are less invasive, typically anchoring to the matrix through 2-to-3 points. Post-implantation culture of mouse embryos starting at zygote was possible until the formation and large expansion of the amniotic cavity, which constituted 2/3 of the whole embryo size, corresponding with the initial steps of gastrulation, longer than any previous protocol Transfer of mouse blastocysts back to receptive mothers resulted in birth of viable litter. Limitations, reasons for caution This study has been performed with human embryos ex vivo using bioengineering technology to implant embryos outside the mother uterus. The bio-compatibility of our serum supplements was validated by live birth of transferred embryos in animal models, but would require further evaluation in clinical trials. Wider implications of the findings Our work stresses the safety and efficiency of supplementing embryo culture and particularly embryo transfer, with human plasma-derived serums of clinical-grade. The correct expression of main molecular markers indicates that this type of supplementation promotes implantation physiologically, which opens the possibility to employ these serums in the IVF laboratory. Trial registration number Not applicable

Surface functionalization of poly(dimethylsiloxane) substrates facilitates culture of pre-implantation mouse embryos by blocking non-selective adsorption.

Poly(dimethylsiloxane) (PDMS) is widely used in biomedical settings such as microfluidics for its optical transparency, castability, gas permeability and relative biocompatibility. While PDMS devices with certain modifications or treatments have been used for mammalian pre-implantation embryo culture, it is unclear why native PDMS leads to significant embryo death. In this study, we employ Nile Red as a model hydrophobic small molecule to demonstrate that significant hydrophobic sequestration occurs on native PDMS substrates even with a bovine serum albumin-containing KSOM pre-equilibration. Our results suggest that this small molecule sequestration has detrimental effects on mouse embryo development in PDMS static culture wells, with 0% blastocyst development rates from embryos cultured on native PDMS. We found that prior saturation of the PDMS culture well with water vapour only rescues about 10% of blastocyst development rates, indicating osmolality alone is not responsible for the high rates of embryo arrest. We also present a safe and simple Pluronic F127 pretreatment for PDMS substrates that successfully circumvented the harmful effects of native PDMS, achieving a blastocyst and implantation rate akin to that of our polystyrene controls. Our results call into question how researchers and clinicians can account for the alterations in medium composition and embryo secretions when using hydrophobic substrates, especially in the mammalian embryo culture setting where minimum effective concentrations of peptides and amino acids are commonplace.

Epigallocatechin gallate attenuates tumor necrosis factor (TNF)-α-induced inhibition of osteoblastic differentiation by up-regulating lncRNA TUG1 in osteoporosis

ABSTRACT Promoting osteoblast proliferation and differentiation contributes to the prevention and clinical treatment of osteoporosis. This study was to investigate the effect and mechanism of epigallocatechin gallate (EGCG) on tumor necrosis factor (TNF)-α-caused inhibition of osteoblastic differentiation. First, we cultured mouse embryo osteoblast precursor cells (MC3T3-E1) and induced by TNF-α (0, 2.5, 5, 10 ng/mL). The results revealed that TNF-α significantly inhibited the proliferation, ALP activity and mineralized nodule formation of MC3T3-E1 cells and promoted apoptosis. However, EGCG pretreatment significantly alleviated the inhibitory effect of TNF-α on MC3T3-E1. In addition, TNF-α significantly downregulated the expression of lncRNA TUG1 in MC3T3-E1, while EGCG upregulated the expression of lncRNA TUG1. After overexpression of lncRNA TUG1 in TNF-α-induced MC3T3-E1 cells, it could show similar effects as EGCG. However, interference with lncRNA TUG1 expression diminished the protective effect of EGCG on TNF-α-induced MC3T3-E1 cells. Finally, we found that EGCG inhibited TNF-α-induced activation of the Hippo/YAP signaling pathway, and that low expression of lncRNA TUG1 suppressed this effect. In conclusion, EGCG could suppress Hippo/YAP pathway activity by up-regulating lncRNA TUG1, ultimately improving TNF-α-caused inhibition of osteoblastic differentiation.

DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform.

Electro-Wetting-On-Dielectric (EWOD) based digital operations have demonstrated outstanding potential in actuating and manipulating liquid droplets. Here, we adapted the EWOD for extracting femtogram quantities of cell-free DNA (cf-DNA) from 1 μL of KSOM mouse embryo culture medium. Our group extracted the femtogram quantity of cf-DNA from 1 μL of mouse embryo culture medium in our previous work. Here, we initially explain a modification from our previous extraction protocol, which improves the extraction percentage to 36.74%. Though the modified extraction protocol improves the extraction percentage from our previously reported work, the quantity is still in the femtogram range. The cf-DNA in femtogram quantity is in subcritical/subthreshold concentration for any further analysis, such as sequencing. To the best of our knowledge, we need a minimum of picogram/nanogram DNA quantities for further analysis. We demonstrated a ground-breaking mechanism of this subcritical concentration of cf-DNA amplification to the nanogram range and performed DNA sequencing. Basic Local Alignment Search Tool (BLAST) is used as a sequence similarity search program to confirm the identity percentage between query and subject. More than 97% of nucleotide identities between query and subject sequences have been obtained from the sequencing result. Hence, we can use the methodology to amplify the subcritical concentration of extracted DNA for further analytics. Moreover, as we extract the cf-DNA from the embryo culture medium, the natural growth of the embryo has not been disrupted. This entire mechanism will pave a new path towards the lab-on-a-chip (LOC) concept.

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Vangl2-environment interaction causes severe neural tube defects, without abnormal neuroepithelial convergent extension.

ABSTRACTPlanar cell polarity (PCP) signalling is vital for initiation of mouse neurulation, with diminished convergent extension (CE) cell movements leading to craniorachischisis, a severe neural tube defect (NTD). Some humans with NTDs also have PCP gene mutations but these are heterozygous, not homozygous as in mice. Other genetic or environmental factors may interact with partial loss of PCP function in human NTDs. We found that reduced sulfation of glycosaminoglycans interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause craniorachischisis in cultured mouse embryos, with rescue by exogenous sulphate. We hypothesized that this glycosaminoglycan–PCP interaction may regulate CE, but, surprisingly, DiO labelling of the embryonic node demonstrates no abnormality of midline axial extension in sulfation-depleted Lp/+ embryos. Positive-control Lp/Lp embryos show severe CE defects. Abnormalities were detected in the size and shape of somites that flank the closing neural tube in sulfation-depleted Lp/+ embryos. We conclude that failure of closure initiation can arise by a mechanism other than faulty neuroepithelial CE, with possible involvement of matrix-mediated somite expansion, adjacent to the closing neural tube.

Human embryonic development: from peri-implantation to gastrulation.

The basic body plan of the mammalian embryo is established through gastrulation, a pivotal early postimplantation event during which the three major germ layers (endoderm, ectoderm, and mesoderm) are specified with cellular and spatial diversity. Despite its basic and clinical importance, human embryo development from peri-implantation to gastrulation remains shrouded in mystery. Recent advances in the elongated in vitro culture of rodent and non-primate embryos and the construction of embryo-like structures have helped to improve understanding of the mechanisms of human early embryonic development. Here, we review the recent advances and possible future directions in the development of in vitro models to better understand human embryogenesis from peri-implantation to gastrulation.

Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator.

Conventional in vitro culture and manipulation of mouse embryos require a CO2 incubator, which not only increases the cost of performing experiments but also hampers the transport of embryos to the other laboratories. In this study, we established and tested a new CO2 incubator-free embryo culture system and transported embryos using this system. Using an Anaero pouch, which is a CO2 gas-generating agent, to increase the CO2 partial pressure of CZB medium to 4%-5%, 2-cell embryos were cultured to the blastocyst stage in a sealed tube without a CO2 incubator at 37°C. Further, the developmental rate to blastocyst and full-term development after embryo transfer were comparable with those of usual culture method using a CO2 incubator (blastocyst rate: 97% versus 95%, respectively; offspring rate: 30% versus 35%, respectively). Furthermore, using a thermal bottle, embryos were reliably cultured using this system for up to 2 days at room temperature, and live offspring were obtained from embryos transported in this simple and very low-cost manner without reducing the offspring rate (thermal bottle: 26.2% versus CO2 incubator: 34.3%). This study demonstrates that CO2 incubators are not essential for embryo culture and transportation and that this system provides a useful, low-cost alternative for mouse embryo culture and manipulation.