The role of metabolism in cardiac development.

Metabolism is the fundamental process that sustains life. The heart, in particular, is an organ of high energy demand, and its energy substrates have been studied for more than a century. In recent years, there has been a growing interest in understanding the role of metabolism in the early differentiation of pluripotent stem cells and in cancer research. Studies have revealed that metabolic intermediates from glycolysis and the tricarboxylic acid cycle act as co-factors for intracellular signal transduction, playing crucial roles in regulating cell behaviors. Mitochondria, as the central hub of metabolism, are also under intensive investigation regarding the regulation of their dynamics. The metabolic environment of the fetus is intricately linked to the maternal metabolic status, and the impact of the mother's nutrition and metabolic health on fetal development is significant. For instance, it is well known that maternal diabetes increases the risk of cardiac and nervous system malformations in the fetus. Another notable example is the decrease in the risk of neural tube defects when pregnant women are supplemented with folic acid. These examples highlight the profound influence of the maternal metabolic environment on the fetal organ development program. Therefore, gaining insights into the metabolic environment within developing fetal organs is critical for deepening our understanding of normal organ development. This review aims to summarize recent findings that build upon the historical recognition of the environmental and metabolic factors involved in the developing embryo.

Effect of arachidonic acid on pre- and post-hatching in vitro bovine embryo development.

CONTEXT: Arachidonic acid (AA) is the precursor of prostaglandins, which may play autocrine roles during early embryo development. AIMS: To test the developmental effects of addition of AA to pre- and post-hatching culture media on in vitro -produced bovine embryos. METHODS: Pre-hatching effects of AA were tested by culturing bovine zygotes in synthetic oviductal fluid (SOF) supplemented with 100 or 333µM AA. Post-hatching effects of AA were tested by culturing Day 7 blastocysts in N2B27 supplemented with 5, 10, 20 or 100µM AA up to Day 12. KEY RESULTS: Pre-hatching development to blastocyst was completely abrogated at 333µM AA, whereas blastocyst rates and cell numbers were not altered at 100µM AA. Impaired post-hatching development was observed at 100µM AA, whereas no effect on survival rates was noted at 5, 10 and 20µM AA. However, a significant reduction in Day 12 embryo size was observed at 10 and 20µM AA. Hypoblast migration, epiblast survival and formation of embryonic-disc-like structures were unaffected at 5-10µM AA. AA exposure downregulated the genes PTGIS , PPARG , LDHA and SCD in Day 12 embryos. CONCLUSIONS: Pre-hatching embryos are mostly irresponsive to AA, whereas AA was observed to have negative effects during early post-hatching development. IMPLICATIONS: AA does not improve in vitro bovine embryo development and is not required up to early post-hatching stages.

α-Ketoglutarate Improves Meiotic Maturation of Porcine Oocytes and Promotes the Development of PA Embryos, Potentially by Reducing Oxidative Stress through the Nrf2 Pathway.

α-Ketoglutarate (α-KG) is a metabolite in the tricarboxylic acid cycle. It has a strong antioxidant function and can effectively prevent oxidative damage. Previous studies have shown that α-KG exists in porcine follicles, and its content gradually increases as the follicles grow and mature. However, the potential mechanism of supplementation of α-KG on porcine oocytes during in vitro maturation (IVM) has not yet been reported. The purpose of this study was to explore the effect of α-KG on the early embryonic development of pigs and the mechanisms underlying these effects. We found that α-KG can enhance the development of early pig embryos. Adding 20 µM α-KG to the in vitro culture medium significantly increased the rate of blastocyst formation and the total cell number. Compared with to that of the control group, apoptosis in blastocysts of the supplement group was significantly reduced. α-KG reduced the production of reactive oxygen species and glutathione levels in cells. α-KG not only improved the activity of mitochondria but also inhibited the occurrence of apoptosis. After supplementation with α-KG, pig embryo pluripotency-related genes (OCT4, NANOG, and SOX2) and antiapoptotic genes (Bcl2) were upregulated. In terms of mechanism, α-KG activates the Nrf2/ARE signaling pathway to regulate the expression of antioxidant-related targets, thus combating oxidative stress during the in vitro culture of oocytes. Activated Nrf2 promotes the transcription of Bcl2 genes and inhibits cell apoptosis. These results indicate that α-KG supplements have a beneficial effect on IVM by regulating oxidative stress during the IVM of porcine oocytes and can be used as a potential antioxidant for IVM of porcine oocytes.

New Wenshen Shengjing Decoction Improves Early Embryonic Development by Maintaining Low Levels of H3K4me3 in Sperm.

BACKGROUND: New Wenshen Shengjing Decoction (NWSSJD), a traditional Chinese compound medicine, has significant effect on spermatogenesis disorder and can significantly improve sperm quality. Many components in NWSSJD can induce epigenetic modifications of different types of cells. It is not yet known whether they can cause epigenetic modifications in sperm or early embryos. OBJECTIVE: This study investigated the effect of NWSSJD on mouse early embryonic development and its regulation of H3K4me3 in mouse sperm and early embryos. METHODS: Spermatogenesis disorder was induced in male mice with CPA (cyclophosphamide). NWSSJD was administrated for 30 days. Then, the male mice were mated with the female mice with superovulation, and the embryo degeneration rate of each stage was calculated. Immunofluorescence staining was used to detect the expression of H3K4me3 in sperm and embryos at various stages. Western blotting was performed to detect methyltransferase SETD1B expression. The expressions of development-related genes (OCT-4, NANOG, and CDX2) and apoptosis-related genes (BCL-2 and p53) were measured with qRT-PCR. RESULTS: Compared with the CPA group, NWSSJD significantly reduced the H3K4me3 level in sperms, significantly increased the number of normal early embryos (2-cell embryos, 3-4-cell embryos, 8-16-cell embryos, and blastocysts) per mouse, and reduced the degeneration rate of the embryos. The expression levels of H3K4me3 and methyltransferase SETD1B in early embryos were significantly elevated by NWSSJD. Additionally, NWSSJD significantly promoted BCL-2 expression, while reducing p53 expression, thus inhibiting embryonic cell apoptosis. Moreover, the expressions of development-related genes OCT-4 and CDX2 were significantly increased by NWSSJD, but NANOG expression had no significant difference. CONCLUSION: NWSSJD may promote early embryonic development possibly by maintaining low H3K4me3 levels in sperms and normal H3K4me3 modification in early embryos and by inhibiting embryonic cell apoptosis.

Progestogen supplementation during superovulation leads to higher embryo viability and TGFB1 gene expression in sheep.

This study aimed to compare the effect of the administration of either medroxyprogesterone acetate (MPA) or progesterone (P4) in superovulation (SOV) treatments applied during the first follicular wave on follicular development, embryo yield, and the expression of genes related to pluripotency maintenance, differentiation of the trophectoderm, cell growth and differentiation, apoptosis and energy metabolism in sheep embryos. The estrous cycle of 36 multiparous ewes was synchronized with a short protocol, and the animals were randomly allocated to three groups. At the beginning of SOV, 12 ewes per treatment received an intravaginal sponge impregnated with 60 mg of MPA (TMPA), or an intravaginal device containing 0.33 g of P4 (TP4), or received no progestogen treatment (CON). The device was kept until the fifth dose of FSH. Ewes were mated with five fertile rams. Gene expression was performed by RT-qPCR using grade I and II blastocysts. The numbers of corpora lutea, total structures and viable embryos recovered per ewe were similar (P > 0.05) among groups. However, the viability rate was higher in TP4 (71.9 ± 16.3%) compared to CON (24.4 ± 16.8%; P = 0.01) and similar to TMPA (49.9 ± 16.3%; P = 0.2). Similarly, when compared with CON, treatment with P4 or MPA positively regulated the TGFB1 transcript involved in cell proliferation and differentiation (P = 0.01 and P = 0.03, respectively). In conclusion, supplementation with P4 during the first follicular wave of the estrous cycle improves embryo viability and alters the expression of the TGFB1 gene.

Transcriptome sequencing reveals Gastrodia elata Blume could increase the cell viability of eNPCs under hypoxic condition by improving DNA damage repair ability.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodia elata Blume (GEB), known as Tianma in China, is a traditional medicinal herb that has been reported to have various pharmacological effects and neuroprotection, has long been used for treating dizziness, epilepsy, stroke. However, explanation of its underlying mechanisms remains a great challenge. AIM OF THE STUDY: The neuroprotective mechanism of GEB on hypoxia-induced neuronal injury in cultured mouse embryonic neural progenitor cells (eNPCs) was investigated, with emphasis on the eNPCs proliferation and DNA damage repair. MATERIALS AND METHODS: In this study, hypoxia was focused, which may be caused by stroke or acute cerebral ischemia and is considered as one of the important factors contributing to the Central Nervous System diseases. CoCl2 was adopted to construct a hypoxic/ischemic condition in eNPCs. eNPCs proliferation analysis validated GEB neuroprotective effect under hypoxic/ischemic condition. Transcriptome and weighted gene co-expression network analysis (WGCNA) screened the special gene-network module correlated with what appeared to have significant positive correlation with GEB. Then, Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed to explore the biological functions of selected genes in the modules that had high correlation with GEB. RESULTS: GEB has neuroprotective effect and could rescue eNPCs proliferation under hypoxic/ischemic condition induced by CoCl2. Transcriptome and WGCNA unveil the neuroprotective mechanism of GEB on improving DNA damage repair ability by increasing the expression of genes associated with DNA repair and replication. Western blotting and qPCR showed that GEB could improve DNA damage repair ability by increasing the expression of Mcm2, Mcm6, Pold2, Pole, Pole2, Rfc1, Pole4, Dna2 and Rpa2, which were associated with DNA damage and replication. CONCLUSION: Through transcriptome and WGCNA, this study unveiled Gastrodia elata Blume could increase the cell viability of eNPCs under hypoxic condition by improving DNA damage repair ability.

Sodium arsenate induced genotoxicity, morphometric and morphological changes in mice embryo and protective role of Moringa oleifera extracts.

The present study was carried out to find the comparative ameliorative role of Moringa oleifera leaf and flower extracts against sodium arsenate induced genotoxic, morphometric and morphological changes in mice embryo. Seven to eight week old pregnant females (N=44) with body weight of 20-25g at gestation day zero were divided randomly in groups (A, B, C, D, E, F, G, H, I, J and K). Group A was of control while all others were experimental groups and administered with selected doses of sodium arsenate as toxicant (6mg/kg B.W and 12mg/kg/B.W) and Moringa oleifera leaf and flower extracts as antidote (150mg/kg and 300mg/kg B.W). Significant (p<0.05) amelioration at dose 300mg/kg of Moringa oleifera leaf extract was observed against sodium arsenate induced morphological abnormalities like micromelia, excencephally, cryptothalmia, anopthalmia, laproschisis and morphometric changes like fetus weight, head circumference, crown rump and snout length were observed. Significant protection of DNA was showed in Moringa oleifera leaf extract treated groups (27.50±2.51) as compared to sodium arsenate (66.25±2.75). So concluded that sodium arsenate induced teratogenicity can be decreased using Moringa extract especially of Moringa oleifera leaf extract as it contains bioactive compounds like phenolics.

N-3 polyunsaturated fatty acids effectively protect against neural tube defects in diabetic mice induced by streptozotocin.

Folate cannot prevent all neural tube defects (NTD), indicating that other pathogeneses still exist except for the folate deficiency. Maternal diabetes mellitus during pregnancy can increase the risk of offspring NTD. Our previous study showed that polyunsaturated fatty acids (PUFA) were lower in the placenta of human NTD cases than in healthy controls, and the supplementation of fish oil (rich in long-chain (LC) n-3 PUFA, mainly C20:5n-3 and C22:6n-3) had a better prevention effect against sodium valproate induced NTD than corn oil (rich in C18:2n-6) and flaxseed oil (rich in C18:3n-3). The aim of the present study was to investigate whether PUFA could prevent diabetes-induced NTD in mice. Streptozotocin (STZ)-induced diabetic pregnant mice were fed with a normal diet (DMC), a diet containing a low dose of fish oil (DMLn-3), a diet containing a high dose of fish oil (DMHn-3) or a diet rich in corn oil (DMn-6). Healthy pregnant mice were fed with a normal diet (HC). Compared with the DMC group, the rate of NTD was significantly lower in the DMHn-3 group (4.44% vs. 12.50%), but not in the DMLn-3 (11.11%) or DMn-6 group (12.03%). The NTD rate in the DMHn-3 group was comparable with that in the HC group (1.33%) (p = 0.246), and lower than that in the DMn-6 group (p = 0.052). The NTD rate in DMLn-3 and DMn-6 groups was significantly higher than that in the HC group. No significant difference was observed in NTD rate between DMLn-3 and DMHn-3 groups, and between DMLn-3 and DMn-6 groups. Compared with the HC group, the DMC group had a significantly lower C22:6n-3 in both serum and embryos. Fish oil supplementation ameliorated neuroepithelial cell apoptosis, and the apoptotic rate was comparable between DMHn-3 and HC groups. Although the apoptotic rate was significantly lower in the DMn-6 group than the DMC group, it was still much higher than that in the HC group. The proteins P53 and Bax in embryos were higher, while the proteins Bcl-2 and Pax3 were lower in the DMC group than in the HC group. The disturbance of Pax3, P53 and Bax induced by diabetes was abolished in DMLn-3, DMHn-3 and DMn-6 groups. Importantly, Bcl-2 in embryos was restored to the normal level only in the DMHn-3 group but not in the DMLn-3 or DMn-6 group. In conclusion, LC n-3 PUFA enriched fish oil has a protective effect against NTD in diabetes induced by STZ through improving neuroepithelial cell apoptosis, and the mechanism may be by increasing the anti-apoptosis protein Bcl-2 independently of Pax3 and P53.

Iron overload compromises preimplantation mouse embryo development.

We and others have previously shown that abnormal pelvic environment plays an important role in the unexplained infertility of endometriosis. However, whether iron overload caused by ectopic periodic bleeding found in patients with endometriosis participates in endometriosis-associated reproductive failure is unknown. This study aimed to investigate effects of iron at level relevant to pelvic iron overload on the development of preimplantation mouse embryo. Two-cell embryos were collected, and cultured to blastocysts in G1/G2 medium supplemented with iron alone or in combination with iron chelator. The development rates, ATP level, mitochondrial membrane potential (MMP), reactive oxygen species level (ROS), and apoptotic and ferroptotic indices were compared between control and iron treatments across each specific developmental stage. Prolonged exposure to iron remarkably impaired early embryo development in vitro by hampering blastocyst formation (P < 0.001), which could be partly restored by iron chelator (P < 0.001). The arrest of embryo development was linked with iron-initiated mitochondrial dysfunction with reduction of ATP generation and MMP (P < 0.05 and P < 0.001, respectively). Impaired mitochondria altered ROS accumulation post-iron exposure at morula stage and blastocyst stage (P < 0.05). Moreover, Iron-exposed blastocyst stage embryos showed higher apoptotic and ferroptotic rates (P < 0.001 and P < 0.05, respectively). Our results highlight that pathologically relevant level of iron compromises preimplantation mouse embryo development by disrupting mitochondrial function and triggering both apoptosis and ferroptosis, which implicates that excess iron found in peritoneal fluid of women with endometriosis likely participates in endometriosis-associated reproductive failure.

Maternal iron deficiency perturbs embryonic cardiovascular development in mice.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.

Iron-dependent apoptosis causes embryotoxicity in inflamed and obese pregnancy.

Iron is essential for a healthy pregnancy, and iron supplementation is nearly universally recommended, regardless of maternal iron status. A signal of potential harm is the U-shaped association between maternal ferritin, a marker of iron stores, and risk of adverse pregnancy outcomes. However, ferritin is also induced by inflammation and may overestimate iron stores during inflammation or infection. In this study, we use mouse models to determine whether maternal iron loading, inflammation, or their interaction cause poor pregnancy outcomes. Only maternal exposure to both iron excess and inflammation, but not either condition alone, causes embryo malformations and demise. Maternal iron excess potentiates embryo injury during both LPS-induced acute inflammation and obesity-induced chronic mild inflammation. The adverse interaction depends on TNFα signaling, causes apoptosis of placental and embryo endothelium, and is prevented by anti-TNFα or antioxidant treatment. Our findings raise important questions about the safety of indiscriminate iron supplementation during pregnancy.

Solution NMR in human embryo culture media as an option for assessment of embryo implantation potential.

NMR offers the potential to holistically screen hundreds of metabolites and has already proved to be a powerful technique able to provide a global picture of metabolic changes in a wide range of biological systems underlying complex and multifactorial matrixes. This review covers the literature until May 2020 centered on the early prediction of the viability of in vitro developed embryos using several analytical techniques, including NMR. Nowadays, the predominant non-invasive technique for selecting viable embryos is based on morphology, where variables associated with the rate of cleavage and blastocyst formation are evaluated by the embryologist following standardized criteria that are somewhat subjective. This morphological approach is therefore inadequate for the prediction of embryo quality, and several studies have focused on developing new non-invasive methods using molecular approaches based particularly on metabolomics. This review outlines the potential of NMR as one of these non-invasive in vitro methods based on the analysis of spent embryo culture media.

Effects of fatty acid supplementation during vitrification and warming on the developmental competence of mouse, bovine and human oocytes and embryos.

RESEARCH QUESTION: Does fatty acid supplementation in vitrification and warming media influence developmental competence in oocytes after vitrification and warming? DESIGN: Mouse oocytes and four-cell embryos were vitrified and warmed with solutions supplemented with fatty acid and cultured to the blastocyst stage. To study lipid metabolism after vitrification, quantitative real-time polymerase chain reaction was used to analyse the expression of genes related to beta oxidation in mouse embryos vitrified and warmed with or without fatty acids. The effects of fatty acid supplementation in the warming solutions on the developmental competence of bovine and human embryos were analysed. Blastocyst outgrowth assay was used to evaluate the potential of human blastocysts for adhesion to fibronectin. RESULTS: The neutral lipid content of mouse oocytes in the fatty acid 1% supplementation group was significantly higher than in the fatty acid 0% group (P = 0.0032). The developmental rate to the blastocyst stage was significantly higher in the fatty acid 1% group than in the fatty acid 0% group in mice (P = 0.0345). Fatty acid supplementation in warming solution upregulated Acaa2 and Hadha in mouse embryos. Fatty acids significantly improved the developmental ability of bovine embryos to the blastocyst stage (P = 0.0048). Warming with 1% fatty acid supplementation significantly increased the proportion of human blastocysts with morphological grade A inner cell mass (P = 0.0074) and trophectoderm (P = 0.0323). CONCLUSIONS: Fatty acid supplementation in the warming solutions improved the developmental competence of vitrified-warmed mouse oocytes by activating the beta-oxidation pathway. Fatty acid supplementation enhanced the developmental rate of bovine embryos to the blastocyst stage and improved morphological characteristics of human embryos vitrified at the cleavage stage.

A subpopulation of embryonic microglia respond to maternal stress and influence nearby neural progenitors.

The interplay between hypothalamic neurons and microglia as they integrate stressors to regulate homeostasis is of growing interest. We asked if microglia in the embryonic hypothalamus were likewise stress responsive and, if so, whether their precocious activation perturbs nearby neural stem cell (NSC) programs. We performed single-cell transcriptomics to define embryonic hypothalamic microglia heterogeneity and identified four microglial subsets, including a subpopulation adjacent to NSCs that was responsive to gestational cold stress. Stress exposure elevated CCL3 and CCL4 secretion, but only in male brains, and ex vivo CCL4 treatment of hypothalamic NSCs altered proliferation and differentiation. Concomitantly, gestational stress decreased PVN oxytocin neurons only in male embryos, which was reversed by microglia depletion. Adult offspring exposed to gestational stress displayed altered social behaviors, which was likewise microglia dependent, but only in males. Collectively, immature hypothalamic microglia play an unappreciated role in translating maternal stressors to sexually dimorphic perturbation of neurodevelopmental programs.

Spatiotemporal evaluation of the mouse embryonic redox environment and histiotrophic nutrition following treatment with valproic acid and 1,2-dithiole-3-thione during early organogenesis.

Redox regulation during metazoan development ensures that coordinated metabolic reprogramming and developmental signaling are orchestrated with high fidelity in the hypoxic embryonic environment. Valproic acid (VPA), an anti-seizure medication, is known to increase markers of oxidation and also increase the risk of neural tube defects (NTDs) when taken during pregnancy. It is unknown, however, whether oxidation plays a direct role in failed neural tube closure (NTC). Spatial and temporal fluctuations in total glutathione (GSH) and total cysteine (Cys) redox steady states were seen during a 24 h period of CD-1 mouse organogenesis in untreated conceptuses and following exposure to VPA and the Nrf2 antioxidant pathway inducer, 1,2-dithiole-3-thione (D3T). Glutathione, glutathione disulfide (GSSG), and Cys, cystine (CySS) concentrations, measured in conceptal tissues (embryo/visceral yolk sac) and fluids (yolk sac fluid/amniotic fluid) showed that VPA did not cause extensive and prolonged oxidation during the period of NTC, but instead produced transient periods of oxidation, as assessed by GSH:GSSG redox potentials, which revealed oxidation in all four conceptal compartments at 4, 10, and 14 h, corresponding to the period of heartbeat activation and NTC. Other changes were tissue and time specific. VPA treatment also reduced total FITC-Ab clearance from the medium over 3 h, indicating potential disruption of nutritive amino acid supply. Overall, these results indicated that VPA's ability to affect cellular redox status may be limited to tissue-specific windows of sensitivity during the period of NTC. The safety evaluation of drugs used during pregnancy should consider time and tissue specific redox factors.

The effect of lutein and Urtica dioica extract on in vitro production of embryo and oxidative status in polycystic ovary syndrome in a model of mice.

BACKGROUND: Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrinopathies in women during the reproductive age. Herbal medicines are used increasingly alone or in supplement with chemical medicines for the treatment of different diseases and dysfunctions. This study was aimed to evaluate the effects of lutein and nettle (Urtica dioica) extract on the biochemical parameters and the reproductive function in the PCOS model of mice. METHODS: Following the induction of PCOS by dehydroepiandrosterone (DHEA), the mice (n = 98) were randomly assigned into seven groups, each consisting of fourteen mice; the groups were included control group (received solvent), PCOS group (received 6 mg/100 g B.W/day IP, DHEA for 21 days), PCOS+ Nettle extract (200 and 400 mg/kg), PCOS+ Lutein (125 and 250 mg/kg), and PCOS+ NL (200 mg/kg nettle extract and 125 mg/kg lutein). The nettle extract and lutein were administrated using gavage for 30 consecutive days after PCOS induction. Malondialdehyde (MDA), total antioxidant capacity (TAC), and estrogen were measured in serum, ovary, and uterus samples by the ELISA method. The total number of oocytes, oocyte quality, fertilization rate, 2-cell blastocyst, and arrested embryos (type I, type II, and type III) were also investigated. RESULTS: A combination treatment of the nettle and lutein produced the lowest concentration of MDA in comparison to other groups which affected by the PCOS. The lowest level of TAC was observed in the PCOS group without treatment. The number of oocytes, oocyte quality, fertilization rate, and 2-cell blastocyst were significantly higher in the control group, but the lowest values were observed in the PCOS group without any treatment. CONCLUSIONS: The most favorable findings include improving antioxidant capacity, oocyte and embryo quality were observed in the PCOS+ 125 L group.

Toxicity of Methanolic Extracts of Seeds of Moringa stenopetala, Moringaceae in Rat Embryos and Fetuses.

Moringa stenopetala is a medicinal plant that has been used in Ethiopian traditional medicine as a remedy for the treatment of hypertension, diabetes, and stomach pain. The study is aimed at assessing the toxicity of the methanol extracts of the seeds of Moringa stenopetala on the developing embryo and fetuses of rats. The seeds of Moringa were extracted by maceration using 80% methanol. The extract (250-1000 mg/kg) was orally administered to pregnant Swiss albino rats from days 6 to12 of gestation. Embryos and fetuses were recovered by laparotomy on gestational day 12 and day 20, respectively, and were assessed for developmental anomalies. On day 20, significant prenatal growth retardation such as reduced litter weight and crown-rump length were observed in near term fetuses of 1000 mg/kg treated rats. Litter weight in 1000 mg/kg and pair-fed control groups was 2.41 g ± 0.108 and 3.08 g ± 0.093, respectively. Delay in the development of an otic, optic, and olfactory system, as well as a reduction in a number of branchial bars, occurred on day 12 embryos of 1000 mg/kg treated rats. The rate of fetal resorption in 1000 mg/kg and pair-fed control groups was 1.6 ± 0.55 and 0.42 ± 0.52, respectively. There was also a high incidence of fetal death in the 1000 mg/kg treated group but it was not statistically significant. The offspring's of Moringa-treated rats did not show gross external malformations at all doses. These findings suggest that the methanol seed extract of Moringa stenopetala is not safe to rat embryos and fetuses. Its toxic effects were evidenced by a significant delay in embryonic and fetal development and an increase in fetal resorptions and fetal death.

Inhibition of MTOR signaling impairs rat embryo organogenesis by affecting folate availability.

Mechanistic target of rapamycin (MTOR) is essential for embryo development by acting as a nutrient sensor to regulate cell growth, proliferation and metabolism. Folate is required for normal embryonic development and it was recently reported that MTOR functions as a folate sensor. In this work, we tested the hypothesis that MTOR functions as a folate sensor in the embryo and its inhibition result in embryonic developmental delay affecting neural tube closure and that these effects can be rescued by folate supplementation. Administration of rapamycin (0.5 mg/kg) to rats during early organogenesis inhibited embryonic ribosomal protein S6, a downstream target of MTOR Complex1, markedly reduced embryonic folate incorporation (-84%, P < 0.01) and induced embryo developmental impairments, as shown by an increased resorption rate, reduced embryo somite number and delayed neural tube closure. These alterations were prevented by folic acid administered to the dams. Differently, although an increased rate of embryonic rotation defects was observed in the rapamycin-treated dams, this alteration was not prevented by maternal folic acid supplementation. In conclusion, MTOR inhibition during organogenesis in the rat resulted in decreased folate levels in the embryo, increased embryo resorption rate and impaired embryo development. These data suggest that MTOR signaling influences embryo folate availability, possibly by regulating the transfer of folate across the maternal-embryonic interface.

Factors of the human embryo culture system that may affect media evaporation and osmolality.

STUDY QUESTION: Which lab-related factors impact the culture system's capacity to maintain a stable osmolality during human embryo culture? SUMMARY ANSWER: Incubator humidity, the volume of mineral oil, the type of culture media and the design of time-lapse dishes have been identified as important parameters that can cause an impact on media evaporation and consequently osmolality during culture. WHAT IS KNOWN ALREADY: Culture medium is a critical component in human embryo culture. Minimizing its evaporation during culture is an adequate strategy to stabilize osmolality and, as a result, improving culture conditions and clinical outcomes. STUDY DESIGN, SIZE, DURATION: The studied variables included media composition and supplementation; volume of mineral oil; incubator humidification; and the type of dish and incubator used. Additionally, six time-lapse dish models were compared in their ability to prevent evaporation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Dishes were incubated in parallel to analyze osmolality during culture between groups: synthetic oviductal medium enriched with potassium versus human tubal fluid medium; protein versus no protein supplementation; dry versus humid atmosphere; high versus low volume of mineral oil. Additionally, media evaporation was compared between six models of time-lapse dishes with distinct designs, cultured in a joint incubator. Two of them were retested in their corresponding incubator to analyze the dish-incubator fit. Daily osmolality measurements were compared between groups. Linear regression was performed to analyze evaporation rates. MAIN RESULTS AND THE ROLE OF CHANCE: Protein supplementation did not significantly affect evaporation. Contrarily, humidity levels inside the incubators, the volume of mineral oil and the type of culture media, played an important role in osmolality stabilization. The design of time-lapse dishes and their recommended preparation protocol heavily influenced their evaporation rates, which were further altered by each incubator's characteristics. Media with initially high osmolalities had a bigger risk of reaching hypertonic levels during culture. LIMITATIONS, REASONS FOR CAUTION: While numerous, the studied variables are limited and therefore other factors could play a role in osmolality dynamics, as well. Incontrollable atmospheric factors could also result in some variation in the observed results between different centers and laboratories. WIDER IMPLICATIONS OF THE FINDINGS: Published literature has extensively described how hypertonic media may impair embryo development and negatively affect clinical outcomes; therefore, maintaining a stable osmolality during culture should be considered essential. This work is of interest both for embryologists when analyzing their culture system and methodologies, as well as manufacturers in charge of designing IVF consumables. STUDY FUNDING/COMPETING INTEREST(S): This study was privately funded. TRIAL REGISTRATION NUMBER: N/A.

Effect of retinoic acid signaling on Ripply3 expression and pharyngeal arch morphogenesis in mouse embryos.

BACKGROUND: Pharyngeal arches (PA) are sequentially generated in an anterior-to-posterior order. Ripply3 is essential for posterior PA development in mouse embryos and its expression is sequentially activated in ectoderm and endoderm prior to formation of each PA. Since the PA phenotype of Ripply3 knockout (KO) mice is similar to that of retinoic acid (RA) signal-deficient embryos, we investigated the relationship between RA signaling and Ripply3 in mouse embryos. RESULTS: In BMS493 (pan-RAR antagonist) treated embryos, which are defective in third and fourth PA development, Ripply3 expression is decreased in the region posterior to PA2 at E9.0. This expression remains and its distribution is expanded posteriorly at E9.5. Conversely, high dose RA exposure does not apparently change its expression at E9.0 and 9.5. Knockout of retinaldehyde dehydrogenase 2 (Raldh2), which causes more severe PA defect, attenuates sequential Ripply3 expression at PA1 and reduces its expression level. EGFP reporter expression driven by a 6 kb Ripply3 promoter fragment recapitulates the endogenous Ripply3 mRNA expression during PA development in wild-type, but its distribution is expanded posteriorly in BMS493-treated and Raldh2 KO embryos. CONCLUSION: Spatio-temporal regulation of Ripply3 expression by RA signaling is indispensable for the posterior PA development in mouse.