Murine blastocysts release mature microRNAs into culture media that reflect developmental status
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PROVIDER: PRJNA575279 | ENA |
REPOSITORIES: ENA
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Project description:Extracellular microRNA sequences have attracted interest for their potential use as accessible biomarkers that may be non-invasively collected from a wide range of biological fluids, including spent culture media. The ongoing milieu between the late-stage preimplantation embryo and the receptive uterus remains an actively investigated area of research and the physiological role of secreted microRNA sequences remains to be determined. Spent culture media used to culture late stage E4.0 murine blastocysts was screened for 641 mature microRNA sequences using a qPCR-based array. We report here the 39 sequences that were exclusively detected in the conditioned media using this approach. The embryonic miR-290 family were among the most abundant extracellular sequences. Notably, implantation-relevant members miR-126a, miR-101a, miR-143 and miR-320 were also detected.
2021-01-16 | GSE138285 | GEO
Project description:Extracellular microRNA (miRNA) sequences derived from the pre-implantation embryo have attracted interest for their possible contributions to the ongoing embryonic-uterine milieu, as well as their potential for use as accessible biomarkers indicative of embryonic health. Spent culture media microdroplets used to culture late-stage E4.0 murine blastocysts were screened for 641 mature miRNA sequences using a reverse transcription-quantitative polymerase chain reaction-based array. We report here 39 miRNAs exclusively detected in the conditioned media, including the implantation-relevant miR-126a-3p, miR-101a, miR-143, and miR-320, in addition to members of the highly expressed embryonic miR-125 and miR-290 families. Based on these results, an miRNA panel was assembled comprising five members of the miR-290 family (miR-291-295) and five conserved sequences with significance to the embryonic secretome (miR-20a, miR-30c, miR-142-3p, miR-191, and miR-320). Panel profiling of developing embryo cohort lysates and accompanying conditioned media microdroplets revealed extensive similarities in relative quantities of miRNAs and, as a biomarker proof of concept, enabled distinction between media conditioned with differently staged embryos (zygote, 4-cell, and blastocyst). When used to assess media conditioned with embryos of varying degrees of degeneration, the panel revealed increases in all extracellular panel sequences, suggesting cell death is an influential and identifiable factor detectable by this assessment. In situ hybridization of three panel sequences (miR-30c, miR-294, and miR-295) in late-stage blastocysts revealed primarily inner cell mass expression with a significant presence of miR-294 throughout the blastocyst cavity. Furthermore, extracellular miR-290 sequences responded significantly to high centrifugal force, suggesting a substantial fraction of these sequences may exist within a vesicle such as an exosome, microvesicle, or apoptotic bleb. Together, these results support the use of extracellular miRNA to assess embryonic health and enable development of a non-invasive viability diagnostic tool for clinical use.
| S-EPMC8193861 | biostudies-literature
Project description:In the field of in vitro embryo production it is generally thought that the culture media are not as good as the oviductal fluid, resulting in detrimental changes of embryonic gene expression. Yet in vitro embryo culture (IVC) is one of the pillars of the assisted reproductive technologies. Therefore, unintended effects on gene expression may impact on the health of ART babies (PMID 27554442). Granted, human ART has many more components than just the culture media, but, the practice of embryo culture superimposes with the phase of life when cellular totipotency is present - that's why embryo culture media receive special attention. Discerning what culture media do is difficult because several confounders are present, including but not limited to oocyte heterogeneity, sperm heterogeneity as well as time of fertilization. We propose to take advantage of experimentally produced parthenogenetic embryos, and thereby remove two confounders (sperm variability, time of fertilization), in order to assess the effects of specific culture media of embryonic gene expression, in the mouse model of human reproduction. After synchronous parthenogenetic activation of MII oocytes, pronuclear oocytes were cultured in parallel in ART medium vs KSOM(aa) medium. Resultant blastocysts were compared and contrasted for gene expression among the parthenotes, as well as against zygotic blastocysts.
2022-02-01 | GSE111112 | GEO
Project description:Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. We counter that this classic view needs to be revised, because we have presented compelling evidence the sister blastomeres are both totipotent in only a subset of 2-cell stage mouse embryos (PMID 28811525). Building on our previous findings, we here ask the question if the differences between sister blastomeres can be modulated experimentally. To this end, we separate the sister blastomeres, yielding monozygotic twins. We culture these twins in two different media (GM501 vs. Sage 1 step), to see if the mRNA ratios of twin blastocysts lie more far apart from each other when culture took place in different media as compared to culture in the same medium (GSE90674). Transcriptome analysis followed by intra-pair mRNA ratio analysis revealed differences between the blastocysts of the same monozygotic pair. Some of the differences were sensitive to the choice of culture conditions, while other differences were insensitive.
2021-12-31 | GSE110735 | GEO
Project description:Analysis of mouse monozygotic twin blastocysts produced in two different culture media
| PRJNA434439 | ENA
Project description:Analysis of gene expression in uniparental mouse blastocysts produced in different culture media
| PRJNA435950 | ENA
Project description:PurposeTo study whether members of the miR-290-295 cluster in spent culture medium (SCM) of embryos are correlated with morphokinetics and apoptosis.MethodsCryopreserved 1-cell stage mouse embryos were cultured to the blastocyst stage, development was monitored by time-lapse, 59 SCM were collected, and miR-291a and miR-294 were detected with polymerase chain reaction (PCR). Blastocysts were immuno-stained for sexing (H2AK119ub) and for apoptosis (TUNEL). Each embryo and SCM were individually processed. Correlations were run between the miRNAs and developmental events (t2, t3, t4, t5, t8, tSB, tB, ECC2, ECC3, s2, s3, dB) and apoptosis (apoptotic cells/total cell number %). MiR-294 SCM and cell levels were compared in 40 blastocysts. Apoptosis was induced in 15 blastocysts with UV radiation and SCM samples were analyzed for miR-294.ResultsMiR-291a and miR-294 are released in variable levels by mouse blastocysts. Their release is similar between male and female embryos. No significant correlations were found between these miRNAs and development. MiR-294 was significantly positively correlated with apoptosis (r = 0.560, p < 0.001). Cellular expression was lower in blastocysts that released miR-294 in high levels compared with null, low, and medium release embryos (p < 0.01). UV radiation caused apoptosis which triggered higher secretion of miR-294 in 15 blastocysts versus 13 control embryos (p < 0.01).Conclusion(s)MicroRNAs are important regulators of preimplantation development. Apoptosis triggers the release of miR-294 by blastocysts which possibly serves a secretory role for embryo-maternal communication. SCM miRNA analysis is possible for individually cultured embryos and future studies can investigate miRNAs as noninvasive markers of embryo quality.
| S-EPMC7376808 | biostudies-literature
Project description:Growth media conditions influence the secretion route and release levels of engineered extracellular vesicles
| PRJNA725794 | ENA
Project description:In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions of IVC. A pro-oxidant agents that act intra-cellularly by inhibiting GSH synthesis (0.4 mM buthionine sulfoximine [BSO]) was added from days 3 to 7, and transcriptomic analysis was then performed in resulting blastocysts. Precisely, after in vitro maturation and fertilization, bovine zygotes were culture in vitro in normal condition, then at day 3, embryos were allocated into culture in control or supplemented with BSO (0.4 mM) until day 7. At this time, blastocysts were harvested and analyzed. Our hypothesis was that BSO treatment will affect blastocyst survival and gene expression associated with low embryo quality
2012-11-14 | GSE42280 | GEO
Project description:In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions of IVC. A pro-oxidant agents that act extra-cellularly by promoting ROS production (0.01 mM 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) was added from days 3 to 7, and transcriptomic analysis was then performed in resulting blastocysts. Precisely, after in vitro maturation and fertilization, bovine zygotes were culture in vitro in normal condition, then at day 3, embryos were allocated into culture in control or supplemented with AAPH (0.01 mM) until day 7. At this time, blastocysts were harvested and analyzed. Our hypothesis was that AAPH treatment will affect blastocyst survival and gene expression associated with low embryo quality
2012-11-14 | GSE42279 | GEO