Biology subjects

Sampaio, R. V.

Publications and source records attributed to Sampaio, R. V..

6 recordsLinked to original sources

bioRxiv2026

Epigenetic Remodeling Increases Plasticity of Bovine Fibroblasts Without Improving Embryo and Stem Cell Development

Epigenetic memory, reinforced by redundant heterochromatic networks, is a major barrier to somatic cell reprogramming. In cattle, single-agent chromatin-modifying treatments show inconsistent efficacy, likely due to compensatory crosstalk among repressive marks. Here, we evaluated a multi-target small-molecule cocktail, GUSTC, combining GSK343 (EZH2 inhibitor), UNC0638 (G9a/GLP inhibitor), Sodium Butyrate (HDAC inhibitor), Trichostatin A (HDAC inhibitor), and Chaetocin (SUV39H1 inhibitor) to simultaneously target facultative and constitutive heterochromatin in bovine fibroblasts. At optimized, non-cytotoxic concentrations, GUSTC treatment reduced H3K9me2, H3K9me3, H3K27me3, and HP1-alpha levels while increasing H3K9 acetylation by immunofluorescence. Using GUSTC-treated fibroblasts as donor cells for somatic cell nuclear transfer did not significantly change cleavage (83.6% vs. 92.2%; p = 0.15), blastocyst rate (28.4% vs. 39.0%; p = 0.20), bovine embryonic stem cell line derivation efficiency (26.3% vs. 33.3% of seeded blastocysts; p = 0.57), or the core pluripotency marker profile (OCT4, NANOG, SOX2) of the resulting lines. In a separate, serum-free, pluripotency-permissive culture system, GUSTC-treated cells adopted a colony-like morphology and showed increased POU5F1 and NANOG expression, along with increased DNMT1, EHMT2, HDAC1, HDAC2, and SUV39H1 transcripts. GUSTC thus reduces repressive heterochromatin marks in donor fibroblasts and is associated with a potentially more reprogramming-permissive state, without detectable improvement in preimplantation developmental competence. It provides a starting point for chemical reprogramming strategies in cattle.

developmental biology↗

bioRxiv2025

Rapid Derivation of Cloning-Competent Cells from Peripheral Blood Advances Conservation Biobanking

I.Establishing viable cell lines from endangered species is essential for conservation, yet traditional fibroblast derivation from skin biopsies faces challenges including variable success rates, contamination risk, and extended culture timelines. We demonstrate that endothelial progenitor cells (EPCs) and pericytes isolated from peripheral blood represent superior alternatives for biobanking across three mammalian genera (Canis, Bison, and Equus). Blood-derived cells exhibited 2-3 fold faster doubling rates (15-20 hours versus >35 hours for fibroblasts) and reduced time to generate banked lines from 3-4 weeks to 1.5-2 weeks. Proteomic profiling of 32 canonical markers confirmed EPCs and pericytes represent distinct populations with lineage-specific molecular signatures. Optical genome mapping demonstrated equivalent genomic stability across all cell types with no detectable structural variants or aneuploidies. Critically, interspecific somatic cell nuclear transfer (iSCNT) experiments confirmed both EPCs and pericytes generate viable embryos with efficiency meeting or exceeding fibroblasts. Gray wolf blood-derived cells produced six viable fetuses with 15% implantation rate, while bison EPCs showed higher blastocyst formation (7%) than fibroblasts (3%) from the same individual. Blood collection during routine veterinary procedures offers minimally invasive sampling with reduced contamination compared to skin biopsies. These findings support integrating blood-derived cell banking into conservation programs, enabling opportunistic genetic preservation during standard management activities and expanding options for genetic rescue through assisted reproductive technologies.

cell biology↗

bioRxiv2023

Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin: In vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo Vs in vitro and in vivo Vs cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). miRNA cargo was similar between all three groups (426 common cargo) differences between in vivo and cloned (8 miRNAs), and in vivo and in vitro (6 miRNAs) observed. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changes to the proteomic content of target cells (96, and 85 respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy. SIGNIFICANCE STATEMENTDuring the peri-implantation period of pregnancy in mammals, the endometrium acts as a biosensor for the developmental competency of the embryo. However, the mechanism by which biosensor capability of the endometrium is established, remains elusive. In this study, we show that embryos of different developmental competencies have distinct microRNA cargo contained in their extracellular vesicles (EVs). Exposure of the endometrium to these conceptuses alters the transcriptional response of the endometrium during the process of pregnancy recognition. This differential response is mediated in part, by the delivery and action of the these differentially abundant microRNAs into EVs. Here we propose differences in EV-mediated miRNA cargo are responsible in part for this biosensor capability of the endometrium.

developmental biology↗

bioRxiv2023

Haploid androgenetic development in bovines reveals imbalanced WNT signaling and impaired cell fate differentiation.

Haploid embryos have contributed significantly to our understanding of the role of parental genomes in development and can be applied to important biotechnology for human and animal species. However, development to the blastocyst stage is severely hindered in bovine haploid androgenetic embryos (hAE). To further our understanding of such developmental arrest, we performed a comprehensive comparison of the transcriptomic profile of morula-stage embryos, which were validated by qRT-PCR of transcripts associated with differentiation in haploid and biparental embryos. Among numerous disturbances, results showed that pluripotency pathways, especially the wingless-related integration site (WNT) signaling, were particularly unbalanced in hAE. Moreover, transcript levels of KLF4, NANOG, POU5F1, SOX2, CDX2, CTNNBL1, AXIN2, and GSK3B were noticeably altered in hAE, suggesting disturbance of pluripotency and canonical WNT pathway. To evaluate the role of WNT on hAE competence, we exposed early day-5 morula stage embryos to the GSK3B inhibitor CHIR99021. Although no alterations were observed in pluripotency and WNT-related transcripts, exposure to CHIR99021 improved their ability to reach the blastocysts stage, confirming the importance of the WNT pathway in the developmental features of bovine hAE. Summary statementThis study shows the importance of the WNT pathway on bovine haploid androgenetic development by walking through transcriptomics and pluripotency markers associated with cell fate determination during early development.

developmental biology↗

bioRxiv2021

Characterization of Histone Lysine β-hydroxybutyrylation in bovine tissues, cells, and cumulus-oocyte complexes

Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through the histone post-translational modifications. The ketone body {beta}-hydroxybutyrate (BHB) was shown to cause a novel type of epigenetic modification, Histone Lysine {beta}-hydroxybutyrylation (Kbhb), associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation and its effects on cellular epigenome are largely unknown. To unravel these effects, we sought and identified that Kbhb is present in bovine tissues in vivo and further confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. We also demonstrated that the maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation neither to develop until blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but this modification was only faintly detected in oocytes. Profiling the transcriptome in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The down-regulated genes are mainly involved in glycolysis and ribosome assembly pathways, while the up-regulated genes are involved in mitochondrial metabolism and oocyte development. The specific genes and pathways altered by BHB treatment will provide entry points to carry out functional experiments aiming to mitigate problems and improve fertility in cattle suffering metabolic disorders. A key goal for future work will be to understand mechanistically how BHB transmits signals from the environment to affect cellular functions and the bovine epigenome. Summary sentenceBeta-hydroxybutyrate induces Histone Lysine {beta}-hydroxybutyrylation in fibroblasts and cumulus-oocyte complexes, it alters the transcriptome in cumulus cells, but does not affect oocytes competence to resume meiosis and develop until blastocyst stage.

developmental biology↗

bioRxiv2019

Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming.

Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone metyltransferases EHMT1 and EHMT2. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 causes diverse modifications to the chromatin during early development, suggesting an intense epigenetic crosstalk during nuclear reprogramming.

developmental biology↗