Biology subjects

Meirelles, F. V.

Publications and source records attributed to Meirelles, F. V..

9 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

Mammal placental phenotypes are predictable from microRNA repertoires.

Similar placental morphologies evolved multiple times independently in the history of mammal evolution1,2. Yet the genetic architecture that repeatedly guides distinct mammal lineages towards similar complex placental phenotypes has remained elusive. MicroRNAs (miRNAs), despite their diversity in mammals3-7 and known roles as developmental regulators8-10, remain under-examined as drivers of morphological innovation. We identified presence-absence patterns for 429 miRNA gene families across 398 mammalian genomes and discovered that placental phenotype is highly predictable from genomic miRNA repertoires (classification accuracy 74.5-95.8%). We identified 42 miRNA gene families significantly associated with placentation type, whose gene targets are enriched for developmental processes. Notably, convergent placental morphologies consistently involve identical miRNA families, revealing that evolution of this trait is constrained to predictable genetic pathways. We demonstrate that MIR-11986, uniquely associated with cotyledonary placentation, has tissue-specific expression in key reproductive tissues. MiRNA-mediated regulation therefore constrains placental morphological diversification into reproducible programs, offering insights into how genetic architecture shapes the predictability of convergent evolution. This striking pattern reveals a fundamental principle of evolution: that the miRNA regulatory networks available to control and guide complex placental morphological innovation are constrained and predictable.

genomics↗

bioRxiv2023

Assessment of Total Oocyte Transcripts Representation through Single Ooplasm Biopsy in Bovine with High Reliability

Understanding the entire transcriptional and epigenetic landscape is facilitated by the application of omics in a number of ways. Today, omic instruments are more affordable and easier to implement. In human research, for instance, single-omics are a reality and are used extensively to generate vast quantities of data. This method permits the comprehensive reconstruction of transcriptome and epigenetic markers removing bias from pooled samples. In tandem with the evolution of machines and protocols, algorithms and genome annotation have undergone continuous improvement. The genome annotation of domestic animals is inferior to that of humans, rodents, and less complex organisms. In the case of heifers, the reference is incomplete, with significant gaps and only a portion of the noncoding transcripts. The purpose of this study is to validate our compartmentalized single oocyte biopsy by comparing a small fraction of bovine oocytes, 1%, to the entire oocyte at the Metaphase II stage. In addition, we examined the use of four database sources (NCBI, ENSEMBL, UCSC, and NONCODE) to produce a merged non-redundant gene alignment and counting in order to enhance gene detection and normalization, resulting in a more accurate method to comprehend the entire landscape. This study is a continuation of our research titled "Retrospective model utilizing biopsies, granulosa cells, and polar body to predict oocyte competence in bovine" in which this method was used to retrospectively compare biopsy oocytes collected during the MII phase. With the addition of NONCODE information, gene normalization was significantly enhanced. In addition, our analysis identified 4560 noncoding genes from NONCODE references. ENSEMBL and NCBI have nearly the same number of annotated genes (16,423 vs. 17,804), but using ENSEMBL as a reference, 2356 genes were able to be normalized and identified. Proceeding to biopsy x oocyte analysis, we were able to detect a greater number of genes in oocytes than in biopsy, where the preponderance was from NONCODE sources (68). Despite these minor differences, the high correlation of expression between them (89%) was consistent and proved to be a valuable instrument for studying the oocyte without destroying it.

bioinformatics↗

bioRxiv2023

Retrospective model utilizing biopsies, granulosa cells, and polar body to predict oocyte competence in bovine

Developmental competence is obtained by a series of morphological and molecular changes during mammalian oocyte growth within the ovulatory follicle. This entails the accumulation of cytoplasmic transcripts that will be used throughout the early stages of development prior to embryonic genome activation, a process known as ooplasm maturation. Furthermore, during follicular growth, epigenetic maturation occurs, which is essential for appropriate embryo development. We believe that transcripts and DNA methylation differ between blastocyst oocytes and those that cleaved but were arrested on day three. We devised a retrospective technique to identify transcripts in oocyte, cumulus, and granulosa cells, as well as DNA methylation connected with oocyte competence, in this work. We dissected and harvested ovarian follicles to achieve this purpose. We extracted and flash frozen the granulosa cells after rupturing them. The oocytes were put in maturation media droplets, and the cumulus cells and polar body were removed and kept the following day. To prevent spermatozoon interference, we chemically activated the oocytes and tracked their development (until they reached the blastocyst stage). We went back to their biopsies, cumulus cells, and polar bodies and did RNA-seq (biopsies and cumulus) and single polar body WGBS (polar bodies) when we collected the results 7 days later, i.e. 1-) embryos that cleaved but stopped development (termed CL) or 2-) embryos that cleaved and progressed until the blastocyst stage (termed BL). Additionally, after transcriptome results from oocyte-biopsy and cumulus cells, the granulosa cells from their individual oocytes were sequenced as a noninvasive strategy. This study is a follow-up to our previous work, "Assessment of Total Oocyte Transcripts Representation in bovine Using Single Ooplasm Biopsy with High Reliability." Following sequencing, we discovered that the two groups, BL x CL, were transcriptionally different in granulosa and biopsy samples, although cumulus cells were a poor predictor of oocyte competence. By analyzing the differentially expressed genes, we discovered multiple genes and pathways related to oocyte competency, demonstrating the efficacy of our method. Despite no change in morphology, these alterations in pathways and genes show that the oocyte CL group was transcriptionally and epigenetically delayed, with ferroptosis and necroptosis processes activated. The oocyte BL group demonstrated numerous molecular signaling, oocyte meiosis, GnRH signaling, G-protein cascade, and RNA stability pathways. In network analysis, we discovered GNAS, an imprinted gene and one of the most important essential genes. The transcripts from granulosa cells confirm the oocyte results. Nonetheless, transcriptional variations in granulosa cells were far greater than those in oocytes (97% x 34% variance), implying a completely distinct transcriptome in the follicular niche. In terms of the WGBS, we discovered differentially methylated areas linked with oocyte competency, as well as transcriptome results confirming the structures ability to predict outcome. These findings might be beneficial in clinical settings for those undergoing infertility therapy. In the oocyte, we discovered a complex transcriptional and epigenetic regulation network. Furthermore, mature cumulus transcription produced information that differed from the true content of the MII oocyte and granulosa cells before maturity. Our findings underscore the significance of maternal transcripts and epigenetic maturation in early parthenogenesis, as well as the use of granulosa cells as early indicators of competence.

genomics↗

bioRxiv2023

An exploratory data analysis from ovine and bovine RNA-seq identifies pathways and key genes related to cervical dilatation

The present study developed a review and exploration of data in public and already validated repositories. The main objective is to identify the pathways involved in ruminants cervical dilatation, which are conserved between cattle and sheep in the follicular and luteal phases of the reproductive cycle. In cattle, 1961 genes were found to be more expressed in the follicular phase and 1560 in the luteal phase. 24 genes were considered exclusively expressed from these 18 genes were in the follicular phase and 6 genes were in the luteal phase. In sheep, 2126 genes are more expressed in the follicular phase and 2469 genes are more expressed in the luteal phase. Hoxb genes were identified in both species and are correlated with the PI3K/Akt pathway. PI3K/Akt was also found in both cattle and sheep, appearing prominently in the follicular and luteal phases of both species. Our analyzes have pointed out that the PI3K/Akt pathway and the Hoxb genes appear in prominence, in modulating mechanisms that involve estrus alterations in the cervix. PI3K/Akt appears to be an important pathway in the cervical relaxation process.

genomics↗

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↗

bioRxiv2018

Mitofusin 1 is required for the oocyte-granulosa cell communication that regulates oogenesis

Mitochondrial function, largely regulated by the dynamics of this organelle, is inextricably linked to oocyte health. While the proteins that modulate mitochondrial fusion, Mitofusin 1 (MFN1) and 2 (MFN2), are required for embryogenesis, their role in oocyte development remains unclear. Here we show that the oocyte-specific deletion of Mfn1, but not Mfn2, results in a complete loss of oocyte growth and ovulation due to a block in folliculogenesis at the preantral-to-antral follicle transition. We pinpoint the loss of oocyte ovulation to disrupted oocyte-somatic cell communication - Mfn1-null oocytes are deficient for the production of the important somatic cell signaling factor GDF9. Unexpectedly, the double loss of Mfn1 and Mfn2 mitigates the effects on oocyte growth and ovulation, which is explained by a partial rescue of oocyte-somatic cell communication and folliculogenesis. Together, this work demonstrates that mitochondrial function influences communication of oocyte with follicular somatic cells and suggests that the balanced expression of modulators of mitochondrial dynamics is critical for proper oocyte development.

developmental biology↗