Biology subjects

Biase, F. H.

Publications and source records attributed to Biase, F. H..

4 recordsLinked to original sources

bioRxiv2024

Identification of novel genes in cattle (Bos taurus) and biological insights into their function in embryo development

Appropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes. We identified 274,342 transcript sequences, and 3,033 of those transcripts do not match a gene present in an annotation, thus are potential new genes. Notably, 63.67% (1,931/3,033) of potential novel genes exhibited coding potential. Also noteworthy, 97.92% of the putative novel genes overlapped annotation with transposable elements. Comparative analysis of transcript abundance identified that 1,840 novel genes (recently added to the annotation) or potential new genes were differentially expressed between developmental stages (FDR<0.01). We also determined that 522 novel or potential new genes (448 and 34 respectively) were upregulated at eight-cell embryos compared to oocytes (FDR<0.01). In eight-cell embryos, 102 novel or putative new genes were co-expressed (|r|>0.85, P<1x10-8) with several genes annotated with gene ontology processes related to pluripotency maintenance and embryo development. CRISPR-Cas9 genome editing confirmed that the disruption of one of the novel genes highly expressed in eight-cell embryos reduced blastocyst development (ENSBTAG00000068261, P=1.55x10-7). In conclusion, our results revealed several putative new genes that need careful annotation. Many of the putative new genes have dynamic regulation during pre-implantation development and are important components of gene regulatory networks involved in pluripotency and blastocyst formation.

genomics↗

bioRxiv2023

Extensive rewiring of the gene regulatory interactions between in vitro-produced conceptuses and endometrium during attachment

Pregnancy loss is a significant problem when embryos produced in vitro are transferred to a synchronized uterus. Currently, mechanisms that underlie losses of in vitro-produced embryos during implantation are largely unknown. We investigated this problem using cattle as a model of conceptus attachment by analyzing transcriptome data of paired extraembryonic membrane and endometrial samples collected on gestation days 18 and 25, which spans the attachment window in cattle. We identified that the transfer of an in vitro-produced embryo caused a significant alteration in transcript abundance of hundreds of genes in extraembryonic and endometrial tissues on gestation days 18 and 25, when compared to pregnancies initiated by artificial insemination. Many of the genes with altered transcript abundance are associated with biological processes that are relevant to the establishment of pregnancy. An integrative analysis of transcriptome data from the conceptus and endometrium identified hundreds of putative ligand-receptor pairs. There was a limited variation of ligand-receptor pairs in pregnancies initiated by in vitro-produced embryos on gestation day 18, and no alteration was observed on gestation day 25. In parallel, we identified that in vitro production of embryos caused an extensive alteration in the co-expression of genes expressed in the extraembryonic membranes and the corresponding endometrium on both gestation days. Both the transcriptional dysregulation that exists in the conceptus or endometrium independently, and the rewiring of gene transcription between the conceptus and endometrium are a potential component of the mechanisms that contribute to pregnancy losses caused by in vitro production of embryos. SIGNIFICANCE STATEMENTThe successful establishment of pregnancies following the transfer of an in vitro produced embryo is essential for cattle production and assisted human reproduction. Most of the pregnancies initiated by the transfer of an in vitro produced embryo fail, in part because of dysfunctional interaction between the embryo and endometrium during pregnancy establishment. Our study identified that conceptuses produced in vitro and their corresponding endometrium have massive dysregulation in gene activity during the peri-implantation window, which affects crucial biological functions necessary for pregnancy. These gene expression alterations are a major contributor to the high rates of pregnancy loss following the transfer of an in vitro produced embryo. Our findings have implications for improving assisted reproduction in both agriculture and biomedicine.

developmental biology↗

bioRxiv2023

Ablation of OCT4 function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART)

CRISPR-Cas ribonucleoproteins are important tools for gene editing in pre-implantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes using CRISPR-Cas editing technology. Two electroporation sessions with Cas9D10A ribonucleoproteins targeting exon 1 and the promoter of OCT4 produced biallelic deletions in 91% of the embryos tested. In most cases, the deletions were longer than 1000 nucleotides long. Electroporation of Cas13a ribonucleoproteins prevents the production of the corresponding proteins. We electroporated Cas9D10A ribonucleoproteins targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a ribonucleoproteins targeting OCT4 mRNAs in the second session to ablate OCT4 function in cattle embryos. A lack of OCT4 resulted in embryos arresting development prior to blastocyst formation at a greater proportion (13%) than controls (31.6%, P<0.001). The few embryos that developed past the morula stage did not form a normal inner cell mass. Transcriptome analysis of single blastocysts, confirmed to lack exon 1 and promoter region of OCT4, revealed a significant (FDR<0.1) reduction in transcript abundance of many genes functionally connected to stemness, including markers of pluripotency (CADHD1, DPPA4, GNL3, RRM2). The results confirm that OCT4 is key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle. Significance StatementCRISPR-Cas mediated DNA editing can revolutionize agriculture and biomedicine due to its simplicity of design and use. Modifications induced in embryos, though challenging to accomplish, are beneficial for the advancement of livestock production and the study of biological function. Here, we developed an approach using CRISPR-Cas enzymes to remove DNA segments of the cattle genome in one-cell embryos. Our results show major advancement in the efficiency of producing large deletions in the genome of cattle embryos. Using our approach, we removed the function of the OCT4 gene. Our results confirmed OCT4 as a major regulator of pluripotency genes during embryo development and its requirement for the formation of an inner cell mass in cattle.

developmental biology↗

bioRxiv2022

A multi-omics analysis identifies molecular features associated with heifer fertility in a case-control design including Angus and Holstein cattle

BackgroundInfertility or subfertility is a critical barrier to sustainable cattle production, including in heifers. The development of heifers that do not produce a calf within an optimum window of time is a critical factor for the profitability and sustainability of the cattle industry. The early identification of heifers with optimum fertility using molecular phenotyping is a promising approach to improving sustainability in beef and dairy cattle production. ResultsUsing a high-density single nucleotide polymorphism (SNP) chip, we collected genotypic data from 575,053 SNPs. We also produced quantitative transcriptome data for 12,445 genes (12,105 protein-coding genes, 228 long non-coding RNAs, and 112 pseudogenes) and proteome data for 213 proteins. We identified two SNPs significantly associated with heifer fertility (rs110918927, chr12: 85648422, P = 6.7x10-7; and rs109366560, chr11:37666527, P = 2.6x10-5). We identified two genes with differential transcript abundance (eFDR [≤] 0.002) between the two groups (Fertile and Sub-Fertile): Adipocyte Plasma Membrane Associated Protein (APMAP, 1.16 greater abundance in the Fertile group) and Dynein Axonemal Intermediate Chain 7 (DNAI7, 1.23 greater abundance in the Sub-Fertile group). Our analysis revealed that the protein Alpha-ketoglutarate-dependent dioxygenase FTO was more abundant in the plasma collected from Fertile heifers relative to their Sub-Fertile counterparts (FDR < 0.05). Interestingly, two proteins did not reach the significance threshold in the model accounting for all samples (Apolipoprotein C-II, APOC2 (FDRglmm = 0.06) and Lymphocyte cytosolic protein 1, LCP1 (FDRglmm = 0.06)), but both proteins were less abundant in the plasma of Fertile Holstein heifers (P < 0.05). Lastly, an integrative analysis of the three datasets identified a series of features (SNPs, gene transcripts, and proteins) that can be useful for the discrimination of heifers based on their fertility. When all features were utilized together, 21 out of 22 heifers were classified correctly based on their fertility category. ConclusionsOur multi-omics analyses confirm the complex nature of female fertility. Very importantly, our results also highlight differences in the molecular profile of heifers associated with fertility that transcend the constraints of breed-specific genetic background.

genomics↗