Investigation of MYST4 histone acetyltransferase and its involvement in mammalian gametogenesis

<p>Abstract</p> <p>Background</p> <p>Various histone acetylases (HATs) play a critical role in the regulation of gene expression, but the precise functions of many of those HATs are still unknown. Here we provide evidence that MYST4, a known HAT, may be involved in early mammalian gametogenesis.</p> <p>Results</p> <p>Although <it>MYST4 </it>mRNA transcripts are ubiquitous, protein expression was restricted to select extracts (including ovary and testis). Immunohistochemistry experiments performed on ovary sections revealed that the MYST4 protein is confined to oocytes, granulosa and theca cells, as well as to cells composing the blood vessels. The transcripts for <it>MYST4 </it>and all-<it>MYST4</it>-isoforms were present in oocytes and in <it>in vitro </it>produced embryos. In oocytes and embryos the MYST4 protein was localized in both the cytoplasm and nucleus. Within testis sections, the MYST4 protein was specific to only one cell type, the elongating spermatids, where it was exclusively nuclear.</p> <p>Conclusion</p> <p>We established that MYST4 is localized into specialized cells of the ovary and testis. Because the majority of these cells are involved in male and female gametogenesis, MYST4 may contribute to important and specific acetylation events occurring during gametes and embryo development.</p

Alterations in transcript abundance of bovine oocytes recovered at growth and dominance phases of the first follicular wave

<p>Abstract</p> <p>Background</p> <p>Oocyte developmental competence is highly affected by the phase of ovarian follicular wave. Previous studies have shown that oocytes from subordinate follicles recovered at growth phase (day 3 after estrus) are developmentally more competent than those recovered at dominance phase (day 7 after estrus). However, the molecular mechanisms associated with these differences are not well elucidated. Therefore, the objective of this study was to investigate transcript abundance of bovine oocytes retrieved from small follicles at growth and dominance phases of the first follicular wave and to identify candidate genes related to oocyte developmental competence using cDNA microarray.</p> <p>Results</p> <p>Comparative gene expression analysis of oocytes from growth and dominance phases and subsequent data analysis using Significant Analysis of Microarray (SAM) revealed a total of 51 differentially regulated genes, including 36 with known function, 6 with unknown function and 9 novel transcripts. Real-time PCR has validated 10 transcripts revealed by microarray analysis and quantified 5 genes in cumulus cells derived from oocytes of both phases. The expression profile of 8 (80%) transcripts (ANAXA2, FL396, S100A10, RPL24, PP, PTTG1, MSX1 and BMP15) was in agreement with microarray data. Transcript abundance of five candidate genes in relation to oocyte developmental competence was validated using Brilliant Cresyl Blue (BCB) staining as an independent model. Furthermore, localization of mRNA and protein product of the candidate gene MSX1 in sections of ovarian follicles at days 0, 1, 3 and 7 of estrous cycle showed a clear fluorescent signal in both oocytes and cumulus cells with higher intensity in the former. Moreover, the protein product was detected in bovine oocytes and early cleavage embryos after fertilization with higher intensity around the nucleus.</p> <p>Conclusion</p> <p>This study has identified distinct sets of differentially regulated transcripts between bovine oocytes recovered from small follicles at growth and dominance phases of the first follicular wave. The validation with independent model supports our notion that many of the transcripts identified here may represent candidate genes associated with oocyte developmental competence. Further specific functional analysis will provide insights into the exact role of these transcripts in oocyte competence and early embryonic development.</p

Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies

Abstract Background Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. Results Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. Conclusion By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos

Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors

Pre- and post- implantation development. Effect of TSA treatment on in vitro and in vivo development of cloned embryos compared to fertilized counterparts. (DOCX 18 kb

Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bvovine oocytes

Background: In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation. Objectives: To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM. Materials & methods: The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass. Results: Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts. Conclusions: These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal

The Resilient Dairy Genome Project - a general overview of methods and objectives related to feed efficiency and methane emissions.

The Resilient Dairy Genome Project (RDGP) is an international large-scale applied research project that aims to generate genomic tools to breed more resilient dairy cows. In this context, improving feed efficiency and reducing greenhouse gases from dairy is a high priority. The inclusion of traits related to feed efficiency (e.g., dry matter intake [DMI]) or greenhouse gases (e.g., methane emissions [CH4]) relies on available genotypes as well as high quality phenotypes. Currently, 7 countries, i.e., Australia [AUS], Canada [CAN], Denmark [DNK], Germany [DEU], Spain [ESP], Switzerland [CHE], and United States of America [USA] contribute with genotypes and phenotypes including DMI and CH4. However, combining data is challenging due to differences in recording protocols, measurement technology, genotyping, and animal management across sources. In this study, we provide an overview of how the RDGP partners address these issues to advance international collaboration to generate genomic tools for resilient dairy. Specifically, we describe the current state of the RDGP database, data collection protocols in each country, and the strategies used for managing the shared data. As of February 2022, the database contains 1,289,593 DMI records from 12,687 cows and 17,403 CH4 records from 3,093 cows and continues to grow as countries upload new data over the coming years. No strong genomic differentiation between the populations was identified in this study, which may be beneficial for eventual across-country genomic predictions. Moreover, our results reinforce the need to account for the heterogeneity in the DMI and CH4 phenotypes in genomic analysis

Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bovine oocytes

BackgroundIn vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation.ObjectivesTo determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM.Materials and methodsThe study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass.ResultsRemoval of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts.ConclusionsThese findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal