DNA methylation haplotype block signatures responding to Staphylococcus aureus subclinical mastitis and association with production and health traits.

BACKGROUND: DNA methylation has been documented to play vital roles in diseases and biological processes. In bovine, little is known about the regulatory roles of DNA methylation alterations on production and health traits, including mastitis. RESULTS: Here, we employed whole-genome DNA methylation sequencing to profile the DNA methylation patterns of milk somatic cells from sixteen cows with naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis and ten healthy control cows. We observed abundant DNA methylation alterations, including 3,356,456 differentially methylated cytosines and 153,783 differential methylation haplotype blocks (dMHBs). The DNA methylation in regulatory regions, including promoters, first exons and first introns, showed global significant negative correlations with gene expression status. We identified 6435 dMHBs located in the regulatory regions of differentially expressed genes and significantly correlated with their corresponding genes, revealing their potential effects on transcriptional activities. Genes harboring DNA methylation alterations were significantly enriched in multiple immune- and disease-related pathways, suggesting the involvement of DNA methylation in regulating host responses to S. aureus subclinical mastitis. In addition, we found nine discriminant signatures (differentiates cows with S. aureus subclinical mastitis from healthy cows) representing the majority of the DNA methylation variations related to S. aureus subclinical mastitis. Validation of seven dMHBs in 200 cows indicated significant associations with mammary gland health (SCC and SCS) and milk production performance (milk yield). CONCLUSIONS: In conclusion, our findings revealed abundant DNA methylation alterations in milk somatic cells that may be involved in regulating mammary gland defense against S. aureus infection. Particularly noteworthy is the identification of seven dMHBs showing significant associations with mammary gland health, underscoring their potential as promising epigenetic biomarkers. Overall, our findings on DNA methylation alterations offer novel insights into the regulatory mechanisms of bovine subclinical mastitis, providing further avenues for the development of effective control measures.

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, Canada, Denmark, Germany, Spain, Switzerland, and United States) contribute with genotypes and phenotypes including DMI and CH4. However, combining data are 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.

Sperm-borne tsRNAs and miRNAs analysis in relation to dairy cattle fertility.

Sperm small non-coding RNAs (sncRNAs), such as microRNAs (miRNAs) and tRNA-derived small RNAs (tsRNAs), have been found to have implications for male fertility and play a role in the intergenerational transmission of specific phenotypes by influencing the early embryo's physiological processes in various animal species. This study postulates that there exists a correlation between sperm small non-coding RNAs (sncRNAs) and bull fertility, which in turn can influence the fertility of offspring through the modulation of early embryo development. To investigate this hypothesis, we generated comparative libraries of sperm sncRNAs from sires exhibiting high (n = 3) versus low bull fertility (n = 3), as well as high (n = 3) versus low daughter fertility (n = 3), as determined by the industry-standard Bull fertility index and Daughter fertility index. In total, 12 tsRNAs carried by sperm (11 down-regulated and 1 up-regulated) were found to be associated with bull fertility, while 19 tsRNAs (11 down-regulated and 8 up-regulated) were found to be associated with daughter fertility (q < 0.05, Log2foldchange>±1.5, base mean > 50). Notably, tRX-Glu-NNN-3811 exhibited potential as a biomarker for predicting fertility in both male and female dairy cattle. Moreover, a total of six miRNAs sperm-borne (two up-regulated and four down-regulated) and 35 miRNAs (27 up-regulated and eight down-regulated) exhibited a significant correlation with both bull fertility and daughter fertility individually (p < 0.05, base mean > 50, log2foldchange>±1.5), two microRNAs, namely miR-2385-5p (down-regulated) and miR-98 (up-regulated), exhibit a significant association (p < 0.05, base mean > 50, log2foldchange>±1.5) with the fertility of both bulls and daughter. The targets of these two microRNAs were subsequently identified and integrated with the transcriptomic database of the embryonic cells at the two-cell stage, which is known to be indicative of embryonic competence. The KEGG analysis revealed a potential correlation between these targets and choline metabolism, a crucial factor in embryonic epigenetic programming. In summary, the findings of this study indicate that sperm-borne small non-coding RNAs (sncRNAs) hold promise as biomarkers for predicting and enhancing fertility in dairy cattle. Furthermore, it is plausible that these sncRNAs may exert their effects on daughter fertility by targeting genes in the early embryo.

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

DNA methylation profiles in bovine sperm are associated with daughter fertility.

The current decline in dairy cattle fertility has resulted in significant financial losses for dairy farmers. In the past, most efforts to improve dairy cattle fertility have been focused on either management or genetics, while epigenetics have received less attention. In this study, 12 bulls were selected from a provided 100 bull list and studied (High daughter fertility = 6, Low daughter fertility = 6) for Enzymatic methylation sequencing in the Illumina HiSeq platform according to the Canadian daughter fertility index (DFI), sires with high and low daughter fertility have average DFI of 92 and 112.6, respectively. And the bull list provided shows a mean DFI of 103.4. 252 CpGs with methylation differences greater than 20% (q < 0.01) were identified, as well as the top 10 promising DMRs with a 15% methylation difference (q < 1.1e-26). Interestingly, the DMCs and DMRs were found to be distributed more on the X chromosome than on the autosome, and they were covered by gene clusters linked to germ cell formation and development. In conclusion, these findings could enhance our ability to make informed decisions when deciding on superior bulls and advance our understanding of paternal epigenetic inheritance.

Genome-wide methylation profile of mitochondrial DNA across bovine preimplantation development.

This study characterized variations in the methylation profile of mitochondrial DNA (mtDNA) during initial bovine embryo development and correlated the presence of methylation with mtDNA transcription. Bovine oocytes were obtained from abattoir ovaries and submitted to in vitro culture procedures. Oocytes and embryos were collected at various stages (immature oocyte, IM; mature oocyte, MII; zygote, ZY; 4-cells, 4C; 16-cells, 16C and blastocysts, BL). Total DNA (including mtDNA) was used for Whole Genome Enzymatic Methyl Sequencing and for quantification of mtDNA copy number. Extracted RNA was used for quantification of mitochondrial transcripts using Droplet Digital PCR. We selected ND6, CYTB, tRNA-Phe and tRNA-Gln based on their location in the mitochondrial genome, functionality and/or previous literature associating these regions with cytosine methylation. The number of mtDNA copies per oocyte/embryo was found to be similar, while methylation levels in mtDNA varied among stages. Higher total methylation levels were found mainly at 4C and 16C. In specific gene regions, higher methylation levels were also observed at 4C and 16C (ND6, CYTB and tRNA-Phe), as well as an inverse correlation with the quantity of transcripts for these regions. This is a first description of epigenetic changes occurring in mtDNA during early embryonic development. Our results indicate that methylation might regulate the mtDNA transcription at a local level, particularly around the time of embryonic genome activation.

Gene co-expression in response to Staphylococcus aureus infection reveals networks of genes with specific functions during bovine subclinical mastitis.

Staphylococcus aureus is one of the most prevalent contagious bacterial pathogen of bovine mastitis. The subclinical mastitis it causes has long-term economic implications and it is difficult to control. To further understanding of the genetic basis of mammary gland defense against S. aureus infection, the transcriptomes of milk somatic cells from 15 cows with persistent natural S. aureus infection (S. aureus-positive, SAP) and 10 healthy control cows (HC) were studied by deep RNA-sequencing technology. Comparing the transcriptomes of SAP to HC group revealed 4,077 differentially expressed genes (DEG; 1,616 up- and 2,461 downregulated). Functional annotation indicated enrichment of DEG in 94 Gene Ontology (GO) and 47 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Terms related to the immune response and disease processes were mostly enriched for by upregulated DEG, whereas biological process terms related to cell adhesion, cell movement and localization, and tissue development were mostly enriched for by downregulated DEG. Weighted gene co-expression network analysis grouped DEG into 7 modules, the most important module (colored turquoise by software and here referred to as Turquoise module) was positively significantly correlated with S. aureus subclinical mastitis. The 1,546 genes in the Turquoise module were significantly enriched in 48 GO terms and 72 KEGG pathways, with 80% of them being disease- and immune-related terms [e.g., immune system process (GO:0002376), cytokine-cytokine receptor interaction (bta04060) and S. aureus infection (bta05150)]. Some DEG such as IFNG, IL18, IL1B, NFKB1, CXCL8, and IL12B were enriched in immune and disease pathways suggesting their possible involvement in the regulation of the host response to S. aureus infection. Four modules (Yellow, Brown, Blue, and Red) were negatively correlated (significantly) with S. aureus subclinical mastitis, and were enriched in functional annotations involved in the regulation of cell migration, cell communication, metabolic process, and blood circulatory system development, respectively. Application of sparse partial least squares discriminant analysis to genes of the Turquoise module identified 5 genes (NR2F6, PDLIM5, RAB11FIP5, ACOT4, and TMEM53) capable of explaining the majority of the differences in the expression patterns between SAP and HC cows. In conclusion, this study has furthered understanding of the genetic changes in the mammary gland and the molecular mechanisms underlying S. aureus mastitis, as well as revealed a list of candidate discriminant genes with potential regulatory roles in response to S. aureus infection.

Genome-Wide DNA Methylation and Transcriptome Integration Associates DNA Methylation Changes with Bovine Subclinical Mastitis Caused by Staphylococcus chromogenes.

Staphylococcus chromogenes (SC) is a common coagulase-negative staphylococcus described as an emerging mastitis pathogen and commonly found in dairy farms. This study investigated the potential involvement of DNA methylation in subclinical mastitis caused by SC. The whole-genome DNA methylation patterns and transcriptome profiles of milk somatic cells from four cows with naturally occurring SC subclinical mastitis (SCM) and four healthy cows were characterized by next-generation sequencing, bioinformatics, and integration analyses. Comparisons revealed abundant DNA methylation changes related to SCM, including differentially methylated cytosine sites (DMCs, n = 2,163,976), regions (DMRs, n = 58,965), and methylation haplotype blocks (dMHBs, n = 53,098). Integration of methylome and transcriptome data indicated a negative global association between DNA methylation at regulatory regions (promoters, first exons, and first introns) and gene expression. A total of 1486 genes with significant changes in the methylation levels of their regulatory regions and corresponding gene expression showed significant enrichment in biological processes and pathways related to immune functions. Sixteen dMHBs were identified as candidate discriminant signatures, and validation of two signatures in more samples further revealed the association of dMHBs with mammary gland health and production. This study demonstrated abundant DNA methylation changes with possible involvement in regulating host responses and potential as biomarkers for SCM.

DOHaD: A Menagerie of Adaptations and Perspectives: The interplay between early embryo metabolism and mitoepigenetic programming of development.

In brief: This review discusses advances in the knowledge of epigenetic mechanisms regulating mitochondrial DNA and the relationship with reproductive biology. Abstract: Initially perceived simply as an ATP producer, mitochondria also participate in a wide range of other cellular functions. Mitochondrial communication with the nucleus, as well as signaling to other cellular compartments, is critical to cell homeostasis. Therefore, during early mammalian development, mitochondrial function is reported as a key element for survival. Any mitochondrial dysfunction may reflect in poor oocyte quality and may impair embryo development with possible long-lasting consequences to cell functions and the overall embryo phenotype. Growing evidence suggests that the availability of metabolic modulators can alter the landscape of epigenetic modifications in the nuclear genome providing an important layer for the regulation of nuclear-encoded gene expression. However, whether mitochondria could also be subjected to such similar epigenetic alterations and the mechanisms involved remain largely obscure and controversial. Mitochondrial epigenetics, also known as 'mitoepigenetics' is an intriguing regulatory mechanism in mitochondrial DNA (mtDNA)-encoded gene expression. In this review, we summarized the recent advances in mitoepigenetics, with a special focus on mtDNA methylation in reproductive biology and preimplantation development. A better comprehension of the regulatory role of mitoepigenetics will help the understanding of mitochondrial dysfunction and provide novel strategies for in vitro production systems and assisted reproduction technologies, as well as prevent metabolic related stress and diseases.

Comparison of cattle derived from in vitro fertilization, multiple ovulation embryo transfer, and artificial insemination for milk production and fertility traits.

The use of assisted-reproduction technologies such as in vitro fertilization (IVF) is increasing, particularly in dairy cattle. The question of consequences in later life has not yet been directly addressed by studies on large animal populations. Studies on rodents and early data from humans and cattle suggest that in vitro manipulation of gametes and embryos could result in long-term alteration of metabolism, growth, and fertility. Our goal was to better describe these presumed consequences in the population of dairy cows produced by IVF in Québec (Canada) and to compare them to animals conceived by artificial insemination (AI) or multiple ovulation embryo transfer (MOET). To do so, we leveraged a large phenotypic database (2.5 million animals and 4.5 million lactations) from milk records in Québec aggregated by Lactanet (Sainte-Anne-de-Bellevue, QC, Canada) and spanning 2012 to 2019. We identified 304,163, 12,993, and 732 cows conceived by AI, MOET, and IVF, respectively, for a total of 317,888 Holstein animals from which we retrieved information for 576,448, 24,192, and 1,299 lactations (total = 601,939), respectively. Genetic energy-corrected milk yield (GECM) and Lifetime Performance Index (LPI) of the parents of cows were used to normalize for genetic potential across animals. When compared with the general Holstein population, MOET and IVF cows outperformed AI cows. However, when comparing those same MOET and IVF cows with only herdmates and accounting for their higher GECM in the models, we found no statistical difference between the conception methods for milk production across the first 3 lactations. We also found that the rate of Lifetime Performance Index improvement of the IVF population during the 2012 to 2019 period was less than the rate observed in the AI population. Fertility analysis revealed that MOET and IVF cows also scored 1 point lower than their parents on the daughter fertility index and had a longer interval from first service to conception, with an average of 35.52 d compared with 32.45 for MOET and 31.87 for AI animals. These results highlight the challenges of elite genetic improvement while attesting to the progress the industry has made in minimizing epigenetic disturbance during embryo production. Nonetheless, additional work is required to ensure that IVF animals can maintain their performance and fertility potential.

Whole-genome DNA methylation analysis of the sperm in relation to bull fertility.

In brief: Bull fertility is an important economic trait, this study identified some DNA methylation biomarkers that are associated with bull fertility. Abstract: Subfertile bulls may cause huge economic losses in dairy production since their semen could be used to inseminate thousands of cows by artificial insemination. This study adopted whole-genome enzymatic methyl sequencing and aimed to identify candidate DNA methylation markers in bovine sperm that correlate with bull fertility. Twelve bulls were selected (high bull fertility = 6; low bull fertility = 6) based on the industry's internally used Bull Fertility Index. After sequencing, a total of 450 CpG had a DNA methylation difference higher than 20% (q < 0.01) had been screened. The 16 most significant differentially methylated regions (DMRs) were identified using a 10% methylation difference cut-off (q < 5.88 × 10-16). Interestingly, most of the differentially methylated cytosines (DMCs) and DMRs were distributed on the X and Y chromosomes, demonstrating that the sex chromosomes play essential roles in bull fertility. Additionally, the functional classification showed that the beta-defensin family, zinc finger protein family, and olfactory and taste receptors could be clustered. Moreover, the enriched G protein-coupled receptors such as neurotransmitter receptors, taste receptors, olfactory receptors, and ion channels indicated that the acrosome reaction and capacitation processes are pivotal for bull fertility. In conclusion, this study identified the sperm-derived bull fertility-associated DMRs and DMCs at the whole genome level, which could complement and integrate into the existing genetic evaluation methods, increasing our decisive capacity to select good bulls and explain bull fertility better in the future.

Analysis of MTNR1A Genetic Polymorphisms and Their Association with the Reproductive Performance Parameters in Two Mediterranean Sheep Breeds.

Sheep farming plays an important economic role, and it contributes to the livelihoods of many rural poor in several regions worldwide and particularly in Tunisia. Therefore, the steady improvement of ewes' reproductive performance is a pressing need. The MTNR1A gene has been identified as an important candidate gene that plays a key role in sheep reproduction and its sexual inactivity. It is involved in the control of photoperiod-induced seasonality mediated by melatonin secretion. The aim of this study was to identify SNPs in the MTNR1A gene in two Tunisian breeds, Barbarine (B) and Queue Fine de l'Ouest (QFO). DNA extracted from the blood of 77 adult ewes was sequenced. Selected ewes were exposed to adult fertile rams. A total of 26 SNPs were detected; 15 SNPs in the promoter region and 11 SNPs in the exon II were observed in both (B) and (QFO) breeds. The SNP rs602330706 in exon II is a novel SNP detected for the first time only in the (B) breed. The SNPs rs430181568 and rs40738822721 (SNP18 and SNP20 in our study, respectively) were totally linked in this study and can be considered a single marker. DTL was associated with SNP18 and SNP20 in (B) ewes (p < 0.05); however, no significant difference was detected between the three genotypes (G/G, G/A, and A/A) at these two SNPs. Fertility rate and litter size parameters were not affected by SNP18 and SNP20. There was an association between these two polymorphisms and (B) lambs' birth weights (p < 0.05). Furthermore, the ewes with the A/A genotype gave birth to lambs with a higher weight compared to the other two genotypes for this breed (p < 0.05). There was not an association between SNP 18 and SNP20 and (QFO) ewes' reproductive parameters. These results might be considered in future sheep selection programs for reproductive genetic improvement.

Gestational and health outcomes of dairy cows conceived by assisted reproductive technologies compared to artificial insemination.

Herd gestation and health management are key aspects of effective dairy farm operations and animal welfare improvement. Unfortunately, very little is known about the developmental divergences induced by assisted reproduction technologies (ART) and their consequences once the animal is mature. Indeed, the gestational and health outcomes of this subset of the Holstein population is yet to be characterized. In this study, the intergenerational impacts of ART conception were assessed by looking at the gestation and health outcomes of a large cohort of cows (n = 284,813) for which the conception methods were known. Our results showed that cows conceived by multiple ovulation embryo transfer (MOET) and in vitro fertilisation (IVF) displayed longer gestations: +0.37 ± 0.079 and +0.65 ± 0.21 day compared to cows conceived by artificial insemination (AI). Surprisingly, animals conceived by all methods experienced a similar 1-day decline in average gestation length from 2012 to 2019. Cows conceived by IVF were not more likely to experience stillbirths but were affected by common diseases such as ovarian cysts, mastitis, and uterine diseases in different proportions compared to cows conceived by other methods. This study provides new and unique information on ART animals regarding perinatal mortality and general health outcomes.

Methylome and transcriptome data integration reveals potential roles of DNA methylation and candidate biomarkers of cow Streptococcus uberis subclinical mastitis.

BACKGROUND: Mastitis caused by different pathogens including Streptococcus uberis (S. uberis) is responsible for huge economic losses to the dairy industry. In order to investigate the potential genetic and epigenetic regulatory mechanisms of subclinical mastitis due to S. uberis, the DNA methylome (whole genome DNA methylation sequencing) and transcriptome (RNA sequencing) of milk somatic cells from cows with naturally occurring S. uberis subclinical mastitis and healthy control cows (n = 3/group) were studied. RESULTS: Globally, the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns. The DNA methylation levels at the promoter, first exon and first intron regions were negatively correlated with the expression level of genes at a whole-genome-wide scale. In general, DNA methylation level was lower in S. uberis-positive group (SUG) than in the control group (CTG). A total of 174,342 differentially methylated cytosines (DMCs) (FDR < 0.05) were identified between SUG and CTG, including 132,237, 7412 and 34,693 DMCs in the context of CpG, CHG and CHH (H = A or T or C), respectively. Besides, 101,612 methylation haplotype blocks (MHBs) were identified, including 451 MHBs that were significantly different (dMHB) between the two groups. A total of 2130 differentially expressed (DE) genes (1378 with up-regulated and 752 with down-regulated expression) were found in SUG. Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with significant changes in their methylation levels and/or gene expression changes (MetGDE genes, MethGET P-value < 0.001). Functional enrichment of genes harboring ≥ 15 DMCs, DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response to S. uberis infection, especially cytokine activities. Furthermore, discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers, including 6 DE genes, 15 CpG-DMCs and 5 dMHBs that discriminated between SUG and CTG. CONCLUSION: The integration of methylome and transcriptome of milk somatic cells suggests the possible involvement of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due to S. uberis. The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance.

The genomic response of human granulosa cells (KGN) to melatonin and specific agonists/antagonists to the melatonin receptors.

Melatonin is a known modulator of follicle development; it acts through several molecular cascades via binding to its two specific receptors MT1 and MT2. Even though it is believed that melatonin can modulate granulosa cell (GC) functions, there is still limited knowledge of how it can act in human GC through MT1 and MT2 and which one is more implicated in the effects of melatonin on the metabolic processes in the dominant follicle. To better characterize the roles of these receptors on the effects of melatonin on follicular development, human granulosa-like tumor cells (KGN) were treated with specific melatonin receptor agonists and antagonists, and gene expression was analyzed with RNA-seq technology. Following appropriate normalization and the application of a fold change cut-off of 1.5 (FC 1.5, p ≤ 0.05) for each treatment, lists of the principal differentially expressed genes (DEGs) are generated. Analysis of major upstream regulators suggested that the MT1 receptor may be involved in the melatonin antiproliferative effect by reprogramming the metabolism of human GC by activating the PKB signaling pathway. Our data suggest that melatonin may act complementary through both MT1 and MT2 receptors to modulate human GC steroidogenesis, proliferation, and differentiation. However, MT2 receptors may be the ones implicated in transducing the effects of melatonin on the prevention of GC luteinization and follicle atresia at the antral follicular stage through stimulating the PKA pathway.

Metabolism of fatty acids in follicular cells, oocytes, and blastocysts.

Fatty acids (FA) are one of the substrates that can be oxidized for energy production. The blood concentration of all types of FA varies according to different nutrition conditions, and follicular fluid levels are generally in line with serum levels. Elevated levels of FA, especially non-esterified fatty acids (NEFA), are commonly found in females with metabolic issues, which are often related to subfertility in many species including humans, pigs, cattle, and mice. Long-time exposure to an excessive quantity of fatty acids impairs the cell structure and functions causing injuries in tissues and organs, resulting in lipotoxicity and eventually hampering health and fertility. High levels of saturated NEFA can have detrimental effects on granulosa cells, oocyte quality, and embryo development. Although the harmful effects of FA are established in reproductive tissues, how granulosa cells and cumulus cells respond and cooperate with oocytes when exposed to NEFA requires further understanding. This review provides a summary of the adverse impacts of exposure to NEFA during in vitro maturation on oocytes, follicular cells, and embryos. A comprehensive understanding of the effects of NEFA on oocytes in vitro would improve our understanding of the impacts of natural exposure in vivo. Lay summary: Exposure to excess FAs affects the health of eggs, early embryos, and children born from these. The way different cell types react to excess FAs has not been studied very extensively, especially in pigs which provide a good model to investigate the impact of nutrition on the ovaries in humans. This review also looks at the way cells surrounding the egg react to FAs to help our understanding of the impact of excess fatty acids on female fertility.

IGF2R, KCNQ1, PLAGL1, and SNRPN DNA methylation is completed in bovine by the early antral follicle stage.

Imprinted genes are inherited with different DNA methylation patterns depending on the maternal or paternal origin of the allele. In cattle (Bos taurus), abnormal methylation of these genes is linked to the large offspring syndrome, a neonatal overgrowth phenotype analogous to the human Beckwith-Wiedemann syndrome. We hypothesized that in bovine oocytes, some of the methylation patterns on maternally imprinted genes are acquired in the last phase of folliculogenesis. The pyrosequencing analysis of IGF2R, KCNQ1, PLAGL1, and SNRPN imprinted genes showed no clear progression of methylation in oocytes from follicles 1-2 mm (late pre antral/early antral) and up. Instead, these oocytes displayed complete methylation at the imprinted differentially methylated regions (>80%). Other mechanisms related to imprint maintenance should be investigated to explain the hypomethylation at IGF2R, KCNQ1, PLAGL1, and SNRPN maternally imprinted sites observed in some bovine embryos.

Bovine oocyte exposure to perfluorohexane sulfonate (PFHxS) induces phenotypic, transcriptomic, and DNA methylation changes in resulting embryos in vitro.

Knowledge on the effects of perfluorohexane sulfonate (PFHxS) on ovarian function is limited. In the current study, we investigated the sensitivity of oocytes to PFHxS during in vitro maturation (IVM), including consequences on embryo development at the morphological, transcriptomic, and epigenomic levels. Bovine cumulus-oocyte complexes (COCs) were exposed to PFHxS during 22 h IVM. Following fertilisation, developmental competence was recorded until day 8 of culture. Two experiments were conducted: 1) exposure of COCs to 0.01 µg mL-1 - 100 µg mL-1 PFHxS followed by confocal imaging to detect neutral lipids and nuclei, and 2) exposure of COCs to 0.1 µg mL-1 PFHxS followed by analysis of transcriptomic and DNA methylation changes in blastocysts. Decreased oocyte developmental competence was observed upon exposure to ≥ 40 µg mL-1 PFHxS and altered lipid distribution was observed in the blastocysts upon exposure to 1-10 µg mL-1 PFHxS (not observed at lower or higher concentrations). Transcriptomic data showed that genes affected by 0.1 µg mL-1 PFHxS were enriched for pathways related to increased synthesis and production of reactive oxygen species. Enrichment for peroxisome proliferator-activated receptor-γ and oestrogen pathways was also observed. Genes linked to DNA methylation changes were enriched for similar pathways. In conclusion, exposure of the bovine oocyte to PFHxS during the narrow window of IVM affected subsequent embryonic development, as reflected by morphological and molecular changes. This suggests that PFHxS interferes with the final nuclear and cytoplasmic maturation of the oocyte leading to decreased developmental competence to blastocyst stage.

Comparing the whole genome methylation landscape of dairy calf blood cells revealed intergenerational inheritance of the maternal metabolism.

This study evaluated the hypothesis that the maternal metabolic stressed status could be inherited to their F1 daughters via epigenetic mechanism. The maternal cow blood ß-hydroxybutyric acid (BHB) level (≥0.9 mM/L) was used as an indicator of maternal metabolic stress. Eight newborn daughters' blood cells were used for methylation comparison and analysis. By Whole Genome Bisulphite Sequencing (WGBS), a total of 1,861 Differentially Methylated Regions (DMRs), including 944 differentially methylated cytosines (DMCs), were identified. Most DMRs were distributed in intronic and intergenic regions, and most of the DMR in promoter regions were hypermethylated. Differentially methylated genes (DMGs) with DMR methylation differences higher than 20% were mainly enriched in metabolism-related pathways. These results suggest that newborn calves' metabolic pathways were altered, with 64 DMGs being clustered with metabolic signalling by KEGG analysis. Our study revealed the whole epigenetic landscape of calf blood cells and suggested that the maternal metabolic status can affect the embryo's epigenetic status and metabolic-related pathways in offspring, providing further evidence for epigenetic intergenerational inheritance of metabolic stress in domestic animals. Besides, this study also contributed more evidence to support the Developmental Origins of Health and Disease (DOHAD) theory in large animals.