Project description:Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, and more specifically DNA methylation, in the offspring. Final oocyte maturation and early embryo development coincide with specific methylation changes and both time periods are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect the establishment and maintenance of DNA methylation patterns in oocytes and embryos and subsequently alter transcriptomic profiles and developmental competence of the resultant blastocysts. To do this, bovine oocytes and embryos were exposed to different NEFA concentrations in two separate experiments. In the first experiment, oocytes were matured in vitro for 24 hours in medium containing: 1) physiological (âBASALâ) concentrations of oleic (OA), palmitic (PA) and stearic (SA) or 2) elevated (pathophysiological or âHIGH COMBIâ) concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. The developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray techniques. Data regarding DNA methylation were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to their BASAL-exposed counterparts (19.3 % compared to 23.2 % and 18.2 % compared to 25.3 %, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation profiles and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to an adverse environment. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later stages of life observed in offspring from mothers displaying lipolytic disorders. Two experiments were set up to characterize the epigenomic profile of the resultant day 7.5 blastocysts. In the first (in vitro maturation or IVM) experiment, the effect of elevated NEFA exposure during oocyte maturation (24 h) on DNA methylation profiles was evaluated by exposing oocytes to the BASAL or HIGH COMBI conditions. In the second (in vitro culture or IVC) experiment, the effect of elevated NEFA exposure during embryo culture (6.5 days) on DNA methylation marks was evaluated by exposing embryos to the BASAL or HIGH COMBI conditions. A total of 1039 and 1412 oocytes were used in the IVM and IVC experiment, respectively, equally distributed between treatments in 4 replicates. Resultant day 7.5 blastocysts were snap frozen for subsequent DNA methylation analysis (80 blastocysts in each experiments, equally collected between treatments in 4 replicates).

Project description:Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, and more specifically DNA methylation, in the offspring. Final oocyte maturation and early embryo development coincide with specific methylation changes and both time periods are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect the establishment and maintenance of DNA methylation patterns in oocytes and embryos and subsequently alter transcriptomic profiles and developmental competence of the resultant blastocysts. To do this, bovine oocytes and embryos were exposed to different NEFA concentrations in two separate experiments. In the first experiment, oocytes were matured in vitro for 24 hours in medium containing: 1) physiological (âBASALâ) concentrations of oleic (OA), palmitic (PA) and stearic (SA) or 2) elevated (pathophysiological or âHIGH COMBIâ) concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. The developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray techniques. Data regarding DNA methylation were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to their BASAL-exposed counterparts (19.3 % compared to 23.2 % and 18.2 % compared to 25.3 %, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation profiles and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to an adverse environment. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later stages of life observed in offspring from mothers displaying lipolytic disorders. The effect of elevated NEFA exposure during embryo culture (6.5 days) on gene expression was evaluated by exposing embryos to the BASAL or HIGH COMBI conditions. A total of 1412 oocytes were used, equally distributed between treatments in 4 replicates. Resultant day 7.5 blastocysts were snap frozen for subsequent transcriptome analysis (80 blastocysts in each experiments, equally collected between treatments in 4 replicates).

Project description:The fallopian tube epithelium is one of the potential sources of high-grade serous ovarian cancer (HGSC). The use of estrogen only hormone replacement therapy increases ovarian cancer risk. Despite estrogenâ??s influence in OVCA, selective estrogen receptor modulators (SERMs) typically demonstrate only a 20% response rate. This low response could be due to a variety of factors including the loss of estrogen receptor signaling or the role of estrogen receptor signaling in different potential cell types of origin. The response of fallopian tube epithelium to SERMs is not known, and would be useful when determining therapeutic options for tumors that arise from this cell type, such as high-grade serous cancer. Using normal murine derived oviductal epithelial cells (mouse equivalent to the fallopian tube) estrogen receptor expression was confirmed and interaction with its ligand, estradiol, triggered mRNA and protein induction of progesterone receptor (PR). The SERMs 4-hydroxytamoxifen, raloxifene and desmethylarzoxifene, functioned as estrogen receptor antagonists in the oviductal cells. Cellular proliferation and migration assays suggested that estradiol does not significantly impact cellular migration and increased proliferation in CD1, but not in FVB derived cell lines. Further, using RNAseq, the oviduct specific transcriptional genes targets of estrogen and 4-hydroxytamoxifen signaling were determined and validated. The RNA-seq revealed enrichment in proliferation, anti-apoptosis, calcium signaling and steroid signaling processes. Finally, the ER and PR receptor status of a panel of HGSC cell lines was investigated highlighting the need for better models of estrogen responsive HGSC cell lines. Murine oviductal epithelial cells from the FVB background were hormone starved for 48 hours (with a media change after 24 hours), then treated in triplicate with solvent control (DMSO) (0.1%), 1 nM 17-betaestradiol or 100 nM 4-hydroxytamoxifen for 24 hours. Following treatment, RNA was was isolated, libraries were prepped and sequenced using the Illumina HiSeq 2500 platform.

Project description:Ticks are among the most economically important arthropod parasites due to the heavy infestations they provoke, affecting human and animal health and food production. Ticks are also important vectors of pathogens, mediating the dispersion of livestock and zoonotic diseases. Some examples of livestock diseases vectored by ticks include bovine babesiosis, anaplasmosis, theileriosis, and/or heartwater disease, all of which are enlisted by the World Organization for Animals as possible threats to animals and public health. The family Ixodidae (hard ticks) is of veterinary relevance, and many ixodid tick species within the genera Ixodes, Dermacentor, Amblyomma, Haemophysalis, Hyalomma, and Rhipicephalus, among others, impact farming production worldwide. However, there is no doubt that most pernicious tick species is Rhipicephalus microplus, commonly known as the southern cattle fever tick. This specie is not only the major economically important tick parasite for livestock production, but it is also the vector of Babesia bovis and B. bigemina, the causal agents of babesiosis, a severe disease that affects cattle from tropical and subtropical regions. Currently, global economic losses of the cattle industry attributable to R. microplus are estimated at around $18.7 US billion dollars per year. The economic impact is the leading reason to develop different strategies to constrains R. microplus and its vector-borne pathogens, including chemical, mechanical or environmental treatments, biological control, and/or genetic methods. Chemical, among other methods, are the most frequently applied, but the intense use worldwide, has led to the selection of acaricidal-resistant ticks populations. The molecular adaptations of ticks to overcome the effects of acaricides (e.g., deltamethrin, cypermethrin, amitraz, ivermectin) includes metabolic detoxiﬁcation mechanisms and/or the selection of mutated molecular targets. Recently, a couple of systematic reviews pointed out a significant increment in the number of studies intended to understand the acaricidal resistance in ticks. These reports highlighted the acaricide resistance observed in R. microplus and the wide geographical dispersion of this problem. Whereas many acaricide-resistant R. microplus populations have been reported in Brazil, India, and Mexico, the lack of strict regulatory policies on tick control, leaving the application of acaricides at discretion of farmers. Therefore, the correct implementation of existing strategies together with novel sustainable control methods may be crucial to safeguard livestock production in the context of growing population and global warming. However, until the implementation of such sanitary measures becomes the rule, the main control strategy will still rely on the use of chemical acaricides, such as ivermectin (IVM). Ivermectin is an antiparasitic macrocyclic lactone used to control the populations of R. microplus and other ticks in livestock farming. As occurs in other countries, in Mexico ivermectin is the primary synthetic antiparasitic drug, and its abuse during the last three decades represents an important selection pressure that boosted the development of ivermectin-resistant populations of R. microplus. Therefore, the understanding of the molecular basis of the R. microplus resistance to ivermectin, especially in those cases occurring in tropical and subtropical areas of developing countries, can help to underpin the development of novel strategies to tackle this problem. In this context, omics tools can provide helpful information for the discovery of the molecular mechanisms behind the ivermectin resistance in R. microplus and other related tick species. Previous studies identified the main proteomic components of ovaries of R. microplus, including proteins involved in protein synthesis, post-translational modification, nuclear regulation, cytoskeleton, proteasome, transcriptional regulation, energetic metabolism, detoxification metabolism, or energy storage. Recently, the vitellogenin receptor (VgR) was suggested as a target for tick control because of its essential role in egg formation and maturation. However, molecular information on tick ovaries currently available limits explores the feasibility of VgR or other reproduction-related molecules as promissory targets for tick control.

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Exposure of bovine oocytes and embryos to elevated non-esterified fatty acid concentrations: integration of epigenetic and transcriptomic signatures in resultant blastocysts

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