A novel fibroblast‐like cell line RTHDF was established from hypodermal connective tissue of rainbow trout Oncorhynchus mykiss and telomerase activity was demonstrated early and late in cell line ...development. When RTHDF cells were exposed to bioenergetic stress, i.e. anoxia, activation of the stress activated member of the mitogen‐activated protein kinase family, p38MAPK and induction of heat shock protein (Hsp70) were evident. The time‐course of the p38MAPK activation and the induction of Hsp70 expression in RTHDF were studied in response to chemically induced anoxia. p38MAPK was activated rapidly, with maximal activity after 10 min of anoxia. Hsp70 was induced after 30 min of anoxia, followed by overnight recovery in growth medium at 21° C. Using the p38MAPK‐specific inhibitor SB203580, the enhanced expression of Hsp70 occurred independently of p38MAPK activation in RTHDF. These data suggests that RTHDF can be useful in studying biochemical responses of teleost cells to environmental stress.
A continuous cell line, ZEB2, was developed from zebrafish blastula-stage embryos expressing enhanced green fluorescent protein (GFP). Originally the rainbow trout spleen cell line, RTS34st, was used ...as feeders to initiate and maintain the cells through several passages. ZEB2 was then grown for 2 years without feeders in L-15 with 15% fetal bovine serum (FBS) for 120 population doublings. This new cell line, ZEB2J, was heteroploid, had detectable telomerase activity, and was adherent. After growing into monolayers, some cells continued to grow into mounds. Cultures expressed Pou-2 mRNA and contained many alkaline phosphatase and a few stage-specific embryonic antigen-1-positive cells. In dishes coated with a phospholipid polymer (2-methacryloxyloxyethyl phosphorylcholine, MPC), ZEB2J formed spherical aggregates. Aggregates attached to conventional culture plastic, and most cells that emerged from aggregates had typical epithelial-like shapes of ZEB2J, which suggests that ZEB2J had limited differentiation potential, despite expressing some stem cell properties. The fluorescence of ZEB2J allowed relationships with feeder cells to be studied. In MPC dishes, ZEB2J formed mixed spheroids with RTS34st. In adherent cocultures, RTS34st and other fish cell lines strongly stimulated the ZEB2J growth, which could be quantified specifically because ZEB2J expressed GFP. ZEB2J should be useful for optimizing culture conditions for zebrafish embryonic stem cells.
Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have ...been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
STUDY QUESTION
Is the metabolic composition of the follicular fluid of women undergoing assisted reproductive treatment (ART) related to serum composition and BMI and is it associated with oocyte and ...embryo quality?
SUMMARY ANSWER
We showed that metabolic alterations in the serum are reflected in the follicular fluid and that some of these alterations may affect oocyte quality, irrespective of BMI.
WHAT IS KNOWN ALREADY
Many studies have focused on the effect of metabolic disorders, such as obesity and type 2 diabetes, on assisted reproduction outcomes. There are, however, only few studies focusing on the importance of the correlation between serum and follicular fluid compositions and the composition of the follicular fluid as the oocyte's micro-environment, affecting its development and subsequent embryo quality.
DESIGN, PARTICIPANTS AND SETTING
In this prospective cohort study, patient information, fertility treatment outcome data, follicular fluid and serum were obtained from women undergoing ART. Patients were categorized according to their BMI (kg/m2) as normal (n = 60), overweight (n = 26) or obese (n = 20). Serum and follicular fluid samples were analyzed for urea, total protein, albumin, cholesterol, high-density lipoprotein cholesterol, triglycerides, non-esterified fatty acids, apolipoprotein A1, apolipoprotein B, glucose, lactate, C-reactive protein, insulin-like growth factor -1 (IGF-1), IGF-binding protein 3 (only in follicular fluid), free carnitine and total carnitine. Metabolite concentrations in serum and follicular fluid samples were correlated and were associated with BMI and fertility treatment outcome.
MAIN RESULTS
Most serum metabolite differences between patients were reflected in the follicular fluid (P < 0.05). Follicular fluid apolipoprotein A1 and follicular fluid total protein concentrations negatively affected oocyte quality parameters (P < 0.05). However, overall BMI-related associations were poor.
BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION
In this study, we included every patient willing to participate. Within this cohort, women with a BMI transcending 35 kg/m2 were scarce (n = 2), because extremely overweight women are mostly advised to lose weight before starting ART. Furthermore, the number of patients in each BMI group was different, possibly masking associations between the metabolic composition of serum and follicular fluid and oocyte quality parameters.
GENERALIZABILITY TO OTHER POPULATIONS
There were significant associations indicating that metabolic changes in the serum are reflected in the follicular fluid, potentially affecting oocyte quality, irrespective of the patient's BMI. For ethical reasons, this study only focused on women already in need of artificial reproductive treatment. From a metabolic point of view, we consider this cohort as a representative sample of all women of reproductive age.
STUDY FUNDING
This study was funded by the special research fund, university of Antwerp (BOF UA). None of the authors has any conflict of interest to declare.
Maternal obesity can cause reduced oocyte quality and subfertility. Mitochondrial dysfunction plays a central role here, and most often inbred mouse models are used to study these pathways. We ...hypothesized that the mouse genetic background can influence the impact of high fat diet (HFD)-induced obesity on oocyte quality. We compared the inbred C57BL/6 (B6) and the outbred Swiss strains after feeding a HFD for 13w. HFD-mice had increased body weight gain, hypercholesterolemia, and increased oocyte lipid droplet (LD) accumulation in both strains. LD distribution was strain-dependent. In Swiss mouse oocytes, HFD significantly increased mitochondrial inner membrane potential (MMP), reactive oxygen species concentrations, mitochondrial ultrastructural abnormalities (by 46.4%), and endoplasmic reticulum (ER) swelling, and decreased mtDNA copy numbers compared with Swiss controls (P < 0.05). Surprisingly, B6-control oocytes exhibited signs of cellular stress compared to the Swiss controls (P < 0.05); upregulated gene expression of ER- and oxidative stress markers, high mitochondrial ultrastructural abnormalities (48.6%) and ER swelling. Consequently, the HFD impact on B6 oocyte quality was less obvious, with 9% higher mitochondrial abnormalities, and no additive effect on MMP and stress marks compared to B6 control (P > 0.1). Interestingly, mtDNA in B6-HFD oocytes was increased suggesting defective mitophagy. In conclusion, we show evidence that the genetic background or inbreeding can affect mitochondrial functions in oocytes and may influence the impact of HFD on oocyte quality. These results should create awareness when choosing and interpreting data obtained from different mouse models before extrapolating to human applications.
Maternal lipolytic metabolic disorders result in a lipotoxic microenvironment in the ovarian follicular fluid (FF) which deteriorates oocyte quality. Although cellular stress response mechanisms are ...well defined in somatic cells, they remain largely unexplored in oocytes, which have distinct organelle structure and nuclear transcription patterns. Here we used shotgun proteomic analyses to study cellular responses of bovine oocytes and cumulus cells (CCs) after in vitro maturation under lipotoxic conditions; in the presence of pathophysiological palmitic acid (PA) concentration as a model. Differentially regulated proteins (DRPs) were mainly localized in the endoplasmic reticulum, mitochondria and nuclei of CCs and oocytes, however the DRPs and their direction of change were cell-type specific. Proteomic changes in PA-exposed CCs were predominantly pro-apoptotic unfolded protein responses (UPRs), mitochondrial and metabolic dysfunctions, and apoptotic pathways. This was also functionally confirmed. Interestingly, although the oocytes were enclosed by CCs during PA exposure, elevated cellular stress levels were also evident. However, pro-survival UPRs, redox regulatory and compensatory metabolic mechanisms were prominent despite evidence of mitochondrial dysfunction, oxidative stress, and reduced subsequent embryo development. The data provides a unique insight that enriches the understanding of the cellular stress responses in metabolically-compromised oocytes and forms a fundamental base to identify new targets for fertility treatments as discussed within.
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, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts.
Bovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological ("BASAL") concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological ("HIGH COMBI") 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. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to 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 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 adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders.
Overall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
It has been well documented that the maturing oocyte is very vulnerable to changes in its micro-environment, the follicular fluid (FF). Recent research has focused on different components within this ...FF, like hormones, growth factors and metabolites, and how their concentrations are altered by diet and the metabolic health of the mother. It has been proposed that fatty acids (FAs) are potential factors that influence oocyte maturation and subsequent embryo development. However, a thorough study of the specific FF FA composition per lipid fraction and how this may be affected by BMI is currently lacking. Therefore, we investigated the BMI-related concentration of FAs in the phospholipid (PL), cholesteryl-ester (CHE), triglyceride (TG) and non-esterified (NE) lipid fraction in the FF of women undergoing assisted reproductive treatment (ART).
In this descriptive cross-sectional study, the FF of normal weight (18.5 ≤ BMI < 25.0 kg/m(2), n = 10), overweight (25.0 ≤ BMI < 30.0 kg/m(2), n = 10) and obese (BMI ≥ 30.0 kg/m(2), n = 10) women, undergoing ART, was sampled and analyzed for 23 specific FAs in the PL, CHE, TG and NEFA fraction, using a gas chromatographic analysis method. Differences between BMI-groups were studied by means of univariate general linear models and post hoc Sheffé tests.
Total FA concentrations in the PL and CHE fraction did not differ between BMI groups. Total TG concentrations tended to differ and total NEFA concentrations differed significantly between BMI groups. Interestingly, 42% and 34% of the total FAs was esterified in the PL and CHE fraction, respectively, while only 10% were present in both the TG and NEFA fraction. Only few individual FA concentrations differed in the PL, CHE and TG fraction between BMI groups, whereas abundant BMI-related differences were found in the NEFA fraction.
Our data show that differences in BMI are associated with alterations in the FA composition of the FF, an effect most pronounced in the NEFA fraction. These BMI-related variations could possibly affect granulosa cell viability, oocyte developmental competence and subsequent embryo quality possibly explaining differences in oocyte quality in obese patients described by others.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Jet flavour classification is of paramount importance for a broad range of applications in modern-day high-energy-physics experiments, particularly at the LHC. In this paper we propose a novel ...architecture for this task that exploits modern deep learning techniques. This new model, called DeepJet, overcomes the limitations in input size that affected previous approaches. As a result, the heavy flavour classification performance improves, and the model is extended to also perform quark-gluon tagging.
Increased global industrial activity has exposed humans to a wide variety of chemical substances some of which, called ‘endocrine-disrupting chemicals’ (EDCs) or ‘endocrine disruptors’, can disrupt ...the endocrine system in the body. The ovarian follicle is a very fragile micro-environment where interactions between hormones, growth factors, the oocyte and its surrounding somatic cells are essential to generate a fully competent oocyte. In vitro experiments suggest that EDCs can disturb this finely tuned balance, but very scarse in vivo data are available to confirm this assumption. Therefore, we have investigated if the presence of EDCs in human follicular fluid is a risk factor for the developmental competence of an in vivo exposed oocyte. Furthermore, because of the limited access to human follicular fluid, we verified if follicular fluid contamination can be predicted based on EDC levels in serum.
METHODS
Follicular fluid (n = 40) and serum (n = 20) samples from women undergoing assisted reproductive technology (ART) were analyzed by means of gas chromatography combined with mass spectrometry to examine the presence of different EDCs, such as polychlorinated biphenyls, polybrominated diphenyl ethers and organochlorine pesticides. Statistical models were used to investigate the relation between the characteristics and ART results of the patients and the contamination status of their follicular fluid and to assess the capacity of serum samples to predict follicular fluid contamination.
RESULTS
Chlorinated biphenyl 153 (72 ± 44 and 201 ± 106 pg/ml) and p,p′-DDE (392 ± 348 and 622 ± 406 pg/ml) were the compounds found in the highest concentrations in follicular fluid and serum samples, respectively. A new variable principal component 1, representing the overall contamination status of the follicular fluid samples, is strongly associated with fertilization rate (P < 0.00001) and the proportion of high-quality embryos relative to the amount of retrieved oocytes (P < 0.05), even when the analysis is adjusted for age, estradiol concentration, BMI, fertilization procedure and male subfertility as explanatory variables. The strong correlations between the EDC concentrations in serum and follicular fluid (r ≥ 0.93) allowed us to build regression models, which accurately predict EDC concentrations in the follicular fluid based on serum samples.
CONCLUSIONS
An overall higher EDC contamination in the follicular micro-environment was associated with a decreased fertilization rate and consequently with a lower chance of an oocyte to develop into a high-quality embryo. In addition, EDC concentrations in serum were reliable predictors of the contamination status of the follicular micro-environment.
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