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.

DNA methylation pattern of bovine blastocysts associated with hyperinsulinemia in vitro.

Insulin functions as a regulator of metabolism and plays an important role in reproduction. Hyperinsulinemia is often observed in patients with obesity and diabetes type 2 and is known to impair fertility, but the underlying molecular mechanisms are only partly understood. Metabolic programming through epigenetic mechanisms such as DNA methylation during embryonic development can lead to health implications for the offspring later in life. Our aim was to study the potential effect of hyperinsulinemia on gene expression and DNA methylation of embryos by adding insulin (0.1 µg/ml = INS0.1 or 10 µg/ml = INS10) during in vitro oocyte maturation by using the EmbryoGENE DNA methylation array for a study of the bovine epigenome. Our results showed significant differences between blastocysts originating from insulin-treated oocytes compared with untreated control blastocysts. In total, 13,658 and 12,418 probes were differentially methylated (DM) in INS0.1 and INS10, respectively, with an overlap of 3,233 probes in the DM regions (DMR) for both insulin groups. Genes related to pathways such as lipid metabolism, growth and proliferation, mitochondrial function, and oxidative stress responses were influenced at both the epigenetic and transcriptomic levels. In addition, imprinted genes and genes with functions in the epigenetic machinery were among the DMRs. This study identified DMRs correlated to differential expression of genes involved in metabolic regulation and should help to improve our knowledge of the underlying molecular mechanisms of metabolic imbalance.

Lipid profile of bovine blastocysts exposed to insulin during in vitro oocyte maturation.

Insulin is a key hormone with important functions in energy metabolism and is involved in the regulation of reproduction. Hyperinsulinaemia is known to impair fertility (for example, in obese mothers); therefore, we aimed to investigate the impact of elevated insulin concentrations during the sensitive period of oocyte maturation on gene expression and lipid profiles of the bovine Day-8 embryo. Two different insulin concentrations were used during in vitro oocyte maturation (INS10=10µgmL-1 and INS0.1=0.1µgmL-1) in order to observe possible dose-dependent effects or thresholds for hyperinsulinaemia in vitro. By investigating gene expression patterns by an mRNA microarray in combination with lipid profile analysis by desorption electrospray ionisation-mass spectrometry (DESI-MS) of embryos derived from insulin-treated oocytes, we gained further insights regarding molecular responses of embryos to insulin provocation during the first days of development. Lipid metabolism appeared to be influenced on multiple levels according to gene expression results but the profiles collected in positive-ion mode by DESI-MS (showing mostly ubiquinone, cholesteryl esters and triacylglycerols) did not differ significantly from controls. There are parallels in follicular development of ruminants and humans that make this bovine model relevant for comparative research on early human embryonic development during hyperinsulinaemia.

Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts.

Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL-1 and 0.1 µg mL-1), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation (ATP5D; CYP11A1; NDUFB7; NDUFB10; NDUFS8). Taken together, we hypothesise that the increased proliferation in the insulin-treated groups might impair the developmental potential of the embryos by inducing metabolic stress on the molecular level, which could be detrimental for the survival of the embryo.

Insulin concentrations used in in vitro embryo production systems: a pilot study on insulin stability with an emphasis on concentrations measured in vivo.

BACKGROUND: Insulin has been used as a stimulatory factor for in vitro cell culture since many years. Even for routine in vitro embryo production (IVP), insulin is added to the media during different steps. There is a strong difference in concentrations used in vitro compared to what is measured in vivo in follicular fluid or serum. We performed a pilot study on insulin stability to explain possible reasons for that variation. RESULTS: We measured insulin concentrations before and after bovine oocyte maturation in an experiment by using a quantitative ELISA (Mercodia bovine insulin ELISA immunoassay) and found that concentrations were stable up to 22 h of incubation. We compared our results with eleven in vivo studies measuring insulin in either serum or follicular fluid and nine IVP-protocols using insulin. In all studies, in vitro concentrations were much higher compared with those found physiologically in vivo. Limited knowledge is available concerning the different activity and stability of insulin in vitro versus in vivo. CONCLUSIONS: The concentrations of insulin used in vitro are quite high in comparison to physiological concentrations found in serum or follicular fluid. One explanation may be a different stability or activity of insulin in vitro even if we could measure stable concentrations of insulin in our pilot study. More precise dose-effect studies have to be performed to draw clear conclusions about the consequences of the use of such high doses as they might have negative consequences for the developing embryo. Insulin has direct effects on the regulation of the metabolism and could even influence the epigenetic programming of the metabolism with unknown consequences for the offspring later in life.

Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential.

Metabolic imbalance impairs fertility, because changes in concentrations of metabolites and hormones in the blood and follicular fluid create an unfavourable environment for early embryonic development. Insulin is a key metabolic hormone known for its effects on fertility: insulin concentrations are increased during energy balance disturbances in diabetes or metabolic syndrome. Still, insulin is frequently used at supraphysiological concentrations for embryo in vitro culture with unknown consequences for the developmental potential of the offspring. In the present study we investigated the effects of insulin exposure during in vitro bovine oocyte maturation on developmental rates, embryo quality and gene expression. Supplementation of the maturation media with insulin at 10 or 0.1 µg mL-1 decreased blastocyst rates compared with an insulin-free control (19.8 ± 1.3% and 20.4 ± 1.3% vs 23.8 ± 1.3%, respectively; P < 0.05) and led to increased cell numbers (nearly 10% more cells on Day 8 compared with control; P < 0.05). Transcriptome analysis revealed significant upregulation of genes involved in lipid metabolism, nuclear factor (erythroid-derived 2)-like 2 (NRF2) stress response and cell differentiation, validated by quantitative polymerase chain reaction. To conclude, the results of the present study demonstrate that insulin exposure during in vitro oocyte maturation has a lasting effect on the embryo until the blastocyst stage, with a potential negative effect in the form of specific gene expression perturbations.