The birth of 'Dolly', the first mammal cloned from an adult donor cell, has sparked a flurry of research activities to improve cloning technology and to understand the underlying mechanism of ...epigenetic reprogramming of the transferred somatic cell nucleus. Especially in ruminants, somatic cell nuclear transfer (SCNT) is frequently associated with pathological changes in the foetal and placental phenotype and has significant consequences for development both before and after birth. The most critical factor is epigenetic reprogramming of the transferred somatic cell nucleus from its differentiated status into the totipotent state of the early embryo. This involves an erasure of the gene expression program of the respective donor cell and the establishment of the well-orchestrated sequence of expression of an estimated number of 10 000-12 000 genes regulating embryonic and foetal development. The following article reviews the present knowledge on the epigenetic reprogramming of the transferred somatic cell nucleus, with emphasis on DNA methylation, imprinting, X-chromosome inactivation and telomere length restoration in bovine development. Additionally, we briefly discuss other approaches towards epigenetic nuclear reprogramming, including the fusion of somatic and embryonic stem cells and the overexpression of genes crucial in the formation and maintenance of the pluripotent status. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realising the great potential of SCNT for basic biological research and for various agricultural and biomedical applications.
•A model of cortisol partitioning which considers two CBG pools is constructed.•A new formula is developed for calculating plasma free cortisol concentration.•iCBG cleavage at sites of inflammation ...increases local free cortisol concentration.•The consideration of eCBG improves the estimation of free cortisol concentration.•No significant difference in the cortisol–protein affinity between clinical groups.
Cortisol bound to corticosteroid binding globulin (CBG) contributes up to 90% of the total cortisol concentration in circulation. Therefore, changes in the binding kinetics of cortisol to CBG can potentially impact on the concentration of free cortisol, the only form that is responsible for the physiological function of the hormone. When CBG is cleaved into elastase-cleaved CBG (eCBG) by the activity of neutrophil elastase, its affinity for cortisol is reduced. Therefore, when eCBG coexists with intact CBG (iCBG) in plasma, the calculation of free cortisol concentration based on the formulae that considers only one CBG pool with the same affinity for cortisol may be inappropriate. In this study, we developed in vivo and in vitro models of cortisol partitioning which considers two CBG pools, iCBG and eCBG, with different affinities for cortisol, and deduce a new formula for calculating plasma free cortisol concentration. The formula provides better estimates of free cortisol concentration than previously used formulae when measurements of the concentrations of the two CBG forms are available. The model can also be used to estimate the affinity of CBG and albumin for cortisol in different clinical groups. We found no significant difference in the estimated affinity of CBG and albumin for cortisol in normal, sepsis and septic shock groups, although free cortisol was higher in sepsis and septic shock groups. The in vivo model also demonstrated that the concentration of interstitial free cortisol is increased locally at a site of inflammation where iCBG is cleaved to form eCBG by the activity of elastase released by neutrophils. This supports the argument that the cleavage of iCBG at sites of inflammation leads to more lower-affinity eCBG and may be a mechanism that permits the local concentration of free cortisol to increase at these sites, while allowing basal free cortisol concentrations at other sites to remain unaffected.
Steroidogenic enzymes can be compartmentalized at different levels, some by virtue of being membrane bound in specific intra-cellular compartments. Although both 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 ...isomerase (3β-HSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) are expressed in the endoplasmic reticulum (ER) membrane, these proteins may still be spatially separated within this membrane system. Side chain cleavage cytochrome P450 (P450scc) is anchored to the inner mitochondrial membrane and this organelle is the major source of pregnenolone (P5) feeding steroidogenesis. Furthermore, steroidogenic enzymes can also be partitioned in different cells. Although well recognized, the effect of enzyme compartmentalization on the rate of steroid production and the balance of different steroids is unclear. This study uses mathematical modeling to investigate the effect of enzyme compartmentalization on steroid synthesis in a human-ovine-bovine model of steroid synthesis. The study shows that the spatial separation of steroidogenic enzymes within the ER has a minimal effect on the rate of steroid synthesis. The compartmentalization of the enzymes into different organelles of a cell creates cellular steroid gradients and can affect the balance of the different steroid products. The partitioning of steroidogenic enzymes in different cells reduces the rate of steroid synthesis. The greater is the distance between the cells that contain different enzymes, the more the rate of steroid synthesis is reduced. Additionally, when 3β-HSD is not in the same cell with P450scc (the P5 source) and P450c17, the ratio of the Δ5-pathway products’ concentrations to the Δ4-pathway products’ concentrations is increased. However, none of these levels of compartmentalization of steroidogenic enzymes alter the qualitative behaviors of steroid synthesis in response to variation in an enzyme activity or P5 supply.
•Reaction-diffusion models of steroid synthesis are developed.•The effects enzyme compartmentalization on steroid synthesis is examined.•Steroid gradients can develop within a cell and affect steroid balance.•Partitioning of enzymes into different cells modulates estrogenic output.•Improved understanding of steroid synthesis in placenta and ovarian follicles.
The cloning of animals by somatic cell nuclear transfer (SCNT) has the potential to allow rapid dissemination of desirable traits from elite animals. However, concern has been expressed that aberrant ...epigenetic marks in SCNT-derived animals may be passed onto the next generation, even though the offspring of clones appear to be mainly normal. Here, we compared the DNA methylation patterns at 10 genomic regions in sperm from SCNT bulls with that from normal, naturally conceived bulls and with the nuclear donor somatic cells. Eight of the 10 genomic regions were differentially methylated in sperm compared with the donor cell DNA. All three satellite sequences examined here were less methylated in sperm than in the donor cells, contradicting the belief that the sperm genome is always highly methylated. The DNA methylation patterns at all 10 regions were almost identical between SCNT and control sperm, with only one out of the 175 CpG sites/groups of sites examined showing significant difference. These results provide the first molecular evidence that the donor cell genome is correctly reprogrammed upon passage through the germ line in males, and that any epigenetic aberrations harbored by SCNT bulls are unlikely to be passed onto their offspring.
► Decreasing 3β-HSD activity to a certain point can increase A4 synthesis. ► Decreasing 3β-HSD activity beyond that point will decrease A4 synthesis. ► Increasing P5 supply rate beyond a certain ...point can suppress the synthesis of A4.
The 3β-hydroxysteroid dehydrogenase/Δ
5–Δ
4 isomerase (3β-HSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) enzymes are important in determining the balance of the synthesis of different steroids such as progesterone (P4), glucocorticoids, androgens, and estrogens. How this is achieved is not a simple matter because each of the two enzymes utilizes more than one substrate and some substrates are shared in common between the two enzymes. The two synthetic pathways, Δ
4 and Δ
5, are interlinked such that it is difficult to predict how the synthesis of each steroid changes when any of the enzyme activities is varied. In addition, the P450c17 enzyme exhibits different substrate specificities among species, particularly with respect to the 17,20-lyase activity. The mathematical model developed in this study simulates the network of reactions catalyzed by 3β-HSD and P450c17 that characterizes steroid synthesis in human, non-human primate, ovine, and bovine species. In these species, P450c17 has negligible 17,20-lyase activity with the Δ
4-steroid 17α-hydroxy-progesterone (17OH-P4); therefore androstenedione (A4) is synthesized efficiently only from dehydroepiandrosterone (DHEA) through the Δ
5 pathway. The model helps to understand the interplay between fluxes through the Δ
4 and Δ
5 pathways in this network, and how this determines the response of steroid synthesis to the variation in 3β-HSD activity or in the supply of the precursor substrate, pregnenolone (P5). The model simulations show that A4 synthesis can change paradoxically when 3β-HSD activity is varied. A decrease in 3β-HSD activity to a certain point can increase A4 synthesis by favouring metabolism through the Δ
5 pathway, though further decrease in 3β-HSD activity beyond that point eventually limits A4 synthesis. The model also showed that due to the competitive inhibition of the enzymes’ activities by substrates and products, increasing the rate of P5 supply above a certain point can suppress the synthesis of A4, DHEA, and 17OH-P4, and consequently drive more P5 towards P4 synthesis.
In a large study in 17 countries, an estimated sodium intake that was either higher or lower than the average estimated sodium intake was associated with an increased risk of cardiovascular events. A ...higher-than-average potassium intake was associated with reduced risk.
Most of the global population consumes between 3.0 and 6.0 g of sodium per day (7.5 to 15.0 g of salt per day).
1
,
2
Guidelines on cardiovascular disease prevention recommend a maximum sodium intake of 1.5 to 2.4 g per day, but achieving this target will require a substantial change in diet for most people.
3
–
5
Although clinical trials have shown a reduction in blood pressure with a reduced sodium intake, to our knowledge, no large randomized trial has been conducted to document reductions in the risk of cardiovascular disease with low sodium intake.
6
Prospective cohort studies have shown inconsistent . . .
Amniotic and allantoic fluid volumes and composition change dynamically throughout gestation. Cattle that are pregnant with
somatic cell nuclear transfer (NT) fetuses show a high incidence of ...abnormal fluid accumulation (particularly hydrallantois)
and fetal mortality from approximately midgestation. To investigate fetal fluid homeostasis in these pregnancies, Na, K, Cl,
urea, creatinine, Ca, Mg, total PO 4 , glucose, fructose, lactate, total protein, and osmolalities were measured in amniotic and allantoic fluids collected at
Days 50, 100, and 150 of gestation from NT pregnancies and those generated by the transfer of in vitro-produced embryos or
by artificial insemination. Deviations in fetal fluid composition between NT and control pregnancies were apparent after placental
and fetal organ development, even when no gross morphological abnormalities were observed. Individual NT fetuses were affected
to varying degrees. Elevated allantoic Na was associated with lower K and increased allantoic fluid volume or edema of the
fetal membranes. Total PO 4 levels in NT allantoic and amniotic fluid were elevated at Days 100 and 150. This was not accompanied by hypophosphatemia
at Day 150, suggesting that PO 4 acquisition by NT fetuses was adequate but that its readsorption by the kidneys may be impaired. Excessive NT placental weight
was associated with low allantoic glucose and fructose as well as high lactate levels. However, the fructogenic ability of
the NT placenta appeared to be normal. The osmolality of the fetal fluids was maintained within a narrow range, suggesting
that the regulation of fluid composition, but not osmolality, was impaired in NT pregnancies.
Abstract
The ability of cloned cattle conceptuses to regulate amniotic and allantoic fluid composition is impaired
Cloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are ...thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI).
Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1) showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns.
These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a good predictor of whether the foetus would develop to term or not.
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