Increased uterine uptake and nuclear retention of [3H]oestradiol through the oestrous cycle and enhanced end-organ sensitivity to oestrogen stimulation in vitamin B6 deficient rats

Vitamin B6 deficient female rats showed a significantly earlier, greater and more prolonged uptake of a tracer dose of [3H]oestradiol into the uterus, with increased nuclear accumulation, compared with vitamin B6 supplemented animals. This was most marked at oestrus, with little difference at anoestrus. The responses to low doses of ethynyl-oestradiol were greater in ovariectomized deficient animals than in those receiving the supplemented diet, with an increased uterotrophic response and greater induction of peroxidase. In the deficient animals there was virtually complete suppression of LH secretion at doses of ethynyl-oestradiol that had no effect in controls. At high doses of ethynyl-oestradiol there was no difference between the two groups of animals. The results suggest that increased uterine uptake and accumulation of [3H]oestradiol in vitamin B6 deficiency is associated with enhanced end-organ responsiveness to sub-maximal oestrogen stimulation, and that pyridoxal phosphate may have a coenzyme role in oestrogen action.     

Differential effects of oestrogen on developing and mature uterine sympathetic nerves

Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.     

Differential responsiveness to oxytocin of the uterus and cervix in the ovariectomized ewe

The responsiveness of the uterus and cervix to oxytocin was compared in ovariectomized ewes fitted with intraparietal electrodes and treated with 17β-oestradiol. Before the injection of the steroid, only the cervix presented dose-related responses to oxytocin infusions. Within the 3–5-day period after oestrogen injection, both the uterus and cervix presented almost similar responsiveness to the neurohormonal agent. After the 6th day following oestrogen, the cervix remained reactive to the infusions of oxytocin, whereas the uterus failed to respond. It is suggested that the reactivity of the cervix, which is at least partially independent of oestrogen priming, may be due to oestrogen-independent oxytocin receptors present only in the cervix.     

Changes of oestrogen receptor levels in Leydig cells from mice and rats during culture

Two functional properties of Leydig cells in culture, i.e. LH-stimulated steroidogenesis and nuclear oestrogen receptor levels have been investigated. Leydig cells isolated from testes of immature rats and mature mice maintained their responsiveness to LH during 48-72 h of cell culture, although the mouse Leydig cells appeared to be less responsive to LH after 72 h of culture. In contrast, nuclear oestrogen receptor levels in both types of Leydig cells declined to 10-20% of the initial value after 24 h in culture. In the 48-72 h culture period nuclear oestrogen receptor levels recovered to 75% of the initial value only in Leydig cells from immature rats, whereas the nuclear oestrogen receptor levels in Leydig cells from mature mice remained low. These data demonstrate that during in vitro culture of Leydig cells, preservation of LH responsiveness does not necessarily warrant that other Leydig cell parameters e.g. nuclear oestrogen receptors also remain unaltered.     

Effects of low-zinc status and essential fatty acid deficiency on bone development and mineralization

1. The effects of essential fatty acids (EFAs) and zinc on the development and mineralization of bones were studied in young growing rats. 2. Female weaning rats were maintained on the diets deficient in EFAs, low in zinc (6 ppm) or both deficient in EFAs and low in zinc. 3. The low-zinc status accentuated signs of EFA deficiency including reduction of the growth rate and weights of bones and resulted in greater incidences of dental caries. 4. There were qualitative and quantitative differences in the fatty acid components of lipids extracted from the femur of the rats. 5. The overall effect was that eicosatrienoic (C20:3) and arachidonic (C20:4) acids were accumulated in EFA deficiency and low-zinc state respectively. 6. Bones of rats fed a low-zinc diet containing no EFAs were totally hypomineralized while those maintained on a diet that was either low in zinc or deficient in EFAs was partially hypomineralized. 7. Dietary zinc may have some roles to play in the biosynthesis of prostaglandins from EFAs and the process of bone mineralization.     

Effects of fasting on the distribution of cytoplasmic and nuclear oestrogen receptors in rat liver, uterus, pituitary and hypothalamus before and after exogenous oestrogen administration

The accumulation of oestrogen receptors in the liver cell nuclei of intact female rats 45 min after administration of 100 micrograms 17 alpha-ethynyloestradiol-17 beta i.p., decreased progressively during a 72-h fast from 2550 +/- 860 to 257 +/- 67 fmol/mg DNA, a level not significantly different from that in uninjected animals. Cytoplasmic oestrogen receptor concentrations also decreased, but only to about 60% of the original level (from 84.1 +/- 27.5 to 50.3 +/- 2.09 fmol/mg protein during the fast). Similar differences were found when these parameters were examined in normally fed and 72-h-fasted ovariectomized rats. On the other hand these parameters were unaffected in uterus, pituitary and hypothalamus. Uterine cytoplasmic receptor concentrations remained at about 500 fmol/mg protein during the fasting period, those in the pituitary and hypothalamus at about 230 and 30 fmol/mg protein, respectively. Nor was in vivo translocation in these organs affected by fasting. Regardless of nutritional status, the nuclear oestrogen receptor concentrations in uterus rose from about 500 to 2000 fmol/mg DNA after ethynyloestradiol administration, those in the pituitary and hypothalamus from approximately 250 to 2000 and from 250 to 500 fmol/mg DNA respectively.     

The relationship of the oestrogen and progestin receptors in the abnormal uterus of the adult anovulatory rat. Effects of neonatal treatment with testosterone propionate or clomiphene citrate.

The neonatal administration of testosterone propionate to Wistar rats resulted in anovulatory adults in persistent vaginal oestrus. Clomiphene citrate had a similar effect. In both groups of adults, hyperplasia of the uterine epithelium and occasional metaplasia was observed. The uterine nuclear and cytosol oestrogen and progestin receptors of these anovulatory rats were found to have affinities for their respective ligands similar to those of normal females. The nuclear oestrogen receptor comprised occupied and unoccupied components, as in normal females. The content of the nuclear oestrogen receptor was comparable with that of females in the late dioestrous or pro-oestrous phase. This content was higher in the clomiphene-treated group. Despite the relatively high nuclear oestrogen receptor content the content of progestin receptors, a putative index of the oestrogenic response, was lower in the treated rats than in normal adult females throughout the cycle. Administration of oestradiol to both treatment groups resulted in depletion of cytosol oestrogen receptor content 1 h later, which, however, was not reflected by an increase in the content of nuclear oestrogen receptors. There was no measurable increase in progesterone receptor content in treated rats after daily administration of oestrogen (5 microgram/rat) for 3 days. These changes in sex-hormone-receptor interactions involving an impairment of the normal oestrogenic response may be associated with the abnormal differentiation of the uterus in these sterile, anovulatory animals.     

Replenishment and nuclear retention of oestradiol-17 beta receptors in rat uteri during postnatal development.

1. Uteri of 6--10-day-old rats do not show a late growth response to oestrogen (increase in rate of DNA synthesis and cell division) exhibited by fully competent (20 days or older) uteri. We posed the question whether the lack of the late growth response is due to an inability to replenish the cytoplasmic pool of oestrogen receptors or to curtailed retention of oestrogen binding in the nucleus. Uterine nuclear and cytoplasmic receptors were measured by a [3H]oestradiol-17 beta exchange assay, at 1, 3, 6, 14 and 24 h after oestrogen injection. 2. The replenishment of cytoplasmic oestrogen receptors showed a similar pattern in the uteri of 6 and 10-day-old (partially responsive) and in 20-day-old (fully responsive) rats. 3. Oestrogen was retained longer in uterine nuclei obtained from 5 and 10-day-old rats than in uterine nuclei of 20 and 25-day-old rats. 4. Oestrogen receptors resistant to 0.4 M KCl extraction (residual receptors) were found in uterine nuclei of 6 and 25-day-old rats after oestrogen injection at all the times tested. The concentration of these residual receptors during the late period (6--24 h after injection) was not significantly different in uterine nuclei of 6-day-old and 25-day-old rats. 5. We conclude that neither lack of oestrogen receptor replenishment nor curtailed retention of oestrogen binding in the nucleus is the factor which limits the complete responsiveness to oestrogen in uteri of rats during postnatal development.     

Inhibition of non-genomic responses to oestrogen in the rat uterus by testosterone propionate

Testosterone propionate (50 mg/kg), administered together with oestradiol, inhibited the oestrogen-induced uterine eosinophilia, deep endometrial oedema and the increase in uterine wet weight, 6 h after treatment. The same dose of the androgen decreased the number of eosinophils in the blood and increased their degranulation, explaining the effect of testosterone in the uterus. The high doses of the androgen used were in the range of the doses reported by others to block selectively the oestrogen-induced increase in uterine peroxidase content but not other responses to oestrogen or the cytosolic oestrogen receptor translocation to the nucleus. The dissociation by high doses of testosterone of the oestrogen-induced uterine eosinophilia, wet weight increase and oedema from other responses to oestrogen in the absence of any measurable effect of testosterone upon cytosolic-nuclear oestrogen receptors supports the idea that uterine eosinophilia and oedema are oestrogenic responses regulated by mechanisms different from those of the genomic responses, and is in agreement with the hypothesis of the mediation of uterine oedema by eosinophils.     

The effects of oestrogens and progesterone on oestrogen receptors in female rat liver.

The administration of oestradiol-17 beta or ethynyloestradiol as well as the synthetic progestogen norethisterone acetate resulted in translocation of the oestrogen receptor. Progesterone and the synthetic progestogen (+)-norgestrel were ineffective. The increases in nuclear oestrogen receptor content 1 h after injection of each steroid were similar but different subsequently. The increase with oestradiol-17 beta extended for 3--6 h and for at least 9 h with ethynyloestradiol. With norethisterone acetate, nuclear content was still increased after 24 h. Oestrogen injection resulted in cytosol receptor depletion and a 'deficit' in receptor content extending for 6 h, whereas norethisterone acetate-induced translocation was quantitative. With injections of norethisterone acetate + ethynyloestradiol the increase at 1 h and retention of the nuclear receptors were similar to that with norethisterone acetate alone. In contrast, the depletion of cytosol receptor and its restoration were similar to that seen with ethynyloestradiol alone, suggesting that norethisterone acetate did not interfere with the oestrogen receptor replenishment. Specific binding in vitro of [3H]oestradiol-17 beta in liver cytosols was inhibited by (+)-norgestrel and norethisterone acetate, but not progesterone, at concentrations of 10--100 microM. Nuclear receptors present after norethisterone acetate injection bound oestrogen with high affinity (Kd = 1.52 nM), similar to receptors of oestrogen-injected animals. In the uterus, differential retention of nuclear receptors in response to oestrogens is associated with different cellular responses. The differences in the response of the receptor system in liver to the various steroids suggests that the corresponding tissue responses may also be dissimilar. These results are discussed in relation to the problems of liver dysfunction in oral-contraceptive users.     

Essential fatty acids in tissue phospholipids and triglycerides of the zinc-deficient rat

This study addressed the possibility that zinc deficiency has different effects on the fatty acid composition of triglyceride compared to total phospholipid. Male weanling Sprague-Dawley rats were maintained for 6 weeks on a semisynthetic diet deficient in zinc (3 mg/kg zinc). Control rats (40 mg/kg zinc) were pair-fed. Lipid fractionation and fatty acid analysis were by thin-layer and gas chromatography, respectively. In zinc-deficient rats, the percentage of linoleic acid was increased or that of arachidonic acid was decreased in total phospholipids of plasma, liver, and testis, and in skin total lipids. Saturated and monounsaturated fatty acids were increased in the triglyceride of liver but decreased in the triglyceride of epididymal fat of zinc deficient rats. Essential fatty acids, as a proportion of total fatty acids, were decreased in triglyceride of liver but increased in triglyceride of epididymal fat of zinc-deficient rats. Our fatty acid data from tissue total phospholipids therefore support the concept that linoleic acid desaturation is impaired in zinc deficiency.     

Effects of oestrogen on adenylate cyclase system and glucose output in rat liver.

Effects of chronic oestrogen treatment on catecholamine- and glucagon-sensitive adenylate cyclase activity and glucose output in hepatocytes of castrated male rats were studied. In hepatocytes from male intact or castrated rats, the beta-adrenergic agonist isoprenaline did not stimulate adenylate cyclase activity and glycogenolysis, but glucagon markedly stimulated all these activities. Treatment of castrated animals with 17 beta-oestradiol for 7 days led to the appearance of beta-adrenergic-stimulated increases in both cyclic AMP generation and glucose output. The basal, glucagon- or fluoride-stimulated activities of adenylate cyclase of hepatic membranes prepared from oestrogen-treated rats were similar to those of control animals. Treatment with oestrogen did not influence the number or affinity of beta-adrenergic receptors. In hepatic plasma membranes from control rats, GTP failed to decrease the affinity of beta-adrenergic receptors for agonists, whereas the GTP-induced shift was apparently observed in those from oestrogen-treated animals. These results suggest that oestrogen is able to facilitate the coupling of hepatic beta-adrenergic receptors to the enzyme by increasing the effectiveness of receptor-guanine nucleotide regulation.     

Effect of median raphe nucleus electrocoagulation and serotonin synthesis blockade on oestrogen receptor content of rat uterus

The number of oestrogen receptors (OR) in rat uterus changes in relation to the cyclic function of the ovary. In the dioestrus phase the OR content of rat uterus is significantly greater than in the oestrus phase. Electrocoagulation of the median raphe nucleus causing in rats increased release of gonadotrophins and consequent disturbances in the cyclic ovarian function is associated also with changes in OR content of the uterus. These changes are, however, not analogous to those observed in the normal sexual cycle. In the oestrus phase a very high accumulation of OR is found in the uterus, while in the dioestrus phase OR are barely detectable. Serotonin synthesis blockade with p-CPA, which is associated with an increase in the number of maturating and mature follicles in the ovary and dioestrus phase prolongation leads to a striking increase in the number of OR in the uterus.     

Intracellular relationships of the oestrogen receptor in the rat uterus and hypothalamus during the oestrous cycle.

Simultaneous measurements were made of the specific oestrogen receptor in the nuclear and cytosol fractions prepared from the uterus and hypothalamus of 50--81-day-old female rats undergoing a 4-day oestrous cycle. In the uterus, the content of nuclear receptor fluctuated in concert with known cyclic changes in the secretion of oestrogen, being maximal at pro-oestrus. Over the period of 50--81 days, the nuclear content at all phases increased with age, again corresponding to known age-related increases in ovarian secretion of oestrogen. This age-related increase in nuclear content, averaged from the values of the different phases in each age group, was related to equivalent increases in uterine wet weight, an increase of 1 pmol of receptor being accompanied by an increase of 80--90 mg. The concentration of cytosol receptor was maintained constant, with respect to wet weight, throughout the cycle and with age, irrespective of changes in nuclear content. In the uterus of normal mature females, translocation of receptor into the nucleus did not lead to depletion of cytosol receptor, suggesting a process of continuous replenishment/synthesis. In the hypothalamus, the nuclear content of oestrogen receptor was also maximal at pro-oestrus. In contrast with the uterus, the content of hypothalamic cytosol receptor was minimal at this phase and reflects depletion of the cytosol receptor, possibly as a result of translocation. The extent of translocation was low compared with that in the uterus and did not alter with age during the age-period studied. This low nuclear binding of the receptor in vivo is discussed in relation to the presence of a cytosol factor, present in limiting amounts, which in vitro mediates the binding of cytosol receptor to oligo(dT)-cellulose. The difference in the physiological response of the uterus and of the hypothalamus to oestrogens may be related to the extent of nuclear binding of receptor.     

Activities of liver microsomal fatty acid desaturases in zinc-deficient rats force-fed diets with a coconut oil/safflower oil mixture of linseed oil

The present study was conducted to investigate the effect of zinc deficiency on fatty acid desaturation in rats fed two different types of dietary fat, a mixture of coconut oil and safflower oil (7∶1, w/w, “coconut oil diet”) or linseed oil (“linseed oil diet”). In order to ensure an adequate food intake, all rats were force-fed by gastric tube. Zinc deficiency caused statistical significant reducion of Δ9-desaturase activity in liver microsomes of rats fed coconut oil diet and tendencial reduction (p<0.15) in rats fed linseed oil diet compared with control rats fed diets with the same type of fat. In agreement with this effect, zinc deficiency in the rats fed both types of dietary fat increased the ratio between total saturated and total monounsaturated fatty in liver phospholipids and liver microsomes. Zinc deficient rats on the coconut oil diet had unchanged Δ6-desaturase activity with linoleic acid as substrate and lowered activity with α-linolenic acid as substrate. In contrast, zinc deficient rats on the linseed oil diet had increased Δ6-desaturase activity with linoleic acid as substrate and unchanged activity with α-linolenic acid. Because linoleic acid is the main substrate for Δ6-desaturase in the rats fed coconut oil diet, and α-linolenic acid is the main substrate in the rats fed linseed oil diet, it is concluded that in vivo Δ6-desaturation was not changed by zinc deficiency in the rats fed both types of dietary fat. Activity of Δ5-desaturase was also not changed by zinc deficiency in the rats fed both dietary fats. Levels of fatty acids in liver phospholipids and microsomes derived by Δ4-, Δ5-, and Δ6-desaturation were not consistently changed by zinc deficiency in the rats fed both types of dietary fat. Thus, the enzyme studies and also fatty acid composition data of liver phospholipids and microsomes indicate that zinc deficiency does not considerably disturb desaturation of linoleic and α-linolenic acid. Therefore, it is suggested that similarities between deficiencies of zinc and essential fatty acids described in literature are not due to disturbed desaturation of linoleic acid in zinc deficiency. The present study also indicates that zinc deficiency enhances incorporation of eicosapentaenoic acid into phosphatidylcholine of rats fed diets with large amounts ofn-3 polyunsaturated fatty acids.     

Aorta protein networks in marginal and acute zinc deficiency

Human zinc deficiency is a global problem and may influence the development of cardiovascular disease. Our objective was to determine Zn deficiency affected pathways and protein interactions in rat aorta and their likely influence on stress-induced atherogenesis. In two separate studies, rats were given diets acutely (<1 mg Zn/kg) or marginally (6 mg Zn/kg) deficient in Zn. Both studies included Zn adequate controls (35 mg Zn/kg) and the acute deficiency study included a pair-fed group. After 6 wk, proteins from thoracic aorta were separated by 2-DE. Proteins affected by zinc deficiency were identified by principal component analysis. Multiple correlations of identified proteins indicated protein networks of related function. Proteins clusters decreased in zinc deficiency were related to fatty acid and carbohydrate metabolism. Structurally related proteins, including zyxin and over nine transgelin 1 proteins, were either increased or decreased by acute and marginal deficiencies. PKC alpha was significantly decreased in Zn deficiency suggesting that Zn may regulate the phosphorylation of target proteins. Zn deficiency-related changes in structural, carbohydrate and fatty acid-related proteins may be disadvantageous for maintaining vascular health and are consistent with a protective role for zinc in the development of atherosclerosis.     

Ultrastructural studies on paneth cell apoptosis in zinc deficient rats

Summary An ultrastructural study into the origins of the increased number of apoptotic bodies in the small intestinal crypts of zinc deficient rats was carried out. Two strains of rat were used and each strain was sub-divided into three groups; zinc deficient, pair-fed controls and ad libitum controls. All three groups of one strain were heavily infested with intestinal parasites, both bacteria and flagellated protozoa. Increased numbers of apoptotic bodies were found in the upper crypt/villus region of zinc deficient rats in both the parasitized and parasite free strains. Some of these apoptotic bodies contained structures resembling the electron lucent intestinal epithelial cells found in zinc deficient rats, others contained unidentifiable cell remnants that had undergone advanced degenerative changes. In zinc deficient parasitized rats only, apoptotic bodies were found at the crypt base which contained identifiable remnants of Paneth cells. The majority of these had been ingested by intestinal epithelial cells but some had been ingested by macrophages. The effect of zinc deficiency and parasitic infestation on apoptosis is discussed.J.G. Jones was supported by funds from the Welsh Scheme for the Development of Health and Social Research.     

The effect of zinc deficiency on the body composition of rats

Body composition and the levels of some plasma metabolites were measured in zinc deficient and control rats with the aim of assessing the nature of the metabolic defects resulting from zinc deficiency. Two experiments, lasting 15 and 20 d, were carried out using 52 immature rats. Zinc deficient animals were fed a diet of 1–2 mg Zn/kg. Pair fed andad libitum control rats received the same diet with 100 ppm zinc added to the drinking water. Feed intake and growth rate were measured, and the carcasses were analyzed for protein, fat, and ash. In each experiment, a group of rats were killed on d 1 to provide pretreatment values and to allow for estimates of net deposition of carcass components. Lactate, urea, and zinc were assayed in plasma, as well as zinc concentration in carcasses and liver. The main effect of zinc deficiency was to reduce feed intake and efficiency of feed conversion, resulting in a reduced proportion of carcass wat because of the reduced feed efficiency, zinc deficiencyper se resulted in an increase in the proportion of fat in the carcass. Plasma lactate concentration was unchanged, but urea concentration increased in both pair fed and zinc deficient rats relative toad libitum fed control animals. The results indicate that a defect in protein synthesis and an increase in energy expenditure, perhaps resulting from increased protein turnover, underlies the reduced growth and efficiency of feed conversion of zinc deficiency.     

Glucose tolerance, plasma and pancreatic insulin levels in zinc deficient rats.

Two experiments were conducted to examine the effect of zinc deficiency on glucose tolerance, and on blood and pancreatic insulin concentrations. In the first study, no significant differences in blood glucose or plasma insulin levels were noted between pair-weighted zinc deficient and zinc sufficient rats after an oral glucose load. In the second experiment, the concentration of pancreatic insulin in pair-fed zinc sufficient rats did not differ significantly from that of zinc deficient rats. However, a zinc deficient group fed ad libitum had significantly lower pancreatic insulin levels, suggesting that food restriction may cause increased pancreatic insulin. Thus, zinc deficiency per se had no apparent effect on oral glucose tolerance or pancreatic insulin concentrations.     

Rapid and sensitive detection of oestrogen receptors in cells and tissue sections by autoradiography with 125I-oestradiol

Summary The presence of receptors for steroid hormones in individual cells and tissue sections was assessed within 4–24 h using dry mount autoradiography with radio-iodinated oestradiol. Low affinity and nonspecific binding of steroids were significantly reduced by washing the cells or sections with diluted antiserum to oestradiol.For cells of the MCF-7 cell line variations in grain density were observed, indicating that cells of the MCF-7 cell line are heterogenous with respect to their cellular receptor concentrations of oestrogen receptors. Receptor-negative cells, such as peritoneal macrophages, did not retain oestradiol label.In tissue sections of rat and calf uterus, predominant labelling was observed on the endometrial gland cells and stroma.Oestradiol receptor binding in the uterus cytosol for both radio-iodinated and tritiated oestradiol showed the same qualitative characteristics as determined by sucrose gradient sedimentation profiles and a comparable amount of binding sites was found for both labels. The relative binding affinity of¹²⁵I-oestradiol compared to [³H]oestradiol is about 70–80%.The dry mount autoradiographic technique as presented can be used for rapid screening of heterogeneiety in oestrogen receptor distribution in cells and tissue sections, since this technique reveals differences in receptor concentrations on the single cell level.