B Robinzon’s research while affiliated with Hebrew University of Jerusalem and other places

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11betaHSD enzyme activity against corticosterone, dehydrocorticosterone, 7alpha- and 7beta-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP(+) or NAD(+), but not NADPH and NADH, as pyridine nucleotide cofactor with K(m) values of 12+/-2 and 390+/-2microM, compared to the K(m) for microsomal 11betaHSD1 of 43+/-8 and 264+/-24microM, respectively. The K(m) for corticosterone in the NADP(+)-dependent nuclear oxidation reaction was 102+/-16nM, compared to 4.3+/-0.8microM for 11betaHSD1. The K(cat) values for nuclear activity with NADP(+) was 1687nmol/min/mg/micromol, compared to 755nmol/min/mg/micromol for microsomal 11betaHSD1 activity. Inhibitors of 11betaHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11betaHSD Type 1 and 2.

In mammalian organs involved in sodium reabsorption, the 11-beta hydroxysteroid dehydrogenases (11betaHSDs) oxidize glucocorticoids (GC) from their 11-alcohol form to their 11-keto state and therefore prevent their binding to mineralocorticoid (MC) receptors (MR) and the development of a MC excess syndrome. In birds the information about 11betaHSDs and GC metabolism in such organs is scarce. Herein, we report the expression and enzymatic activity of 11betaHSDs in the kidney and colon of chickens. Both organs express 11betaHSD2-like mRNA. With NAD(+), microsomes from both tissues oxidized corticosterone (CS) into 11-dehydrocorticosterone (DHC) with K(m) of 200 and 20nM and V(max) of 13 and 2pmol/mg protein/min in the kidney and colon, respectively. Thiram, a specific 11betaHSD2 inhibitor, suppressed this oxidation in kidney. The expression and action of the putative 11betaHSD3 were also tested. The chicken colon, and to a greater extent the kidney, expressed 11betaHSD3-like mRNA. Microsomal fractions from both tissues oxidized CS into DHC in the presence of NADP(+) with K(m) of 150 and 4nM and V(max) of 5 and 0.3pmol/mg protein/min for the kidney and the colon, respectively. This oxidation was not affected when NADP(+) conversion into NAD(+) was inhibited by excess pyrophosphate or a phosphatase inhibitor cocktail. In microsomes of chicken's duodenum, where 11betaHSD1-like mRNA expression is high, NADP(+)-dependent oxidation of CS into DHC has a low-affinity K(m) of 1130nM. This study documented the expression and activity of two enzymes that convert CS into DHC, one is 11betaHSD2-like and the other is similar to the putative mammalian 11betaHSD3.

The mammalian 11-beta hydroxysteroid dehydrogenase type 1 (11 betaHSD1) reduces glucocorticoids (GC) at C11 from the 11-keto-GC nonactive form to the 11-hydroxy-GC active form, an action essential for survival. Whereas GC metabolism at C11 and the role of 11 betaHSD1 are studied extensively in mammals, information about these in birds is scattered. Herein, we report the GC bidirectional metabolism in chickens. In hens' liver and duodenal mucosa, 11 betaHSD1-like mRNA expression was detected; and 11 betaHSD1-like immunoreactivity was found linked to membranes of hepatocytes and duodenal enterocytes. With either NADH or NADPH, the membranal fraction of liver and duodenal mucosa converted dehydrocorticosterone (A) into corticosterone (B) with K(m) (1.1-8.7 microM) and V(max) (10-40 pmol/mg protein/min) values similar to those reported for mammalian 11 betaHSD1. In the presence of NADP(+) or NAD(+), these membranal fractions oxidized B into A. With either NADPH or NADH, the cytosol of chicken liver and duodenal mucosa reduced A into B (K(m) of 1.1 - 2.3 microM and V(max) of 260-960 pmol/mg protein/min). These cytosolic fractions did not convert any amount of B into A when incubated with either NADP(+) or NAD(+). This may suggest that chicken liver and duodenal mucosa express 11 betaHSD1 that is a membrane-bound oxoreductase which uses both NADPH/NADP(+) and NADH/NAD(+) as cosubstrates. The substantial reduction of A into B (but no conversion of B into A) found in the cytosol is most likely executed by a unidirectional soluble reductase, different than 11 betaHSD1.

1. This study compared the effect of bilateral electrolytic lesions of the basomedial hypothalamus (HL) in broiler and White Leghorn (WL) males. 2. Hypothalamic lesions were placed in WL at 10 weeks of age (body weight 1.1 kg) and in broilers, either at 6 weeks (body weight 1.5kg) or at 10 weeks of age (body weight 3.4kg). They were fed ad libitum until autopsy at 16 and 17 weeks of age for broilers and WL, respectively. 3. Hypothalamic lesions caused obesity (high percentage weight of abdominal adipose tissue) in both strains. Obese fowls with unimpaired reproductive systems were classified as OB and those with functional castration as FC (functionally castrated) or FCLC (functionally castrated with large comb). 4. All post-HL syndromes-OB, FC and FCLC-were present in WL, whereas all obese broilers (which are immature at this age) were classified as OB. 5. The percentage weight of abdominal adipose tissue in OB broilers was lower than in OB WL (3% vs 5%, respectively). 6. Daily food intake of OB broilers was higher than control at 12 to 15 weeks of age, regardless of time of placement of HL, whereas daily food intake of OB WL was significantly higher than that of control WL only during the first 2 weeks following HL. 7. Body weight of OB broilers at autopsy was 20% higher than control broilers, whereas body weight of OB WL was not significantly affected. 8. An additional group of broilers was reared to sexual maturity under food restriction until 28 weeks of age. HL were placed at 10 weeks of age (body weight 1.7 kg). Autopsy was performed after a 4-week period of ad libitum feeding. 9. There were OB as well as FC and FCLC among the HL, food-restricted broilers. Percentage weight of testes and spleen were reduced in OB fowls of both strains, but more so in OB WL. 10. Hyperphagia and weight gain were not observed during the ad libitum feeding period of those obese broilers after HL, indicating that hyperphagia and weight gain are secondary to obesity.

The Mediterranean tortoise (Testudo graeca) is listed as "Vulnerable" on the IUCN Red List. Reproductive characteristics and means to increase offspring production were studied in T. graeca terrestris in a semi-natural environment. Courtship and mating occurred during early spring for about 4 weeks, followed by a laying season of approximately 2 months, with a second, shorter mating period in the fall. During the first mating, calcified eggs were already present in the uterus; we inferred that sperm from both mating seasons were stored in the oviduct for fertilization of eggs of the second laying cycle and of the following year. Average egg production was 3.8+/-0.3 eggs/year. Most females laid all of their eggs in a single clutch, but 18% laid in a second clutch, 11-21 days later. X-ray radiography revealed calcified eggs in the uterus about 4 weeks before oviposition. All eggs in the uterus were calcified simultaneously and were laid in a single clutch; if a second clutch developed, those eggs were also calcified simultaneously. Based on endoscopic examinations, ovaries were active throughout the entire year. Plasma progesterone concentrations in females were very low and were detected only soon after oviposition ( 440 +/-141 pg/ml). Plasma estradiol concentrations in females varied from 4.1 +/-1.5 pg/ml to 70.2 +/-29.4 pg/ml, with no clear seasonal pattern. Maintaining tortoises at a low environmental temperature (9 +/-1 degrees C versus 28 +/- 1 degrees C) reduced plasma estradiol concentrations. Giving 2mg/kg tamoxifen (TAM) increased plasma estradiol to 220 +/-33 pg/ml when treatment was given in September but not in late October, winter or spring. Treatment with TAM increased the number of eggs laid during the following laying season to 7.3 +/- 1.0 eggs/year, laid in one to three clutches. In males, plasma testosterone concentrations had a seasonal pattern with the onset of a rise in July from 2 to >4ng/ml, a continued increase to a peak of 12.8+/-5.3 ng/ml during November and a decline thereafter. Artificial incubation in sand at 29 +/-1 degrees C shortened the natural incubation time of 103+/-3.1 days to 83.5 +/- 1.3 days, increased hatching rate from 28 to 53%, and increased survival rate from 51 to 71% at 40 weeks of age. In summary, this study provides options for increasing reproductive performance, hatchability and offspring survival in captive Mediterranean tortoises, and may offer new tools for conservation of animals that are on the verge of extinction.

The effects of glucocorticoids (GC) on embryonic mortality and posthatch BW were studied. Cortisol hemisuccinate or corticosterone in 0.1-mL vehicles were injected into the albumen of 7-d-old White Leghorn chicken embryos. Embryonic mortality rates and the age after injection at which death occurred were determined. When 0.02 to 20 μg cortisol per egg were injected in saline, total embryonic mortality rate increased in a doseresponse manner, with a median lethal dose (LD50) at 10 μg. Saline injection alone caused a similar mortality rate to that caused by injection of 2 μg cortisol (around 35%). However, whereas mortality among the cortisol-treated embryos was greatest on Days 16 to 18, most of the saline-treated embryos died around the time of injection. The lethal effect of corticosterone, which is endogenous GC in adult chickens, was compared to that of cortisol by injecting both in the same vehicle (a saline:ethanol mixture) and was found to be similar. However, when 2, 10, or 20 μg of corticosterone was injected in oil, mortality rates were lower than those caused by the matching doses of cortisol in saline, probably due to the lower diffusion rate of the steroid out of the oil carrier. Hatch weight was significantly lower in chicks treated with 10 and 20 microg cortisol, and BW of the latter was lower compared with control throughout the 3-mo observation. In conclusion, cortisol and corticosterone are equally active in causing embryonic mortality. Posthatch BW is affected only by GC doses that are equal to or greater than the LD50.

Prolonged stress inhibits the hypothalamus-pituitary-gonadal (HPG) axis and reduces plasma testosterone (T). However, enhanced secretion of luteinizing hormone (LH) and T has been documented during the initial stages of acute stress in mammals. This study assayed the effect of short-term stress on plasma T and corticosterone (B) in juvenile, pubertal, and adult White Leghorn cockerels. Stress was induced by brief physical restraint of caged juvenile (7 weeks), pubertal (17 weeks), and adult (40 weeks) cockerels, as well as 40-week-old adults reared together in a room lined with wood shavings (group reared). Blood was sampled immediately before restraint (0 time), at the end of a 10-min restraint period, and at 30, 60, and 180 min after 0 time. Restraint resulted in an initial increase in plasma T in all groups, along with a rise in B. Whereas B generally reached its peak level at the end of the restraining period, T peaked 20 min later. The maximum increase of T and B relative to prestress levels (T and B ratios) was similar in all groups, with median T ratio reaching 1.25-1. 5-about half that of the B ratio. Thus, the extent of T and B response to short-term stress was not influenced by basal levels of T, which were highest in adults, and basal levels of B, which were higher in caged adults than in group-reared adults. Injection of ACTH did not induce a greater increase in plasma T than in sham-injected controls. Further, the elevation of T in response to stress was extinguished in castrated adults, indicating that T is secreted from the testes rather than the adrenals in response to stress. When the same regime of blood sampling was applied to adults not subjected to restraint, the T ratio rose by up to 11 times. It can therefore be stipulated that T response depends on the type of stress applied, a factor that should be considered when investigating androgen levels in plasma.

Embryonic and posthatch long-term exposure to the odor of 2-methoxy-3-isobutyl-pyrazine (2M3IP) was examined for its potential physiological consequences as reflected in changes in BW and organ weights in domestic chicks (Gallus gallus domesticus). Experiments were run from Day 1 of incubation to the age of 3 wk with a total of 360 fertile chicken eggs. The experimental design consisted of four treatment groups: PP chicks were exposed to 2M3IP during both incubation and posthatch rearing; PC chicks were exposed to 2M3IP during incubation only; CP chicks were exposed to 2M3IP during rearing period only; CC control chicks were not exposed to 2M3IP. Chicks were weighed immediately after hatch and at 3 wk of age, when they were necropsied. Various organs (thyroid, adrenal, testes, comb, liver, spleen, abdominal fat, and the bursa of Fabricius) were removed and weighed. Body weights of both sexes in the PP group were reduced. This reduction was significant in males relative to both CP and CC groups and in females only relative to the CP group. Effects of 2M3IP exposure on the examined organs were as follows: in males, adrenal gland weight significantly increased in the PP group vs all other groups. No weight differences were found between the other inspected organs among the four treatments. In females, comb weight significantly decreased compared with the rest of the groups when 2M3IP exposure occurred during incubation (PC). Further investigation is needed to study the mechanisms that underlie the differential effects of pyrazine odor on male and female chicks.

Inherited overfeeding and fattiness reduce laying performance in broiler breeder pullets. Although feed restriction is used to compensate for overeating and weight gain, this management practice leads to increases in BW variation, labor cost, and bird stress. Dietary supplementation of anorectic agents, such as fenfluramine, may be an alternative. Anak female prebreeder hens (19 wk of age; n = 10 per group) were treated as follows: daily oral administration of 5, 10, 20, or 40 mg DL-fenfluramine/kg BW or saline with food provided for ad libitum intake or administration of saline and feed restriction. Daily feed intake (FI), laying rate, egg composition, and BW were measured. At 40 wk of age, adipose tissue and ovary weights were measured. Fenfluramine depressed (P < 0.05) BW and FI in a dose-dependent manner, but was less effective in reducing BW than feed restriction. Suppression of FI occurred in two phases: a dynamic phase, coinciding with the rapid growth phase, during which FI declined progressively and a static phase during which FI reached a plateau at a significantly low level until the end of the experimental period. Egg production peaked first in saline-treated hens fed for ad libitum intake, but soon after started to decline. In all fenfluramine-treated and feed-restricted hens, egg production peaked 3 to 4 wk later and remained high until the end of the experiment. There were no differences in egg and egg component weights among the experimental groups. Abdominal adipose tissue weight was reduced by fenfluramine in a dose-related manner, and its weight in the group treated with the highest dose was similar to that of feed-restricted hens. In these two groups, ovarian weight was significantly higher than in the saline-treated hens fed for ad libitum intake, and a small, nonsignificant increase in ovary size was observed in groups treated with the two median doses of fenfluramine. The effect of fenfluramine on egg production was similar to that of feed restriction, but it was not dose-dependent and, thus, not directly related to its leaning effect. In broiler breeder hens, oral fenfluramine may be used for chemical feed restriction and diminution of fattiness without reducing egg production relative to manually feed-restricted hens.