Effect of vitamin A deprivation on the cholesterol side-chain cleavage enzyme activity of testes and ovaries of rats (Short Communication)

The cholesterol side-chain cleavage enzyme activity is decreased considerably at the mild stage of vitamin A deficiency in rat testes and ovaries and the decrease in activity becomes more pronounced with progress of deficiency. Supplementation of the deficient rats with retinyl acetate, but not retinoic acid, restores the enzyme activity to normal values. The cholesterol side-chain cleavage enzyme of adrenals is not affected by any of the above treatments.     

A quantitative analysis of germ cells and the histone variants in the testes of vitamin A-deficient rats and during subsequent repletion with vitamin A

A quantitative analysis of the different types of germ cells present in the seminiferous tubules of vitamin A-deficient-retinoate maintained rats revealed that the number of pachytene spermatocytes and spermatogonia was greatly reduced in the deficient rats. Spermatids were virtually absent in the deficient tubules which contained mostly spermatogonia and preleptotene spermatocytes along with the Sertoli cells. There was no change in the number of Sertoli cells present in the tubules of deficient rats as compared to that of normal rats. Following supplementation of retinyl acetate to vitamin A-deficient-retinoate maintained rats, there was an immediate thinning of the germinal epithelium resulting from the sloughing off of the damaged spermatocytes which were beyond repair. However, after 12 days of vitamin A supplementation fresh batch of pachytene spermatocytes started appearing while by day 16 round spermatids could be seen. Analysis of the acid soluble proteins from nuclei on different types of Polyacrylamide gel electrophoretic systems has revealed that the levels of the testis specific histone variants Hlt, TH2A and TH2B, synthesized predominantly in the pachytene spermatocytes were greatly reduced in the testes of retinoate maintained rats. Following supplementation of retinyl acetate for either 4 days or 8 days the levels of these histone variants further decreased which correlated with the decrease in the number of pachytene spermatocytes. However, by day 12 of supplementation onwards, their levels started increasing and reached near normal levels by day 24 of vitamin A-supplementation     

A method for quantifying rotational symmetry

Here, a new approach for quantifying rotational symmetry based on vector analysis was described and compared with information obtained from a geometric morphometric analysis and a technique based on distance alone. A new method was developed that generates a polygon from the length and angle data of a structure and then quantifies the minimum change necessary to convert that polygon into a regular polygon. This technique yielded an asymmetry score (s) that can range from 0 (perfect symmetry) to 1 (complete asymmetry). Using digital images of Geranium robertianum flowers, this new method was compared with a technique based on lengths alone and with established geometric morphometric methods used to quantify shape variation. Asymmetry scores (s) more clearly described variation in symmetry and were more consistent with a visual assessment of the images than either comparative technique. This procedure is the first to quantify the asymmetry of radial structures accurately, uses easily obtainable measures to calculate the asymmetry score and allows comparisons among individuals and species, even when the comparisons involve structures with different patterns of symmetry. This technique enables the rigorous analysis of polysymmetric structures and provides a foundation for a better understanding of symmetry in nature.     

Muscle reinnervation in rats treated with vitamin E]

During muscle reinnervation, a transitory phase of polyinnervation occurs. In reinnervated muscles of vitamin E deficient rats, sprouting and polyinnervation are increased with respect to reinnervated controls. In this work, polyinnervation was observed in reinnervated extensor digitorum longus (edl) muscle of rats treated with pharmacological doses of vitamin E. Sciatic nerve was crushed and edl muscle was examined electrophysiologically at 30, 40 and 60 days after denervation. The percentage of polyinnervated cells in controls peaked at 30 days and thus it decreased. In muscles of vitamin E treated rats, the time course of percentage of polyinnervated muscle cells was qualitatively the same, but it was decreased at all times.     

Effect of vitamin A on the testes

Experiments were made on adult male mice with experimental unilateral cryptorchism. The animals were divided into three groups. The first group served as control, the second group of animals received mater-soluble retinoic acid in the total dose 0.1 ml (1% solution), and the third group in the total dose 0.3 ml (1% solution). Eight days after operation the tests were taken for histological and morphometry examination. In the course of experiments, spermatogenic processes in the tests had stopped because of degeneration of differential and mature sexual cells. Meanwhile spermatogonia and sustenocytes of the seminiferous tubules and glandulocytes of the tests had been preserved. Administration of the acid in a dose of 0.1 ml did not increase the resistance of the spermatogenic epithelium to the unfavourable conditions, but maintained its capacity for regeneration. After administration of the acid in a dose of 0.3 ml the animals manifested the signs of hypervitaminosis, the resistance of the spermatogenic epithelium to the action of unfavourable factors decreased and the regenerative capacity was inhibited.     

The effects of vitamin B6 deprivation with 4-deoxypyridoxine in meal-fed rats

Weanling male rats were trained to consume a control diet in one 4-hr meal a day. Rats were then fed one of three experimental diets for 7 days: (a) control diet minus B6 (-B6); (b) control diet (B6); or (c) control diet restricted to food intake of -B6 (B6R). Xanthurenic acid excretion was greater before and after a tryptophan load in -B6 than in B6R. Body weight gain, food intake, and food efficiency were not different between -B6 and B6. However, in B6R body weight gain, food intake and food efficiency were lower than that in both -B6 and B6. Serum glucose (12-16 hr after meal) and percentage body fat were similar in all three groups. No differences were found in organ size between the -B6 and both control groups indicating that short-term deprivation and deoxypyridoxine did not affect organ size. No significant differences were observed for serum growth hormone (GH), pituitary GH, serum prolactin (PRL), or pituitary PRL among the three groups. When meal fed, differences were minimized between B6 deprived and unrestricted control (B6) rats in body weight gain, food intake, food efficiency, and body composition.     

Correlation of histological an histometric changes in rats testes treated with chloroquine phosphate.

Histological and histometric changes in the testes of albino Wistar rats were correlated. Wistar rats weighing between 180-240g were randomly divided into three groups of ten rats each. One group served as control and the rats were given normal saline. The second and third groups received 2mg/kg and 4mg/kg body weights of chloroquine phosphate daily for thirty days respectively. Seminiferous tubules of animals treated with chloroquine phosphate were irregular in shape and were also isolated compared to control. Marked disruption of the inter-tubular stroma of testes in the treated groups was also observed. Histometric variations in testicular tissue was observed in the experimental animals following treatment with chloroquine phosphate. The 2mg/kg body weight and 4mg/kg body weight animals recorded significantly lower [P< 0.05] relative germinal epithelial volume of 43.95 % and 32.70 % respectively when compared to the control (51.75 %). The volume of stroma in the third group (49.33 %) was significantly higher [P < 0.05] when compared to the control (16.83 %) and 2mg/kg body weight rats (22.83 %). We observed negative correlation coefficient between lumen and seminiferous tubular volume in the control group compared to the other groups which showed a positive correlation. Correlation between germinal epithelium and seminiferous tubular volume were positive in all groups. These findings have thrown more light on recognized histological changes by accurately grading these changes which offers objectivity and increased precision compared with direct visual appraisal.     

A refined blood collection method for quantifying corticosterone

In many rodent laboratories, blood samples are collected from rats using the tail vein nick procedure and analyzed to quantify blood corticosterone levels as an indicator of stress. The standard method of corticosterone quantification often requires the collection of a relatively large volume of blood, followed by the extraction of the blood plasma. An alternative blood sampling method requires the collection of only a drop of blood on paper (the 'drop' method), minimizing handling of the animals, and does not require plasma extraction. The authors aimed to validate the drop method of blood sampling for use in corticosterone quantification. They induced stress in rats by cerebral ischemia, collected blood samples at various intervals using both the drop method and the plasma extraction method and then quantified corticosterone by radioimmunoassay. Corticosterone levels of the ischemic rats were compared with those of sham-operated rats and those of ischemic rats that had been given metyrapone, a glucocorticoid synthesis inhibitor, prior to vessel occlusion. Blood corticosterone levels in the samples obtained from the same animal using the two different methods were highly correlated for all rats. The authors further provide a regression model that can be used to predict plasma corticosterone values from those obtained from the drop blood samples. Quantification of corticosterone from only a small drop of blood has many practical and ethical advantages and should be considered as an alternative to standard methods.     

A method for quantifying radioactivity associated with protein in silver-stained polyacrylamide gels.

A method is described in which individual proteins labeled with weak beta-emitting radionuclides, separated by polyacrylamide gel electrophoresis, and stained with silver are released from the gel by the use of the periodate soluble cross-linking agent N,N'-dialyltartardiamide. The radioactivity can then be quantitated using liquid scintillation counting. The method is shown to be insensitive to reasonable variations in the intensity of staining as well as the gel volume over a practical range of gel slices. Recovery from the gel is extremely good with 93% of the counts associated with 14C-labeled proteins of known radioactive concentration being recovered. Analysis of a complex mixture of 3H-labeled proteins indicates resolution similar to that obtainable by autoradiography without the problems associated with quenching of autoradiographic signal by the staining procedure. The method is used to determine the amount of fucose and mannose incorporated into a putative cell adhesion protein during development of the cellular slime mold Dictyostelium purpureum.     

Short-latency ionic effects of nerve growth factor deprivation and readministration on ganglionic cells

Nerve growth factor (NGF) is likely to exert its trophic action on dorsal root ganglion (DRG) and on sympathetic ganglion neurons by controlling a crucial function of these cells. This function would in turn regulate other cellular machineries and, ultimately, lead to the traditional NGF consequences, such as survival and neuritic growth. A corollary of this view is that the key to NGF action must lie in short-latency events, occurring within minutes of NGF administration. Chick embryo DRG dissociates have proved to be an effective experimental system to investigate short-latency responses to NGF, in that (1) measurable functional deficits develop over 6 h of NGF deprivation in vitro and (2) delayed presentation of NGF promptly and fully restores the defective function. The first deficit observed in this experimental system, a decline in RNA-labeling capability, led to the recognition that NGF controls the transport of selected exogenous substrates, all of which are Na⁺-coupled and depend on an Na⁺ gradient across the neuronal membrane. Subsequent work showed that NGF controlled such transport systems by actually regulating the neuronal ability to control intracellular Na⁺. Under NGF deprivation, the DRG cells accumulate Na⁺ to levels that reflect, and presumably equate, the extracellular Na⁺ concentrations. Conversely, on delayed NGF administration, the accumulated Na⁺ is actively extruded to an extent and at a speed that depends on the NGF concentration. The Na⁺ response is elicited by both Beta and 7S NGF, but not by other proteins tested. All ganglionic systems that display a requirement for exogenous NGF in culture have also displayed the Na⁺ response to NGF. The Na⁺ response is grossly paralleled by a K⁺ response. DRG dissociates, in which intracellular K⁺ has been pre-equilibrated with extracellular ⁸⁶Rb⁺, lose their ⁸⁶Rb⁺ over 6 h of NGF deprivation and restore it on delayed NGF administration. The regulation by NGF of mechanisms controlling intracellular Na⁺ and K⁺ levels in their target neurons is likely to occupy an early and fundamentl place in the sequence of events underlying the mode of action of this factor.     

A new method for quantifying encephalization in the growing individual

Here we describe a new method for quantifying encephalization in the growing individual and provide a worked example of the methods. The new method is based on the use of conditional SD scores derived from brain and body growth references. These encephalization SD scores control for age, sex and body size effects on brain size, and therefore, control for the confounds associated with allometry as well as growth differences between the brain and body and between the sexes. The methods also control for distribution skewness. Encephalization SD scores derived from pre- and post-natal data may be directly compared and changes in SD score over time assessed. These methods may be applied to a broad range of data where relative size during growth is to be quantified. Derived SD scores may also be applied to correlation and regression analyses where statistical relationships with other variables are of interest.     

A new method for quantifying iodine in a starch-iodine matrix

A rapid and sensitive method for quantifying iodine in intact starch granules using gas chromatography is described with detection limits as low as 0.2% (w/w) iodine in starch. Sample preparation includes NaBH₄ reduction of the various iodine species associated with starch to the colorless soluble iodide ion, followed by its quantitative derivatization to EtI using in CH₂Cl₂. Identification and quantification of EtI is carried out by extraction and injection of the EtI so generated in CH₂Cl₂ into a gas chromatography-mass spectrometer (GC-MS). Routine quantification of EtI was then performed using GC with a flame ionization detector (GC-FID). Results for different iodine:potassium iodide ratios of the initially bound iodine and for seven different starch matrices showed that in all cases regression coefficients for the standards were high (R² >0.96).     

A computational method for quantifying morphological variation in scleractinian corals

Morphological variation in marine sessile organisms is frequently related to environmental factors. Quantifying such variation is relevant in a range of ecological studies. For example, analyzing the growth form of fossil organisms may indicate the state of the physical environment in which the organism lived. A quantitative morphological comparison is important in studies where marine sessile organisms are transplanted from one environment to another. This study presents a method for the quantitative analysis of three-dimensional (3D) images of scleractinian corals obtained with X-ray Computed Tomography scanning techniques. The advantage of Computed Tomography scanning is that a full 3D image of a complex branching object, including internal structures, can be obtained with a very high precision. There are several complications in the analysis of this data set. In the analysis of a complex branching object, landmark-based methods usually do not work and different approaches are required where various artifacts (for example cavities, holes in the skeleton, scanning artifacts, etc.) in the data set have to be removed before the analysis. A method is presented, which is based on the construction of a medial axis and a combination of image-processing techniques for the analysis of a 3D image of a complex branching object where the complications mentioned above can be overcome. The method is tested on a range of 3D images of samples of the branching scleractinian coral Madracis mirabilis collected at different depths. It is demonstrated that the morphological variation of these samples can be quantified, and that biologically relevant morphological characteristics, like branch-spacing and surface/volume ratios, can be computed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Utilization of vitamin A in rats with inflammation

Reductions in the concentration of retinol (vitamin A) in serum, lung and kidney were observed in rats subjected to inflammation-inducing treatments (turpentine oil injection of thermal injury). At the same time, the liver showed an almost normal vitamin A content. Feeding of retinol to vitamin A-depleted rats with inflammation revealed that intestinal absorption of retinol was still active in the inflamed state, and the livers of these rats showed good incorporation of retinol. The livers of normal and vitamin A-depleted rats subjected to the inflammatory treatments showed a normal RBP content (retinol-binding protein) and hepatic release of holo-RBP into the serum was not impaired functionally. These results suggest the possibility that the decreases of vitamin A in the lung, serum and kidney may be due primarily to enhanced local consumption of vitamin A related to the inflammation, rather than to a reduced supply of vitamin A from the liver or to decreased intestinal absorption. In bovine serum albumin (BSA)-sensitized rats produced by direct intubation of BSA into the lungs, the level of vitamin A in the lung decreased prior to that in the liver or serum, supporting the hypothesis that the decrease in vitamin A in the inflamed lungs of these rats may be due mainly to the consumption of vitamin A in the lung in response to inflammation.