The effect of cortisol on rabbit fetal lung maturation in vitro

Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo. The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [³H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10^?7M) or betamethasone (10^?7M), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.     

The effect of LH-RH administration on LH release in the female rabbit.

Intracarotid infusion of LH-RH to female rabbits stimulated a significant increase in plasma LH concentration in the jugular vein. This response varied with the reproductive state of the animal, with a greater release occurring in oestrous (spontaneous or oestrogen-induced) and non-receptive does than in pseudopregnant or ovariectomized animals. If ovariectomized rabbits were pretreated with oestrogen, the pituitary response to LH-RH was restored. These findings suggest that there is little change in pituitary sensitivity to LH-RH infusion between oestrous and non-receptive rabbits, although pseudopregnancy (high physiological levels of progesterone) or ovariectomy inhibit its ability to respond to a releasing-hormone stimulus.     

The recovery process in the fetal rabbit myocardium following mechanical trauma]

Healing processes of the myocardium after an injury during the intrauterine period were studied. After the mechanical trauma of the heart the rabbit fetus is kept in the maternal organism and is born in due course. It was found that the healing of the injured part of the cardiac muscle occurred shortly and was due to the high proliferative activity of the cells. Morphological structure of the tissue appearing at the site of injury is very similar to that of the muscle tissue. However, further studies are necessary for final decision.     

Influence of pregnancy and sex steroids on concentration, motor effect and receptor binding of VIP in the rabbit female genital tract

The influence of sex steroid and pregnancy on the tissue concentration, uterine motor effect and receptor binding of VIP has been studied in the female genital tract of pregnant rabbits and oophorectomized rabbits during progesterone and/or oestrogen substitution. The concentration of immunoreactive VIP was high in the vagina and cervix, and lower in the uterine body of both pregnant and non-pregnant rabbits. A significant decrease in the VIP concentration (pmol/g wet weight) of the uterine body was observed toward term of pregnancy. The total uterine content of VIP, however, seems unchanged. Treatment of oophorectomized rabbits with ovarian steroids had no effect on the VIP concentration. The sensitivity for and potency of VIP on the relaxation of uterine muscle was significantly higher in oophorectomized rabbits treated with a combination of progesterone and oestrogen than in control rabbits. No difference was observed between non-pregnant and pregnant rabbits. The degradation and binding affinity for 125I-labelled VIP was highest in oophorectomized rabbits substituted with both oestrogen and progesterone. In the pregnant rabbits, the amount of receptors was decreased near term. In conclusion, sex steroids are able to influence the motor effect of VIP at the receptor level, but have no effect on the VIP concentration in the female genital tract.     

The effect of K+ concentration on the energy metabolism in perfused rat heart

From experiments at various perfusion pressures in hemoglobin-free perfused rat hearts, oxygen consumption and redox shift of pyridine nucleotide were found to vary linearly with cardiac work. This relation was used for analysis of the energy metabolism associated with ion pumps. Mechanical activities such as left ventricular pressure and heart rate varied with the extracellular K+ concentration. Ion-pump dependent changes in oxygen consumption and redox state of pyridine nucleotide, estimated as the difference of the values at normal (4.7 mM) and various other extracellular K+ concentrations with corrections for the change due to mechanical work, were found to vary linearly with the K+ concentration. The slope for oxygen consumption was about 0.1 mumol/min/g X wet wt per mM K+. Lactate release changed markedly but transiently, about 1 min after changing the extracellular K+ concentration, and its amount varied linearly with the K+ concentration. In the steady state, however, lactate release was almost independent of the extracellular K+ concentration, although oxidized pyridine nucleotide increased with increasing K+ concentration. Coronary flow increased with the extracellular K+ concentration. Heart rate changed little between 1 and 12 mM K+, but decreased sharply above 12 mM K+. At 20 mM K+, heart beat was arrested and approximately 40% of myoglobin was deoxygenated. The intracellular oxygen concentration was estimated to be about 10 microM even during aerobic perfusion. Similarly, Ca2+-free arrested heart was found to be in a hypoxic state. The results showed that oxygen entry into cardiac tissue is facilitated by the cardiac cycle.     

The effect of serum starvation on the efficacy of the nuclear reprogramming of rabbit embryonic fibroblasts

Successful transplantation of mammalian nuclei from differentiated cells has become possible after the application of original methods directed at the synchronization of cell cycles of the donor cell and recipient cytoplasm. We obtained a line of rabbit fetal fibroblasts which was used to study factors affecting the success of reprogramming. The nuclei of fetal fibroblasts (up to the 10th passage inclusive) proved to be capable of reprogramming and ensuring development of the cloned embryos until the preimplantation stages. The influence of synchronization of the cell cycles of the nucleus donor and recipient on the efficiency of reprogramming was studied. The rate of development of the cloned rabbit embryos to the morula-blastocyst stage reached 67% when the nuclei used were from stationary culture cells (G0-phase).     

Quantitative study of serotonin-containing cells in fetal rabbit duodenum]

In order to attempt identification in vitro of parameters involved in the cellular differenciation, it was necessary to possess a standard material with identical content and distribution of serotonin cells; these two properties are investigated in this paper. Falck's technic is the only sensitive and specific method in demonstrating serotonin young cells devoid of argentaffinity. From 21, 22, 23 and 25 days old rabbit foetuses, 10 mm length duodenal pieces were treated according to Falck and sections serially cut at 8 microns. Some of these specimens were divided prior to Falck treatment in two pieces 3 mm away from the pylorus and in each piece 200 paraffin sections were cut at 8 microns beginning by adjacent ends and progressing in opposite direction. The number of EC sections in 400 whole transverse sections were determined for each foetus. Great variations were observed from one foetus to another, along the intestine, with weight and age. The Student-Fisher test applied to two consecutive pieces of the same duodenum gathering foetuses in groups of 5 animals did not show significative differences from a group to another. Thus, this material can be employed for comparisons under experimental conditions.     

The effect of heavy metal salts on the embryonic development of the common frog]

To elaborate methods of biological testing during monitoring of the environment, a study was made of the effect of heavy metal salts on frog embryos and of the influence of temperature on the extent of toxicity of these substances. It was shown that the early stages of embryogenesis were more sensitive to this toxic effect. The differences in the effect of heavy metals are expressed not only in the extent of toxicity, but also in the pattern of change in toxicity as related to concentration. A rise in temperature of the solution containing heavy metals results in a relative increase in the number of abnormal larvae, whereas a decrease in temperature of the solution leads to a higher mortality at the earliest stages of embryogenesis. The latter, apparently, is due to a longer duration of development at low temperature, and thus, to a longer exposure of embryos to toxic effect. This is more pronounced at earlier stages of development, more vulnerable (sensitive) to toxicants. The estimation of toxicity of a substance by the pathological changes induced in embryos and larvae is one of the most sensitive methods, which allows not only to determine the extent of toxicity, but also to give prognosis of its possible effect on the population.     

Androgen receptors in fetal rabbit lung and the effect of fetal sex on the levels of circulating hormones and pulmonary hormone receptors

High affinity (KD = 0.2 nM), low capacity (3.6-5.0 fmol/mg protein), androgen-specific binding proteins with characteristics typical of androgen receptors were identified in the lungs of rabbit fetuses between the 26 and 29th day of gestation and in the lungs of adult rabbits. While androgen receptor concentrations increased significantly from late gestation to adulthood (P less than 0.01), no sex-related differences were observed in either the binding affinities or concentrations of the receptors at any age tested. Similarly, no sex-related differences were found in the levels of progesterone, cortisol and cortisone in the fetal circulation, or in the levels of progesterone receptors, glucocorticoid receptors and beta-adrenergic receptors in the fetal lung at 26 days of gestation. It is concluded that the fetal lung interacts directly with circulating androgens via specific androgen receptors and that the suggested male disadvantage with respect to lung maturation in the perinatal period does not appear to be associated with sex-related differences in the levels of pulmonary androgen, glucocorticoid, progesterone or beta-adrenergic receptors.     

The effect of carnosine on Ca-channels in rabbit skeletal muscle sarcoplasmic reticulum]

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.     

Regulation of rabbit fetal glycogen: effect of in utero fetal decapitation on the metabolism of glycogen in fetal heart, lung, and liver

To understand the control mechanisms involved in the regulation of fetal glycogen, we have studied the effect of in utero fetal decapitations on glycogen metabolism in rabbit fetal heart, lung, and liver. In utero fetal decapitations were performed between days 18 and 21 of gestation. Two to four fetuses on one side of the horn were decapitated. Fetuses were delivered between days 23 and 26 or between days 28 and 30 of gestation. Fetal heart, lungs, and liver were analyzed for DNA, protein, glycogen, glycogen synthase (I and D forms), glycogen phosphorylase (a and b forms), phosphofructokinase, pyruvate kinase, and lactic dehydrogenase. In fetal heart and lung, no difference was observed in any of the above measurements in the intact and decapitated fetuses. In contrast, fetal liver does not appear to develop the glycogen system as indicated by the very low levels of glycogen (0.02 mg/mg DNA) in decapitated fetuses as compared with intact fetuses (0.4 mg/mg DNA). Similarly the levels of glycogen synthase and phosphorylase were two to three times lower in livers from decapitated fetuses as compared with the livers from intact fetuses. The three enzymes phosphofructokinase, pyruvate kinase, and lactic dehydrogenase were not affected by fetal decapitation in all three tissues. These results indicate that the fetal hypothalamic-pituitary-adrenal (thyroid) axis is not required at least after day 18 of gestation for the normal accumulation and subsequent utilization of glycogen in fetal heart and lungs, while it is an absolute requirement for the development of the fetal liver glycogen system.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of energy substrate concentration and amino acids on the in vitro development of preimplantation porcine embryos

As the pig becomes increasingly used for biomedical research, an effective and efficient in vitro culture system is essential. This study aimed to improve the commonly used porcine embryo culture medium, NCSU23, by altering the energy substrates and adding amino acids, using electrically activated diploid parthenotes from oocytes obtained from the ovaries of prepubertal and adult animals. Morphological development to day 6 and blastocyst cell number were examined. Glucose (5.56 mM) was replaced by pyruvate and lactate (0.2 mM and 5.7 mM, respectively) for either the entire culture period or for the first 48 h only. Blastocyst rates were not different between any of the treatments, and were similar for prepubertal and adult oocytes. When the embryos were cultured with pyruvate and lactate for the first 48 h and then glucose, there was a significant increase in blastocyst cell number compared to glucose only. Blastocysts produced using pyruvate and lactate for the entire time tended to have more cells than those exposed to glucose only and less than those who were cultured in pyruvate and lactate for the first 48 h and then glucose. Nonessential amino acids added for the first 48 h and nonessential and essential amino acids added for the remaining time significantly increased blastocyst cell number only when the embryos were grown in pyruvate and lactate followed by glucose. Blastocyst rates were not different between any of the treatments, and this result was the same when using sow or gilt oocytes. The modified medium was then tested using in vitro matured and fertilized embryos from sow oocytes. Blastocyst rates and cell number were significantly increased in the modified medium compared to those grown in unmodified NCSU23. This shows that altering energy substrates and adding amino acids can increase the quantity and cell number of IVP blastocysts compared with NCSU23.     

The effect of dissolved oxygen tension on the energy metabolism of yeasts]

The specific ATP generation rate in yeasts was examined on the glycolytic pathway and on the respiratory chain as a function of the dissolved oxygen tension of the culture medium. Two different strains were used: Saccharomyces cerevisiae sensitive to the glucose effect and Kluyveromyces fragilis insensitive to the catabolite respression when growing on lactose. The oxidative ATP generation rate followed by these two strains a Michaelis Menten kinetics against the dissolved oxygen concentration. Dissolved oxygen tension only influenced the glycolytic ATP generation rate in Kluyveromyces fragilis. Thus glucose and Pasteur effects are two mutually exclusive regulatory mechanisms of the energy yielding metabolism of the yeasts.