Thomas R. Koszalka

The embryonic rat parietal yolk sac: Changes in the morphology and composition of its basement membrane during development☆

Abstract The basement membrane (Reicherts membrane) of the entire capsular portion of the parietal yolk sac of rat embryos was examined both morphologically and chemically at various stages of gestation. The overall microscopic and compositional analyses showed Reicherts membrane to be typical of basement membranes isolated from other tissues and species. However, with increasing gestational age (from 11.5 to 17.5 days) a number of changes involving Reicherts membrane were noted: 1. The thickness increased rapidly then declined, while the surface area increased tenfold; 2. The total protein content increased twenty-fold while the collagen content increased eight-fold. As a result, the relative collagen content declined significantly; 3. The changes in the amino acid and carbohydrate composition were consistent with the latter finding. The observations listed above were evaluated in light of their possible relevance to an understanding of the morphogenesis of basement membranes during development, and to the possible mechanisms involved in pathogenesis of basement membrane dysfunction.

Production of congenital malformations using tissue antisera.

Abstract Rabbit anti-rat Reicherts membrane (RM) serum, when injected into pregnant rats on the ninth day of gestation, produced embryolethality and stunting but a surprisingly low incidence of malformations. Immunofluorescent studies revealed that the maternally injected RM antiserum localizes in RM and in the basement membrane of some maternal kidney tubules but not in the maternal glomerulus or in the cytoplasm of the visceral yolk sac (VYS) cells as had teratogenic VYS and kidney antisera. When antiserum against rat RM was injected into mice, localization occurred in the glomerular basement membranes, thus confirming the findings of Pierce and co-workers. When teratogenic term VYS antiserum is adsorbed with RM, it still localizes in the VYS, does not localize in RM, and is still teratogenic. When this same antiserum is adsorbed with VYS, the antiserum is no longer teratogenic. It appears that the localization of antibodies in the VYS cells is the major contributor to the production of congenital malformations because localization of antisera in RM alone was associated with a very low incidence of teratogenesis. Since both structures participate in a multitude of biochemical processes, which include adsorption, transport, catabolism, and anabolism of nutrients, nonnutrients, and waste products, there are many plausible hypotheses to explain the nature of the parietal and visceral yolk sac dysfunction.

Sources of amino acids for protein synthesis during early organogenesis in the rat. 1. Relative contributions of free amino acids and of proteins

Rat conceptuses on the 10th day of gestation were cultured for 27 h in whole rat serum. An addition of either [3H]leucine or [3H]leucine-labelled rat serum proteins was made once during the culture period, and the acid-soluble and acid-insoluble radioactivities of embryo and visceral yolk sac measured at harvesting. The extent of radiolabel incorporation into embryonic and yolk-sac proteins increased linearly with the duration of exposure of the conceptus to the radiolabelled leucine or radiolabelled serum proteins, indicating roughly constant rates of incorporation, per unit mass of tissue, throughout the culture period. The incorporation rates, expressed as clearances, were 0.73 and 0.78 microliter/mg tissue protein/h for embryo and yolk sac, respectively, when the source was [3H]leucine; and 1.8 and 1.3 microliters/mg tissue protein/h, for embryo and yolk sac, respectively, when the source was [3H]leucine-labelled serum proteins. It is estimated, from the known leucine and protein concentrations in serum, that protein contributed over 99 per cent of the leucine supplied to the conceptus for its protein synthesis. In parallel experiments, measurements were made on cultures conducted in the presence of an antiserum against rat visceral yolk sac (100 micrograms/ml). Antiserum profoundly inhibited incorporation of radioactivity into embryo and yolk-sac proteins, when the source was 3H-labelled protein, a result consistent with the known ability of the antiserum to inhibit pinocytosis in the yolk sac. Antiserum also decreased incorporation from [3H]leucine in the yolk sac, suggesting that a proportion of the free leucine entering the yolk sac does so by pinocytosis. The failure of antiserum to affect incorporation of [3H]leucine into the embryo probably indicates that leucine can enter the embryo without the mediation of yolk-sac pinocytosis. The primacy of protein, as a source of amino acids for the organogenesis-stage embryo, is consistent with the serious effects, in terms of embryonic death and malformation, that result from the interruption of amino acid supply when either pinocytosis or lysosomal proteolysis in the yolk sac is inhibited.

Organ cultures of the embryonic rat parietal yolk sac: I. Morphologic and autoradiographic studies of the deposition of the collagen and noncollagen glycoprotein components of basement membrane

Abstract The synthesis, deposition, and turnover of [ 3 H]proline- and [ 3 H]glucosamine-labeled basement membrane components have been studied in a system using organ cultures of embryonic rat parietal yolk sac tissues on a nutrient agar substrate. The morphologic and autoradiographic studies described in this report were correlated with the biochemical studies described in the companion paper (Minor et al., Develop. Biol. 48, 1976). These studies showed that: (1) basement membrane (BM) was the only extracellular matrix synthesized in the cultures, (2) 10- to 30- μm thick layers of new BM were deposited during a 6-day culture period, (3) it was only the parietal endodermal cells that synthesized BM, (4) new BM was deposited only on the surface of existing BM, which was in contact with endodermal cells, (5) the amounts of new BM that accumulated were increased by a twice daily feeding schedule and decreased by the presence of trophoblast, (6) there was a notable difference in the redistribution of labeled components during a 6-day chase, and (7) [ 3 H]glucosamine tended to localize in the Golgi complex, whereas [ 3 H]proline was distributed throughout the cytoplasm of the endodermal cells.

Effect of Insulin on the Uptake of Creatine-1-14C by Skeletal Muscle in Normal and X-Irradiated Rats

Summary The uptake of labeled creatine by skeletal muscle was enhanced in rats treated with 2 units of NPH insulin on each of two successive days and then injected ip with creatine-1-14C. While the uptake of labeled creatine by muscle was impaired in rats exposed to an X-ray dose of 500 rads, this impairment was relieved by treatment with insulin after X-irradiation. The level of radioactivity and the creatine concentration in whole blood was lower in insulin-treated rats injected ip with creatine-1-14C than in untreated controls. In contrast, X-irradiated rats injected ip with labeled creatine exhibited higher levels of radioactivity and a higher creatine concentration in blood than did the sham-irradiated controls. When X-irradiated rats were treated with insulin, the amount of radioactivity and the creatine concentration in the blood 1 hr after the injection of labeled creatine was not significantly different from that in the sham-irradiated controls. These observations are consistent with the view that insulin enhances the transfer of creatine from the circulation into skeletal muscle in normal as well as in X-irradiated rats.

Organ cultures of the embryonic rat parietal yolk sac: II. Synthesis, accumulation, and turnover of collagen and noncollagen basement membrane glycoproteins☆

Synthesis, accumulation, and turnover of basement membrane components have been studied in organ cultures of 13.5- and 14.5-day embryonic rat parietal yolk sac tissues on a nutrient agar substrate. The biochemical studies described in this report were correlated with morphologic and autoradiographic studies described in the companion paper (Minor et al., Develop. Biol. 48, 1976). These studies showed that basement membrane is the only extracellular matrix synthesized, it is only synthesized by the parietal endodermal cells, and its synthesis is maintained for at least 6 days. In these cultures, synthesis and degradation of collagen and noncollagen proteins varied independently in response to environmental changes, such as the frequency of feeding or presence of trophoblast. The turnover of basement membrane collagen was much slower than that of the noncollagen proteins and this difference in the rate of turnover of the components had a major role in determining the composition of the newly synthesized basement membrane.

Materno-Fetal Transport of Creatine in the Rat

The transfer of 14C-creatine to the rat fetus was studied following continuous i.v. infusion into the mother. In the presence of a relatively constant maternal plasma 14C-creatine concentration, creatine was accumulated by the chorioallantoic placenta and visceral yolk sac to concentrations higher than that found in maternal or fetal plasma. The ability of the extraembryonic membranes to accumulate creatine changed during gestation; nevertheless, these membranes concentrated creatine against a gradient throughout the period studied (14-22 days of gestation). Neither 14C-creatine nor 14C-urea were concentrated in the placentae or fetal plasma when compared to maternal plasma. Simultaneous infusion of beta-guanidinopropionic acid with 14C-creatine reduced both movement and accumulation of creatine into the fetoplacental unit. It is concluded that the accumulation of creatine by the chorioallantoic placenta and by the visceral yolk sac is an active process with creatine diffusing down its concentration gradient into the fetal circulation.

Placental Transport of Creatine in the Rat

Summary Pregnant rats near term were injected iv with creatine-l-14C or creatinine-1-14C and the distribution of radioactivity was studied in maternal and fetal blood as well as in the visceral yolk sacs and chorio-allantoic placentae. After injection of crea-tine-l-14C into the dam, the level of radioactivity in fetal blood was somewhat higher than that in maternal blood at 30 min, reached maximal levels after 30 min, and remained essentially constant for at least 4 hr. The radioactivity in the labyrinth and junctional zone portions of the chorioal-lantoic placenta and the yolk sac was maximal after 30-60 min and was always considerably higher than that in the fetal and maternal blood. Furthermore, the level of radioactivity in the labyrinth was several times higher than that in the junctional zone portion of the chorioallantoic placenta and the yolk sac in which the radioactivity remained essentially constant after 1 hr. On the other hand, the radioactivity in the labyrinth declined rapidly after the first hour and reached levels which were about one-third of the maximal value after 4 hr. When labeled creatine was injected into the dam, 99% of the radioactivity present in the fetal blood and chorioallantoic placenta was found in free creatine as shown by paper chromatography in three different systems. Ancillary studies showed that the creatine concentration in fetal blood was several times higher than that in the maternal blood on the 21st-22nd day of gestation. Creatine concentration in both portions of the chorioallantoic placenta as well as the visceral yolk sac was higher than that in the fetal blood, indicating that a downhill concentration gradient existed between creatine in placental tissues and that in the fetal circulation. When creatinine-l-14C was injected iv into pregnant rats, the level of radioactivity in fetal blood rose to a maximum after 5-15 min, but the radioactivity in the maternal blood was considerably higher than that in either the fetal blood or the chorioallantoic placenta and yolk sac. These studies indicate that creatine is actively accumulated and released by the placental unit to the growing rat fetus, while creatinine is passively transported from mother to conceptus across the placentae, at least during the later stages of rat gestation. We are indebted to Drs. Richard K. Miller and Barbara Davis for their constructive criticism of the manuscript and to Carole L. Andrew for her excellent technical assistance.

Characterization of cytosolic glucocorticoid receptor of fetal rat epiphyseal chondrocytes

The cytosolic glucocorticoid receptor of 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 x 10(-8) M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 +/- 1.43 x 10(-9) M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinities of steroid for receptor were found to be triamcinolone acetonide greater than corticosterone greater than hydrocortisone greater than progesterone greater than medroxyprogesterone acetate much greater than 17 alpha-hydroxyprogesterone much greater than testosterone greater than 17 beta-estradiol. Cytosolic preparations activated in vitro by warming (25 degrees C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4 degrees C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.

Carrier-mediated uptake of hexoses by the rat visceral yolk sac

The rat visceral yolk sac is shown to possess a sodium-independent, phloretin-sensitive, and phlorizin- and ouabain-insensitive transport system for hexoses. The rate of uptake of (3H)2-deoxy-D-glucose was measured in vitro and shown to be greatest on the 12th day, decreasing progressively with increasing gestational age up to the 20th day. Little uptake of 3-O-methyl-D-glucose, alpha-methylglucoside or L-glucose occurred. On uptake by the visceral yolk sac, 2-deoxy-D-glucose was phosphorylated, leading to considerable accumulation of this sugar. Several sugars inhibited 2-deoxy-D-glucose uptake as follows: D-glucose = mannose greater than fructose greater than galactose greater than xylose greater than fucose.