Effects of chondroitin-4-sulphate on survival, sizes and ultrastructures of cultured WI-38 fibroblasts and fibroblasts from progeria patients.

WI-38 fibroblasts from 'normal' individuals and skin fibroblasts from patients displaying classical symptoms of progeria (accelerated aging) were maintained in tissue culture with and without periodic supplementation of 0.25 mg/ml of chondroitin-4-sulphate (C-4-S) during the ultimate phase of slowed division (phase 3). When C-4-S was not present in the culture medium, cell counts (not necessarily indicative of relative rates of cell division) and mean cell volumes were lower, and intracellular aberrations were higher, in both types of fibroblasts (normal and progeria) at, and even before, the 47th-50th and 13th-16th passages, respectively. The authors emphasise effects of C-4-S in preserving normal ultrastructure, pointing out that C-4-S does not fulfil the criteria of a mitogen and that any possible increases in rates of mitosis following supplementation with this substance are probably secondary to an enhanced metabolic environment.

Morphological and metabolic responses of embryonic hearts to administration of exogenous L-thyroxine.

Injection of 1 microgram L-thyroxine (T4) into the yolk sacs of embryonated chicken eggs at 3 to 6 days of incubation not only induced cardiomegaly but also instigated more rapid differentiation of the heart as an organ and of the individual myocytes per se. Myocytes showed evidence of responding to this dose of exogenous T4 as early as 5 to 6 days of incubation, even though endogenous T4 was not normally forthcoming (in amounts sufficient to provoke organ changes) until 11 to 12 days of incubation. By 7 days of incubation the hearts, conditioned by a single 1 microgram dose of T4, exhibited larger areas occupied by myofibrillar material than controls. Measurements, beginning at 9 days of incubation, indicated the presence of greater amounts of RNA, total non-lipid solids and total lipids. Early increases in DNA in T4-conditioned embryos, compared with controls, indicated that hypertrophic hearts had reached this condition, at least in part, by increased cell division. By 12 days of incubation, hearts pre-treated with T4 showed conversion of many mitochondria to vesicles resembling smooth endoplasmic reticulum. No evidence of classical sarcoplasmic reticulum was seen through hatching.

Changes in fatty acid compositions of mitochondria during embryonic development.

1. A general trend among biomembranes of hepatocytes in the developing avian embryo is to display increasing percentages of unsaturated fatty acids, especially oleic acid (C18:1). 2. However, once increasing amounts of thyroxine appear in the plasma, mitochondria begin to exhibit increasing percentages of saturated fatty acids, primarily stearic acid (C18:0). 3. Increasing saturation of mitochondrial membrane lipids can be inhibited by exposure of embryonated eggs to 500 R of X-irradiation. 4. Injection of embryonated eggs with estrone increases the proportion of oleic acid (C18:1) in mitochondrial membranes but a balancing increase in palmitic acid (C16:0) enables their lipids to remain more saturated than unsaturated.

The glycosaminoglycan chondroitin-4-sulfate alters progesterone secretion by porcine granulosa cells.

Porcine granulosa cells from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) antral follicles were cultured in monolayer for 2 to 3 days with 0 to 3 mg of chondroitin-4-sulfate (C-4-S)/ml in the presence or absence of 0.5 microgram follicle-stimulating hormone (NIH-FSH-S13)/ml. Testosterone (1.4 microgram/ml) was added to some cultures as substrate for estrogen synthesis. Progesterone and estrogen secreted into the media were measured by radioimmunoassay. Concentrations of C-4-S similar to concentrations of chondroitin sulfates (CS) reported for small antral or atretic follicles inhibited both basal and FSH-stimulated progesterone secretion. Progesterone secretion was not inhibited by C-4-S when pregnenolone was added to the media. Thus 3 beta-hydroxysteroid dehydrogenase activity was not inhibited by C-4-S. Estrogen secretion was also not inhibited by even the highest concentration of C-4-S tested. Testosterone did not influence C-4-S inhibition of progesterone secretion. Granulosa cells from medium-sized follicles were more sensitive to C-4-S than cells from small follicles. Granulosa cells from large follicles were completely resistant to C-4-S inhibition of progesterone secretion. These observations suggest that C-4-S may play a role in altering gonadotrophin-stimulated and basal progesterone secretion in follicles during differentiation of granulosa cells.

Redifferentiation and subsequent dedifferentiation of the livers of roosters resulting from acute estrogen challenges.

1. Histologic and metabolic changes take place in livers of rooster receiving challenges consisting of acute doses of estrone. 2. During initial Growth and Redifferentiation livers rapidly increase in size by division of hepatocytes within most lobules, changing from cordlike to acinar configurations. 3. No new lobules appear and degeneration of some cells within lobules takes place even as cell divisions predominate. 4. Cells within lobules assume secretory features. 5. Vitellogenins, very low density lipoproteins, calcium and alkaline phosphatase increase greatly in plasma. 6. Within 35-40 days of cessation of estrogen, livers have returned to near normal sizes and plasmas exhibit normal parameters.

Effects of thyroxine on mitochondrial morphology and uptake of 35S in embryonic chicken livers.

Ultrastructural analyses of reactions of mitochondria in hepatocytes of chicken embryos to low levels of exogenous thyroxine (T4) reveal that such reactions (overall swelling accompanied by disruption of crest geometry) first take place at about 10 days of incubation, T4 having been administered on the 6th day. Physically altered mitochondria may be seen after 11-12 days of incubation but are no longer evident by 13 days. Correlated with the initial evidence of T4 effects on mitochondria at 10 days of incubation is a spurt in hepatocyte proliferation. The time correlation observed between T4 induced mitochondrial changes in morphology and abrupt increases in rates of cell proliferation, suggests that liver nuclear receptors for thyroxine are unavailable prior to 9-10 days of incubation. Golgi complexes within the hepatocytes appear to be especially active in the production of electron-opaque vesicles from at least the 8th day of incubation to 11-12 days. Uptake of 35S (probably into chondroitin sulphates) was found to be fifteen times greater on the 8th day of incubation than at 15 days. This correlates with the period of heightened activity of the Golgi complex. In livers exposed to T4 on the 6th day, uptake of 35S was higher on the 9th and 10th days of incubation as compared to controls.

Effects of chondroitin-4-sulphate on ultrastructure of ovarian granulosa cells.

Granulosa cells from medium sized porcine ovarian follicles exhibit inhibition of progesterone secretion after both 3 and 6 days of incubation in the presence of 2 mg chondroitin-4-sulphate (C-4-S)/ml. After 3 days of incubation some relatively small and undifferentiated granulosa cells are present in the above cultures. Six days following initiation of incubation in the presence of 2 mg C-4-S/ml many of the larger granulosa cells taken from medium sized follicles reveal lysis and/or resorption of material (probably lipoprotein) from their originally dense staining bodies as well as other indications of stress and degeneration.

Serum lipids and lipoproteins in hypothyroidism.

Obese strain (OS) chickens hatched in the NIU vivarium were observed, primarily with respect to serum lipid and lipoprotein parameters, over a period of 36 weeks. The hypothyroid birds began to show differences in total serum lipids as compared to non-OS controls as early as 4 weeks post-hatching. All lipaemic chickens displayed lack (or deficiency) of thyroid tissue. Although all the thyroid deficient chickens showed increases in serum lipids, they were individually highly variable with respect to both quantity of serum lipid and time of onset of severe lipaemia. Six of twenty four OS birds became severely hyperlipaemic. One of these displayed a level of serum lipid amounting to 323 mg/ml. The increased serum lipid consisted primarily of triglyceride and phospholipid and was not accompanied by corresponding increases in apoproteins B or A-I. However, because of its high content of palmitic acid, oleic acid and phospholipid, the bulk of the serum lipid in OS birds appeared to be derived from the liver rather than directly from the diet. It is concluded that sufficient phospholipid was present in the plasma to form micelles, thus reducing requirements for association of solubilizing apoproteins. Livers were considerably enlarged in hyperlipaemic birds, possibly to compensate for decreases in lipid synthesizing enzymes per unit mass of tissue. However, results of gamma-irradiation of OS chickens (showing exacerbation of hyperlipaemia) indicated that hyperlipaemia in OS birds may be largely due to widespread failure of the body's cells to utilize foodstuffs and of adipose tissue to store excessive amounts of fatty acids as triglyceride.

Production of VLDL and vitellogenin(s) in oestrogen-challenged hypothyroid chickens.

Compared with normal control birds of the same breed, obese strain (OS) White Leghorn chickens, exhibiting classical symptoms of hypothyroidism, were generally half as responsive to an oestrogen challenge in terms of synthesis of VLDL and vitellogenins, despite having livers which were, on the average, twice as large. Even the few OS birds which were laying eggs and therefore enjoying a degree of thyroid function, failed to exhibit normal responses to hyperdynamic doses of oestrogens. However, simultaneous injections of triiodothyronine (T3), in the physiological range, and hyperdynamic doses of oestrogenic substances (Ayerst Co., mainly oestrone) into OS chickens resulted in serum levels of VLDL and vitellogenins which were more than double those achieved without thyroid hormone treatment. Untreated OS birds were found typically to exhibit a unique hyperlipaemia (i.e. increased synthesis of serum triglyceride and phospholipid without corresponding increases in synthesis of apoprotein B (normally associated with VLDL and LDL) or apoprotein A-1 (normally associated with HDL). This type of hyperlipaemia resulted in formation of large lipid micelles which imparted turbidity to the serum. Injection of oestrogenic substances into two OS chickens especially hyperlipaemic in this unique fashion resulted in rapid liver production of apoprotein B. By 2 days post injection the levels of apoprotein B in the sera were increased fifteen times over original levels. The initially turbid sera were completely cleared by 36 h post oestrogen treatment. Since the VLDL present during the first 2 days floated at faster rates than those present at 6 days following injection of oestrogen, it appears that newly synthesized apoprotein B (very likely as lipoprotein B) can combine with existing lipid micelles in the plasma, thus ultimately dispersing the larger lipid micelles into lipoproteins of a size consistent with the loss of turbidity.

Calcium transport in nonmammalian vertebrates.

The title of this paper commemorating the contributions made by Professor Urist has an interesting bearing upon basic skeletal tissue biology. This is because the calcium-binding proteins (vitellogenins), upon which Professor Urist and Schjeide have focused much of their attention in non-mammalian vertebrates, although produced by the liver and present in the blood plasmas of non-mammalian vertebrates during vitellogenesis, are, in effect, substitutes for the protein casein present in the milk of mammalian vertebrates. Vitellogenins (180,000-250,000 daltons) appear to be produced solely for deposition in the yolk of the egg so that the calcium they carry (considerably more than is associated with casein of milk) and the amino acids of which they are comprised can be utilized during embryonic development. In many instances the progeny of non-mammalian vertebrates emerge from the shell as miniatures of the adult, capable of rapid movement and thus requiring a well developed skeletal as well as muscular system. Vitellogenins are not found in any other cells (phagocytes excepted) other than hepatocytes wherein they are made, nor are they present in the intercellular matrix of developing or remodeling bone. In non-vitellogenic females and in males of non-mammalian vertebrates, they are absent from the blood plasma altogether, so that nonionized calcium therein is solely bound to such proteins as albumin and the lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Precocious synthesis of oestrogen-induced vitellogenin by embryos pre-treated with thyroid hormones.

Treatment of 9-day-old chicken embryos with oestrogen results in only barely detectable amounts of induced vitellogenin being present in their sera after 13 days of incubation. Readily detectable amounts of vitellogenin are normally first seen in sera at 16 days of incubation (oestrogen having been administered at 12 days of incubation). However, pre-treatment of such embryos (those above) with thyroxine (T4) at 6-8 days of incubation results in substantial amounts of vitellogenin being present in sera of 14-day-old embryos, with slight increases being detected on the 13th day. Pre-treatment of 6 to 8 day-old chicken embryos with T4 also permits induction by oestrogenic substances of substantial amounts of very low density lipoproteins (VLDL) in sera after 13 days of incubation. Without pre-treatment with T4, oestrogen given at 9 days of incubation provokes only a moderately increased increment of VLDL by 13 days of incubation.

Thyroid hormone-induced precocious growth and maturation of the embryonic chicken liver.

The effects of thyroid hormones thyroxine (T4) or triiodothyronine (T3) on the ontogeny of chicken embryonic liver were studied. Two micrograms of T4 administered to chicken embryos, prior to day 11 of incubation, was found to be least toxic and effective in increasing liver weights, total protein and DNA and RNA, over those of controls. A non-toxic dose of T4 (0.1 microgram) had no effect on embryonic chicken liver. Injection of 125I-labelled T4 or T3 into chick embryos showed that T4 was taken up in greater amounts by the liver than was T3. Uptake of both hormones by the liver increased dramatically around day 9 of incubation. Induction of hypothyroidism by methimazole (a goitrogen) suppressed the natural increase in liver weight.

Changes in expressions of red blood cell antigens following gamma irradiation of chicken embryos.

Red blood cells (RBC) from chicken embryos receiving 450 R of gamma-irradiation on the 6th day of incubation displayed various differences from controls with respect to agglutination properties in the presence of antibodies for three blood group antigens as assayed over a period extending from 12 days of incubation to after hatching. An alloantigen (B21), which normally can be detected prior to 10 days of incubation, showed increased agglutinability following radiation exposure at 6 days. Likewise, an 'embryonic' blood cell antigen, which normally would decrease rapidly after 12 days of incubation, persisted in the blood of irradiated embryos. On the other hand, an alloantigen (B19), which normally appears only after 10 days of incubation, was severely depressed in cases where the embryos received 450 R of gamma-irradiation. The authors propose that in the first two instances gene activation (or induction) for the respective RBC antigens had been accomplished prior to radiation exposure, whereas in the last instance induction had not been accomplished prior to the radiation insult and the gamma-rays had strongly interfered with this process.

Ultrastructural variations following use of several weakly protic alcohols as dehydration agents.

Evaluation has been made of ultrastructural variations which obtain following dehydration and infiltration of glutaraldehyde plus osmium fixed CHO cells with the weakly protic alcohols (methanol, ethanol, ethylene glycol, 1-propanol, 2-propanol, propylene glycol and glycerine), acetone alone, and with use of acetone and propylene oxide as transition solvents subsequent to dehydration with ethanol, methanol and ethylene glycol. It appears that the weakly protic alcohols, applied without transition solvent, in addition to some extraction by themselves, variously condition the components of membranes, etc, for possible further extraction by the embedment resin. Extraction by embedment consists mainy of removal of aggregate or 'corpuscular' units from regions assumed to have originally consisted of endomembrane. Relatively discrete extraction pits in membranes are noted in the cases of methanol and ethylene glycol dehydration without transition solvent; whereas use of the other alcohols such as 1-propanol and 2-propanol accentuate the visualization and staining of mitochondrial ribosomes. Cytoplasmic ribosomes stain more strongly when acetone or ethylene glycol are employed as dehydration agents, but some size (length) differences obtain. Apparently, acetone or propylene oxide transition following dehydration with ethanol or methanol (but not the glycols) assists in, or fixes, alterations of endomembrane components into the familiar 'unit membrane' orientation upon which the embedding substances have little discernible effect. Glycol dehydration and infiltration regimens (especially ethylene glycol alone and as a dehydration agent in the application of Bernhard's (1969) staining technique) result in lesser electron density of heterochromatin while accentuating RNP particles. The increased electron density of heterochromatin while accentuating RNP particles. The increased electron density of the RNP particles and the decrease in density of the heterochromatin result in improved visualization of the relationships among nucleolar RNP particles and the nuclear pore complexes.

Time of appearance of cellular antigens on chicken RBC.

At least twelve genetically distinct systems of RBC membrane alloantigens have thus far been detected in the chicken. Earlier studies have indicated that some of these are 'latent' in that the full development of surface antigen appears only gradually during ontogeny. The present experiments show that the alloantigens B2, B19, E1 and I2 are among those which develop more slowly in the RBC plasma membrane system. These might be utilized as indicators of the state of membrane differentiation during various experimental treatments or disease states.

Uptake of melanosomes and increased melanin formation by cultured melanoma cells after treatment with isolated melanosomes.

Melanosomes isolated from a subcutaneous Harding-Passey mouse melanoma and purified by density gradient centrifugation were labelled in vitro with 14C-tyrosine or 3H-dihydroxyphenylalanine in the melanin portion. Incubation of monolayer cultures of Harding-Passey melanoma cells during exponential growth phase (wherin cells contained relatively few melanosomes) with isolated and labelled melanosomes during a time-period of up to 3 days resulted in rapid cellular uptake of label (reaching a saturation level after about half a day). Following a lag period of several hours, the melanin content rose near-linearly in the course of 3 days. Comparison of curves of uptake of radioactivity and melanin concentration indicates that the latter rise is due primarily to newly formed melanin. Ultrastructural studies revealed a strikingly increased number of melanosomes in melanosome-treated cells. Some of these appeared to be the result of phagocytotic uptake. In fact, invagigations of the plasma membrane containing exogenous melanosomes were observed. Since most intracellular melanosomes were localized directly in the cytoplasmic matrix, dissolution of the phagosome membrane appeared to have taken place. Aggregates of melanosomes either surrounded by a membrane or free in the cytoplasm were also observed. These bodies might represent phagolysosomes or/and centres for the formation of new melanosomes. The combined biochemical and ultrastructural findings suggest stimulated melanogenesis induced by phagocytized melanosomes.

Uptake of iodide into growing chicken oocytes.

Sodium 125iodide was injected intraperitoneally into laying hens and two modes of experimentation utilized to determine the nature of its distribution into plasma and yolk compartments.

Apolipoproteins and lipoprotein families in chicken embryos and egg yolk.

1. Turkey anti-LP-A and anti-LP-B cross react strongly with whole adult chicken serum. 2. Similar reactions occur with whole chicken embryo sera (10-21 days of incubation) and egg yolk fluid. 3. Ultracentrifugation of sera from 14-day old chicken embryos, after reaction with anti-LP-B, reveal complete precipitation of the VLDL and LDL of embryo serum (indicating that adult-type LP-B is the major apolipoprotein of these density classes). 4. All of the chicken embryo adult-type apo-LP-A is in the HDL fraction. 5. However, egg yolk and hen serum VLDL contain apo-LP-A as well as apo-LP-B.

Detection of sulphated carbohydrates in primitive yolk granules of the hen's oocyte.

Application of several staining methods specific for sulphated carbohydrates have indicated the presence of one or more of these macromolecular species in primitive yolk granules of the hen oocyte. The rationale for the various methods used are detailed in the Materials and methods section. Discussion centres on whether the substances taking up stain may be chondroitin sulphates or other sulphated glycosaminoglycans and the possible roles of such molecules in early development.

Effect of "restricted ovulator" gene on uptake of yolk-precursor protein.

The bulk of the yolk proteins and lipoproteins constituting the yolks of mature oocytes in birds are synthesized by the liver and transported via the plasma to the oocytes where they are incorporated by micropinocytosis. Evidence is presented indicating that oocytes of hens possessing a mutation identified by Jones, Briles, and Schjeide as a "restricted ovulator gene" fail to incorporate normal amounts and proportions of low density lipoproteins, lipovitellin and possible other proteins making up the bulk of the yolk material. Plasma albumin is taken into the yolks but the other proteins synthesized by the liver for deposition within the oocytes accumulate in the plasma, attaining very high levels. The possible nature of the "lock" preventing normal deposition of the excluded yolk proteins is discussed.