Fourth Day Of Culture Research Articles

Direct induction by estradiol of vitellogenin synthesis in organ cultures of male xenopus laevis liver

Organ cultures of liver from untreated male Xenopus respond to 17β-estradiol in the culture medium by synthesizing and secreting the yolk protein precursor vitellogenin. Vitellogenin synthesis, as a primary response, is first detectable on the fourth day of culture, and comprises up to 12% of the protein synthesized on the eight day. Estradiol is required during the lag period of 4 days. Tissue from male Xenopus injected with 1 mg of estradiol 30 days before the start of culture responds more rapidly and to a greater extent to estradiol in the medium than tissue from uninjected males. During such a secondary response, vitellogenin is first detectable in the medium on the second day of culture, and becomes up to 24% of the protein synthesized on day 6. The rate of amino acid incorporation into total protein also increases in response to estradiol, but the rate of synthesis of albumin decreases rapidly in culture whether or not estradiol is present, in both the primary and secondary responses. A maximal response is seen with 10 −8 M estradiol. Progesterone, testosterone, dexamethasone, and insulin neither induce vitellogenin synthesis in culture nor modify the response to estradiol. DNA synthesis inhibitors do not prevent the response to estradiol in vitro, suggesting that cell division is not required for the initial response leading to vitellogenin synthesis.

Effect of 5-bromodeoxyuridine on ultrastructure of developing limb-bud cells in vitro

Limb-bud cells differentiate into chondrocytes, myocytes, fibrocytes and epithelial cells in vivo. While detailed morphological descriptions of developmental events have been presented, mechanisms governing differentiation of cells in vivo are not yet apparent. Limb-bud cells can be grown in tissue culture and will differentiate into the same cell types as occur in vivo. Chondrogenesis in vitro appears to mimic differentiation in vivo as evaluated both biochemically and ultrastructurally. Incorporation of H 2 35SO 4 into glycosaminoglycans increases markedly on the fourth day in culture. At the same time, cells assume the morphology of mature chondrocytes and synthesize extracellular matrix composed of 250–400Å proteoglycan granules and poorly cross-banded collagen fibers. Myocytes can be detected as early as 21 hr after initiation of cell cultures and contain giant polysomes and thick and thin filaments. When early embryonic limb-bud cells are treated with 5-bromo-2′-deoxyuridine (BrdUrd), myogenesis is diminished and differentiation into chondrocytes is prevented irreversibly. Incorporation of H 2 35SO 4 remains depressed throughout the culture period despite removal of the drug after the second day of growth. Development of organelles associated with synthesis of extracellular material (rough endoplasmic reticulum and Golgi apparatus) is not prevented by treating limb-bud cells with BrdUrd. Extracellular matrix consists primarily of bundles of collagen fibers.

Choline acetyltransferase and acetylcholinesterase in cultured brain cells from chick embryos.

Abstract—Dissociated cells from brains of 7‐day chick embryos were grown in primary culture for as long as 20 days. Many of the plated cells grew out long processes. Others, which proliferated rapidly, formed a confluent layer of flat cells after 4‐6 days. Total DNA and protein increased five‐fold, and activity of choline acetyltransferase (EC2.3.1.6) increased about 40‐fold in 20 days. Acetylcholinesterase (EC3.1.1.7) increased three‐fold by the fourth day of culture and then declined. The pattern of increase for choline acetyltransferase was similar to that for the in vivo development of the enzyme. l‐Thyroxine, cyclic AMP (adenosine‐3′,5′‐monophosphate) or theophylline promoted increased levels of both enzymes by 30‐200 per cent. l‐Thyroxine also increased the activity of acetylcholinesterase in vivo by 40 per cent. When overgrowth by flat cells was prevented by the addition of 10‐3m‐5‐flourouracil, there was a decrease in the activity of choline acetyltransferase and an increase in the activity of acetylcholinesterase in comparison to control activities. The addition of 10‐3m‐morphine or cocaine produced a 30 per cent elevation in the activity of choline acetyltransferase, but this effect could be mimicked with equimolar concentrations of ammonium ion.

Mammalian testes in organ culture

Testes of 14-day-old rats were grown as organ cultures for periods of up to 6 months. The effect of various culture media, gas phases, pH's and incubation temperatures was investigated. Tubular structure with healthy Sertoli cells and mitoses was well maintained under various culture conditions, including growth in chemically defined medium. Spermatocytes survived approximately four weeks without differentiating into spermatids. A cell type, resembling primitive germ cells of younger animals, consistently appeared in seminiferous tubules on the fourth day of culture. Since these cells are not normally observed in the testes of 14-day-old rats, their possible origin in culture is discussed.

STUDIES ON GROWTH IN ORGAN CULTURE OF TESTICULAR TISSUE FROM RATS OF VARIOUS AGES.

AbstractTesticular fragments from newborn, 4, 14, 24, 37‐day old and mature rats were cultured using a modification of Trowell's method. Eagle's medium supplemented with amino acids, Na‐pyruvate and 10% calf serum was employed. Tissue fragments were fixed at time intervals for histologic study. In cultures of fragments from newborn and four‐day rats, gonocytes persisted and showed mitotic activity. In fragments from older but still immature animals, gonocyte‐like cells occasionally undergoing mitosis, appeared in the germinal epithelium after the fourth day in culture. Spermatocytes survived for approximately four weeks. More rapid degeneration of the germinal elements beyond spermatogonia was observed in cultures of mature testes. During the first six days in culture, cells resembling gonocytes, some in mitosis, appeared in the tubules. The studies have shown that under the conditions employed, Sertoli cells and gonocyte‐like could be maintained for at least four months in cultured fragments of testes from animals of various ages. In fragments from older animals, gonocyte‐like cells appeared shortly after initiation of culture while the more mature germinal elements ultimately degenerated.