Effects of gonadotropins and steroids on the subsequent fertilizability of extrafollicular bovine oocytes cultured in vitro

Effects of gonadotropins (2 i.u./ml follicle stimulating hormone, FSH and 10 μg/ml luteinizing hormone, LH) and steroids (1 μg/ml oestradiol, E and progesterone, P) on the fertilizability of extrafollicular bovine oocytes cultured in vitro and transferred in either the rabbit oviduct (Experiment I) or glass test tubes (Experiment II) were investigated. Bovine oocytes collected from follicles of 2–5 mm in diameter were cultured in vitro for 27 h in a medium containing Ham's F-12, 20% (v/v) bovine fetal serum and antibiotics. The combination of the hormones added to the medium was as follows; (1) none (control), (2) E, (3) LH, (4) LH + E, (5) FSH + LH + E, and (6) FSH + LH + E + P. All oocytes were recovered 24 h after insemination and examined for the presence of the pronuclei and a sperm tail with the midpiece in the oocyte cytoplasm, and the extrusion of the second polar body.In Experiment I, 630 of 704 transferred oocytes (85.7%) were recovered from the rabbit oviduct. The maturation rates of these oocytes (overall 61.1%) were not significantly affected by gonadotropins and steroids. Of the 741 of 920 oocytes recovered from test tubes in Experiment II, the maturation rates of them (overall 64.2%) were significantly increased (P < 0.05) by addition of LH (72.9%) and FSH + LH + E + P (74.1%) as compared with controls (55.4%). Fertilization rates were increased (P < 0.05) by the addition of FSH + LH + E compared with the controls in both Experiments I (31.1% and 14.0%) and II (36.2% and 20.8%). Furthermore, the proportion of fertilized eggs in hormone treated groups was the highest in each experiment. The present study indicates that the addition of FSH, LH and E to a medium has improved the fertilizability of extrafollicular bovine oocytes cultured in vitro.     

Effects of substance P and neurotensin on growth hormone and thyrotropin release in vivo and in vitro

Conscious ovariectomized (OVX) rats bearing a cannula implanted in the third ventricle were injected with 2 μl of 0.9% NaCl containing varying doses of substance P (SP) or neurotensin (NT) and plasma GH and TSH levels were measured by RIA in jugular blood samples drawn through an indwelling silastic catheter. Control injections of physiologic saline iv or into the third ventricle did not modify plasma hormone levels. Intraventricular injection of SP or NT at doses of either 0.5 or 2 μg elevated plasma GH concentrations within 5 min and they remained elevated for 60 min. Third ventricular injection of similar doses of SP or NT had no effect on plasma TSH. An intermediate dose of 1 μg of SP or NT given iv had no effect on plasma GH but NT elevated plasma TSH. Incubation of hemipituitaries from OVX rats with varying doses of SP or NT did not alter GH release into the medium but TSH release was enhanced with NT at doses of 100 or more ng/ml of medium. It is suggested that SP acts centrally to stimulate growth hormone-releasing factor (GRF) or to inhibit somatostatin release and thereby enhance GH release and that NT acts directly on the pituitary to stimulate TSH release.     

Collagen in developing chick muscle in vivo and in vitro

Because of the known role of collagen in chick skeletal muscle differentiation the collagen synthesized by embryonic chick muscle was studied. The major collagen synthesized by this muscle was found to be type I collagen. In addition, the effectiveness of types I, II, III and IV collagens in promoting myoblast fusion in vitro was compared. These collagens were found to be equally effective as in vitro substrates.     

Autophagocytosis: in vitro induction by microtuble poisons.

In the rat liver in vivo autophagy has been shown to be induced by glucagon, cyclic AMP (cAMP), dibuturyl-cyclic AMP (db-cAMP) and vinblastine administration. Vinblastine and demecolcine were shown in this study to be potent inducers of autophagy also in Ehrlich ascites tumor cells in vitro. It is suggested that this effect is rather dependent on direct effect on membranes of endoplasmic reticulum than on the breakdown of microtubules by these compounds.     

Effects of polyamines on in vitro phosphorylation and acetylation of histones of the cerebral cortex of rats of various ages

$\text{In}\text{vitro}$ phosphorylation and acetylation of histones and their modulation by spermine and spermidine were studied using slices of cerebral cortex of female rats of various ages. Phosphorylation and acetylation of individual histones decrease with increasing age. Spermine and spermidine have stimulatory effects on both the modifications of specific histones in immature rats. These effects decrease with increasing age. Such changes in covalent modifications of histones may alter gene expression and contribute to the aging process.     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

Characterization of Ehrlich ascites tumor cell messenger RNA specifying ribosomal proteins by translation in vitro

We have examined the messenger RNA which codes for the ribosomal proteins in Ehrlich ascites tumor cells. Poly(A)-containing mRNA was isolated from polysomes and fractionated into 11 size classes whose average molecular weights were between 1.8 × 10⁵ and 24 × 10⁵. These mRNAs were used to direct protein synthesis in a fractionated translational system that was derived completely from Ehrlich ascites tumor cells. More than 90% of the ribosomal proteins which we could identify were coded for by mRNAs averaging in size between Mr = 180 × 10³ and 320 × 10³. The small size of these mRNAs indicates that the cytoplasmic mRNAs which specify the ribosomal proteins are monocistronic. We could detect the synthesis of 36 of 48 ribosomal reference proteins as well as 20 additional polypeptides which had characteristics similar to ribosomal protein. The ribosomal proteins were identified on the basis of their positive charge, small size, electrophoretic properties on two-dimensional polyacrylamide gels and chromatographic characteristics on carboxymethyl-cellulose.     

The non-selective junctional communication phenotype of normal and transformed human epidermal keratinocytes in vitro

The ability of cells to transfer tritium-labelled nucleotides to other cells is a measure of gap-junction-mediated communication based on metabolic cooperation. Here we report that human epidermal keratinocytes (HuK) transfer radiolabel to a variety of cell types including human skin fibroblasts (HuDF), human mammary fibroblasts (HuMF) and calf lens epithelial cells (CaLE). The metabolic cooperation pattern of HuK shows them to be non-selective communicators and in this respect they differ from another human epithelial cell type mammary epithelial cells (HuME), which communicates with CaLE but not HuDF or HuMF. SV40 transformation is known to modulate metabolic cooperation between some human cells in vitro. Here we further report that SV40 transformation of HuK has no obvious effect on their non-selective communication phenotype. Possible mechanisms involved in selective and non-selective junctional communication and their relevance to epithelial/stromal interactions in vivo are discussed.     

The effects of prostacyclin PGI2 on prolactin secretion in vitro

Continuously superfused rat anterior pituitary cells were used to study the effects of exogenous prostaglandins (PGs) and thromboxanes (TXz) on the secretion of prolactin (PRL). No change in hormone release was observed upon superfusion with TXB₂ (10⁻⁵M) or the TX synthesis inhibitor, imidazole (1.5 mM). PGs A2, B2, d2, e1, e2, f1α, F2α, and endoperoxide analogs, U-44069 and U-46619, also had no effect on PRL secretion (all at 10⁻⁵M), In contrast 10⁻⁵M PGI2 was repeteadly found to stimulate PRL release to a level at least 125% above control, while producing no apparent change in the amount of hormone secreted in response to TRH. Somatostatin (SRIF), at a dose of 10⁻M, maximally inhibited TRH-induces PRL output, but failed to alter the PRL response to PGI₂. These studies indicate that PGI₂ may have a direct effect on the anterior pituitary to modify PRL secretion.     

Binding of the carcinogens 5-fluoro-7-hydroxymethyl-12-methylbenz(a)anthracene and its acetate ester to DNA in vitro

Binding of 5-fluoro-7-hydroxymethyl-12-methylbenz(a)anthracene to calf thymus DNA was negligible (1.2 μmole hydrocarbon/mole DNA-P) in the absence of microsomal enzymes whereas in the presence of liver microsomes from unpretreated rats or from rats pretreated with 3-methylcholanthrene binding was greatly enhanced (11.6 and 16.2 μmole hydrocarbon/mole DNA-P respectively). In contrast, the acetate ester of 5-fluoro-7-hydroxymethyl-12-methylbenz(a)anthracene readily bound to DNA non-enzymatically (9.1 μmole hydrocarbon/mole DNA-P). In the presence of a 3′-phosphoadenosine-5′-phosphosulfate (PAPS) generating system, the binding of 5-fluoro-7-hydroxymethyl-12-methylbenz(a)anthracene was independent of sulfate ion. ATP enhanced non-enzymatic binding of 5-fluoro-7-hydroxymethyl-12-methylbenz(a)anthracene to DNA whereas CTP, β,γ-methylene-ATP, and ADP were much less effective suggesting a certain specificity for adenosine in addition to a high energy triphosphate for high binding. These observations suggest that 5-fluoro-7-hydroxymethyl-12-methylbenz(a)anthracene may be converted to a phosphate ester which, like 5-fluoro-7-acetoxymethyl-12-methylbenz(a)anthracene, readily binds to DNA.     

Effect of human high density lipoproteins, anti-apolipoproteins CII and CIII, and hydrolysis of very low density lipoprotein (VLDL) cholesterol ester on VLDL catabolism in vitro

Lipolysis of human very low density lipoproteins (VLDL) by lipoprotein lipase (LPL) was inhibited in the presence of high density lipoproteins (HDL), anti-apolipoprotein (apo) CII, and by increasing the VLDL free cholesterol content but not with anti-apo CIII or lipoprotein-free plasma. The experiments lend direct evidence that the composition of VLDL and their milieu are important determinants of lipolysis by LPL. Apo CIII may not be critical in LPL mediated VLDL catabolism.     

Mg-2,4-divinyl pheoporphyrin a5: the product of a reaction catalyzed in vitro by developing chloroplasts

The major product of an aerobic reaction mixture containing developing chloroplasts, Mg-protoporphyrin IX, S-adenosylmethionine, and other cofactors was isolated and purified. Structural studies using nuclear magnetic resonance confirmed earlier reports, based on fluorescence and absorption spectra, that this compound is Mg-2,4-divinyl pheoporphyrin a5. The molecular weight determined by secondary-ion mass spectroscopy further confirmed the assigned structure. Absorption and fluorescence spectra indicate that this compound is identical to that reported previously by various workers in less-purified biological extracts. The nuclear magnetic resonance spectrum of the Mg-free base also supports the assigned structure.     

Specificity of codon recognition by Escherichia coli tRNALeu isoaccepting species determined by protein synthesis in vitro directed by phage RNA.

Codon-anticodon recognition and transfer RNA utilization for the leucine tRNA isoaccepting species of Escherichia coli have been studied by protein synthesis in vitro directed by sequenced bacteriophage MS2 RNA. We have added radioactive Leu-tRNA^Leu isoaccepting species as tracers, rather than use a tRNA-dependent system, since in the presence of an excess of non-radioactive leucine, there is no transfer of radioactive leucine from one isoaccepting species to another. MS2-specific peptides containing leucine residues encoded by known codons were isolated and identified, and the relative abilities of the Leu-tRNA^Leu isoaccepting species to transfer leucine into these peptides compared. Sequenced tRNA₁^Leu and sequenced tRNA₃^Leu are of roughly equal efficiency in their ability to recognize CUC and CUA codons, while tRNA₃^Leu is highly preferred for the CUU codon; tRNA₄^Leu and tRNA₅^Leu both recognize UUA and UUG codons, with tRNA₄^Leu slightly preferred for the UUA codon. We conclude that: (1) wobble is greater than permitted by the wobble hypothesis; (2) there is still some discrimination in the third code letter, and that the CUX₄ (CUC, CUA, CUU, CUG) portion of the leucine family of six codons is not read by a simple “two out of three” mechanism; (3) a Watson-Crick pair (C · G) between codon and anticodon does not appear to be preferred over an unorthodox pair (C · C) in the wobble position; (4) a standard wobble pair (U · G) between codon and anticodon is preferred over an unorthodox pair (U · C); and (5) the extensive wobble observed in the CUX₄ leucine codon series is not paralleled in the UUX₄ leucine (UUG, UUA) and phenylalanine (UUU, UUC) codon series, where mistranslation would be the consequence of such wobble.     

Characterization of a macrophage chemotactic lymphokine produced by purified protein derivative stimulation in vitro and in vivo

Using an immunoadsorbent column conjugated with anti-macrophage chemotactic factor-c (anti-MCF-c), MCF-c which has been separated and highly purified from a delayed-type hypersensitivity reaction (DHR) site, shares common antigenicity with the major macrophage chemotactic lymphokine released from purified protein derivative (PPD)-stimulated lymphocytes and also macrophage chemotactic lymphokine from phytohemagglutinin (PHA)-stimulated lymphocytes. Using a combination of the immunoadsorbent column and Sephadexgel chromatography these two lymphokines are purified to homogeneity from PPD- or PHA-stimulated guinea pig lymphocyte culture supernatants. These observations, taken in conjunction with the similarity in biological activities, physicochemical properties, and antigenicities, suggest that these two mediators are very similar, or possibly identical. MCF-c with chemotactic activity for macrophages seemed to exist as complexes with serum protein at the skin site of PPD-induced DHR in guinea pigs. The active substance, separated from the complexes under acid conditions, is indistinguishable from the major macrophage chemotactic lymphokine released by PPD stimulation with respect to antigenicity, heat stability, and non-diffusibility. They both have a molecular weight of about 12, 500. The chemotactic lymphokine formed comparable complexes with serum protein under neutral conditions; however, this complex dissociated in acid without loss of activity.     

Guinea pig T-cell in vitro proliferative responses to tyrosine-azobenzenearsonate-pulsed and azobenzenearsonate-coupled macrophages have different specificities

The cell interactions involved in azobenzenearsonate-N-acetyl-tyrosine (ABA-tyr)-induced delayed hypersensitivity in the guinea pig were studied by in vitro blastogenesis. The ABA-sensitive lymphocyte was demonstrated to be a T lymphocyte and its presence in peritoneal exudate cells was shown to be much higher than spleen or lymph node populations. The secondary response of ABA-sensitized lymphocytes to ABA-tyr in culture is dependent on the presence of an accessory cell, with both splenic and peritoneal macrophages being equally effective. ABA coupled directly to macrophages as an immunogen induced strong responses to itself and not to ABA-tyr-pulsed macrophages or ABA-tyr in solution. The reverse was true in animals, immunized with ABA-tyr. ABA conjugated to thymocytes, L₂C leukemia cells, and guinea pig erythrocytes however, did not elicit significant responses. The results obtained in animals immunized with ABA- or ABA-tyr-modified cells was similar whether or not CFA was used. The difference in specificity shown between ABA-coupled and ABA-tyr-pulsed macrophages favors a single receptor hypothesis for T-cell recognition.     

Synthesis and first in vitro cytotoxicity studies of bis(2-chloroethyl) amino group containing polymers. Pharmacologically active polymers: 22

Monomers containing the cytotoxic bis(2-chloroethyl)amino group (nor-nitrogen mustard), linked in deactivated form via urethane and O-acylated hydroxamic acid bonds to polymerizable methacrylic acid derivatives, have been prepared. Besides the homopolymerization, these monomers were copolymerized with hydrophilic comonomers to obtain water-soluble polymers. The linkages used are expected to undergo intracellular enzymatic or hydrolytic cleavage, releasing the cytotoxic bis(2-chloroethyl)amino moiety from the polymeric carrier. Preliminary in vitro cytotoxicity data for the polymers against three rat and mouse experimental tumour lines (Walker 256 carcinosarcoma, L1210 murine leukaemia and ADJ/PC6A plasma cell tumour) are reported. An approximately 10² fold difference in cytotoxic potency was found, depending on the type of the cleavable spacer group.     

In vitro histogenic capacities of limb mesenchyme from various stage mouse embryos

Mesenchyme cells isolated from mouse embryo forelimb buds (stages 15 through 21) and placed in high-density micromass cultures are compared with respect to their in vitro histogenic capacities. Particular emphasis is placed on changes in in vitro chondrogenic capacity. Stage 15 mouse limb cultures form numerous aggregates which uniformly fail to differentiate into cartilage nodules. On the other hand, cartilage nodules are observed in cultures prepared from all subsequent stage limbs, although there is a linear decrease in the size of nodules between stage 16–17 and middle-late stage 21 cultures. This decrease correlates with simultaneous decreases in both the proportion of aggregating cells and the extent of dibutyryl cyclic AMP-stimulated cartilage formation. At the same time, observations indicate that the proportions of nonaggregating and nonchondrogenic mesenchyme, myogenic cells, and, perhaps, fibrogenic mesenchyme are increasing. The only exceptions to these patterns are observed in cultures from middle-late stage 21 limbs, when cartilage differentiation in situ is already extensive. Unlike earlier stage cultures, which form nearly identical numbers of aggregates and nodules, middle-late stage 21 cultures form variable numbers of aggregates, only a few of which differentiate into cartilage nodules. Middle-late stage 21 cultures also contain unexpectedly low numbers of myogenic cells/unit area of culture. Based on changes in the in vitro histogenic capacities, it is concluded that concurrent with a progression of morphogenic events in the limb, there is a progression of changes in the relative proportions of cell subpopulations. Both the existence of the different subpopulations and the changes in their relative proportions can be detected in vitro. Furthermore, it is concluded that cartilage formation in the limbs of both mouse and chick embryos probably occurs according to very similar developmental programs.     

Antigen-inhibited B-lymphocyte differentiation. X. Sedimentation velocity characterization of antigen-binding cells and antibody-forming-cell progenitors in the in vitro microculture immune response of unprimed CBA mice to NIP--POL antigen.

The B lymphocytes serving as antibody-forming-cell (AFC) progenitors have been investigated using two different types of separation methods. Sedimentation velocity fractionation was used to separate subsets of B lymphocytes differing primarily in size. Fractionation on a 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)-gelatin matrix was used to separate NIP-binding cells, a population highly enriched for cells with surface Ig receptors specific for the NIP hapten. Assessment of the functional capacity of the separated B cells was by culture at limiting dilution in the presence of thymus “filler” cells, using the T-independent antigen NIP-POL (polymerized bacterial flagellin) to induce antibody formation. Splenic AFC-progenitors from both adult conventional and neonatal germ-free mice were a physically heterogeneous population, with activity in small, medium, and large lymphocytes. The cells enriched by NIP-gelatin binding, whether isolated and counted directly or isolated and assayed as AFC-progenitors, were no less heterogeneous. These NIP-binding cells resembled in sedimentation characteristics the overall B-cell and overall NIP-specific AFC-progenitor populations, except for some relative enrichment of medium-sized cells (S value, 5.5 mm/hr). The small (S value, 3.4 mm/hr) B-cell region of adult mouse spleen contained both NIP-binding cells and cells responsive as AFC-progenitors in the microculture assay. This contrasts with the results of the in vivo adoptive immune assay, where the smaller B-cell region is unresponsive in unprimed adult animals.     

Fibrin membrane endowed with biological function. VII. An approach to an artificial liver; conversion of ammonia to urea in vitro

As an “artificial liver” for the conversion of ammonia to urea, a group of enzymes in ornithine cycle together with carbamyl phosphate synthetase I and inorganic pyrophosphatase were embedded in a single fibrin membrane. The immobilized enzyme system thus prepared had an ability to convert ammonia to urea not only in a buffer solution but also in human plasma.