Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.     

Expression of creatine kinase isoenzyme during oogenesis and embryogenesis in the mouse

Creatine kinase activity was discovered in the growing mouse oocyte and in the preimplantation embryo. Changes in the enzyme activity during the growth and maturation of the egg and during the development of the embryo up to the blastocyst stage were determined. Close similarity of the protein to the brain-type isoenzyme of creatine kinase was established immunochemically. The kinetic parameters of the brain-type isoenzyme (M. R. Iyengar, C. E. Fluellen, and C. W. L. Iyengar, 1982, J. Muscle Cell Motil. 3, 231–246) and the pattern of development-associated changes in activity suggest a possible role for creatine kinase in maintaining the reported high ATP/ADP ratio (L. Ginsberg and N. Hillman, 1975, J. Reprod. Fertil. 43, 83–90), which is essential for the biosynthetic activities of the embryo.     

Efficient extraction and quantitative determination of nanogram amounts of cellular RNA

An assay for quantitating nanogram amounts of cellular RNA is described. RNA is efficiently extracted from cells, using RNA-free DNA as carrier, by conventional chloroform: phenol procedures and the nucleic acids are precipitated with ethanol. Isolated RNA is hydrolyzed by RNase T2 to ribonucleoside 3′-monophosphates which in turn are converted to 5′-³²P-labeled ribonucleoside 3′,5′-diphosphates in the presence of T4 polynucleotide kinase and [γ-³²P]ATP. Radiolabeled products are separated from remaining [γ-³²P]ATP by chromatography on polyethyleneimine-cellulose, located by autoradiography, excised from the chromatogram, and subjected to liquid scintillation counting to quantitate the amount of RNA. Using mouse liver ribosomal RNA as a standard, the assay is linear over a range of 0 to 64 ng of RNA. The assay has been used to determine the amount of RNA in fully grown mouse oocytes arrested at the dietyate stage of first meiotic prophase. Each oocyte contains 0.61 ± 0.05 ng of RNA and only 25 oocytes have been used for such assays.     

An analysis of the fate of the chick wing bud apical ectodermal ridge in culture

Stages 20 and 25 chick apical ectodermal ridge have been cultured in nutrient medium containing fetal bovine serum and the tissues have been examined for dying cells at 0, 6, 12, 18, and 24 hr. By 12 hr, an average of 43% of the cells were dying. By 24 hr, stage 20 ridge had lost its integrity and stage 25 ridge contained an average of 50% dying cells. These results are in agreement with the observations of R. L. Searls and E. Zwilling (1964, Dev. Biol. 9, 38-55) on isolated stage 20 ridge. In subsequent experiments, ridge ectoderm was cultured in serum-containing medium to which insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenium (5 ng/ml) or insulin (5 micrograms/ml) had been added. Under these conditions the ectoderms remained viable even after 24 hr in vitro.     

Quantitative structure-activity relationships of chymotrypsin-ligand interactions: An analysis of interactions in p3 space.

Fourteen derivatives of l-alanine of the type CH₃CH(NHCO-3-C₅H₄N)COOR₃ have been synthesized and their hydrolysis by chymotrypsin was studied with the object of characterizing enzymic space (?₃) to which R₃ binds. The binding of R₃ ($\text{log}\text{1}\text{K}{}_{\mathrm{m}}$) was shown via correlation analysis to correlate with molar refractivity (MR) of R₃ rather than hydrophobicity (π). The results confirmed our earlier predictions. A correlation equation for the hydrolysis of 77 acyl-amino acid esters of the general formula R₂CH(NHCOR₁)COOR₃ relating $\text{log}\text{(k}{}_{\mathrm{<mtext>cat</mtext>}}\text{k}{}_{\mathrm{m}}\text{)}$ to molar refractivity of R₁, R₂, and R₃ and to $\text{σ}{}^1$ (Taft's polar parameter) of R₃ was formulated. The general picture of ligand interactions with chymotrypsin as seen with correlation analysis is discussed.     

Muciliary transport in newt lungs: the ultrastructure of the ciliary apparatus in isolated epithelial sheets and in functional triton-extracted models

High voltage and conventional electron microscopy were used to investigate the ultrastructure of the ciliary apparatus in intact and in Triton-extracted, reactivated sheets of mucociliary epithelium isolated from newt lung. Each long (about 13 microns) ciliary axoneme terminates on a barrel-shaped basal body which is anchored in the apical cytoplasm by a variety of accessory structures. A basal foot is associated with the midpoint of each basal body and acts as a focal point for numerous microtubules (MTs). In many cases MTs can be seen to interconnect the feet of neighbouring basal bodies. Attached to the proximal end of each basal body and extending in a direction opposite the basal foot is a large 'ciliary root'. Each ciliary root is associated with a distinct bundle of 6-7 nm microfilaments which appear to stain with the specific F-actin probe NBD-phallacidin. A single 3-4 microns long striated rootlet inserts into each ciliary root and extends toward the cell nucleus through an extensive network of microfilaments. At the level of the basal plate 'Y-shaped' structures appear to connect each axonemal outer doublet MT to the plasma membrane. All of these ciliary accessory structures are present in the same relationship in Triton-extracted models. Their morphology and distribution indicates that they serve to anchor the cilia in the apical cytoplasm. In addition some of these structures appear to be responsible for maintaining the structural and functional integrity of the ciliary field in the demembranated and reactivated models.     

Cellular and volumetric changes in relation to the activity cycle in the corpora allata of Diploptera punctata

In the corpora allata (CA) of the viviparous cockroach, Diploptera punctata, a cycle of juvenile hormone (JH) synthesis during ovarian maturation can be correlated with cyclical changes in CA volumes and cell numbers. Uptake of [³H]-thymidine occurs in nuclei of CA cells during periods of increase in cell number. Both members of a pair of CA maintain symmetry of volume, cell number and rate of JH synthesis. After a cycle of CA activity, the CA can be transplanted to a young, allatectomized female, where they support a second wave of oöcyte development.     

Digestion of protein substrates by subtilisin: immobilization changes the pattern of products

Subtilisin BPN' (Bacillus protease strain N') was immobilized on glass-bead carriers of controlled pore size by the glutaraldehyde method. The Vmax and Km values of the synthetic substrate were similar for immobilized and free enzymes. However, the hydrolytic patterns of immobilized and free enzymes toward casein and carboxymethylated lysozyme were different. The free enzyme rapidly hydrolyzed the substrate in the early stage of the reaction to produce peptides of various sizes. The immobilized enzyme, however, slowly digested the casein and lysozyme during digestion; even in the late stage of digestion the original substrates were present in the reaction mixture. The peptide size produced by immobilized enzyme depended on the pore size of the carrier; enzyme immobilized on glass of smaller pore size produced smaller peptide products. These phenomena found with our system of immobilized protease and a protein substrate can be explained by a multiple attack mechanism, in which the substrate that has been forced to enter the matrix is attacked many times by the protease to be completely hydrolyzed, because the substrate and the intermediate-sized product are trapped inside the matrix under reduced diffusion movement. To explain the effective digestion that forms amino acids, we have proposed that a multiple type of attack is responsible for the intracellular protein degradation that takes place in cellular organelles in which hydrolytic enzymes are entrapped.     

Thiols related to mitochondrial ATPase and transports: unmasking upon conformational changes supported by the comparative effects of ethacrynate and dihydroethacrynate.

Comparison between the effects on various rat liver mitochondrial functions of ethacrynate, a thiol reagent inhibitor of oxidative phosphorylations [3, 4] and those of dihydroethacrynate its saturated derivative which is not a thiol reagent, has been performed. Both, ethacrynate and dihydroethacrynate increase oxygen consumption by mitochondria in state 4 (succinate as substrate) in a concentration dependent way (from 1 to 5 X 10(-4) M EA or DHEA). This activation is followed, only with ethacrynate, by an inhibition appearing sooner with higher concentrations. After preincubation or mitochondria with ethacrynate (1 to 5 X 10(-4) M), the stimulation of respiration by (ADP + Pi) is completely inhibited whereas it is only weakly affected by dihydroethacrynate at the same concentrations. Ethacrynate and dihydroethacrynate provoke variations of intramitochondrial Mg2+ and K+ levels which need energy from the respiratory chain. These are affected by Pi or (Pi + ADP) in a different way with ethacrynate and with dihydroethacrynate. After preincubation with mitochondria, ethacrynate and to a smaller extent dihydroethacrynate, inhibit partially ADP translocation; ADP increases the inhibitory effect of EA on translocation and not that of dihydroethacrynate. Ethacrynate increases the oligomycin sensitive ATPase activity and dihydroethacrynate still more. After a ten minutes preincubation with mitochondria, ethacrynate and dihydroethacrynate hardly affect the 2.4 DNP stimulated ATPase activity. Preincubation with succinate or ADP strongly increases the ethacrynate inhibition whereas it decreases dihydroethacrynate inhibition. Ethacrynate and dihydroethacrynate do not affect the efflux of Pi produced by ATP hydrolysis but ethacrynate enforces the inhibitory effect of mersalyl (Mg2+ containing medium). After ten minutes of preincubation with mitochondria, ethacrynate binds 25 nmoles of -SH/mg protein (DTNB titration) and dihydroethacrynate has no effect. These results show an effect of ethacrynate on two types of thiols linked with energy conservation mechanisms and ADP translocation. These thiols could be unmasked or made accessible by conformational modifications of the inner membrane upon energization or addition of ADP.     

Biosynthetic approach to the structure of F1-ATPase (BF1 factor) from Micrococcus lysodeikticus membranes: in vivo labeling of the polypeptides and study of their relationship

The F₁-ATPase or BF₁ factor was purified from Micrococcus lysodeikticus substrain B grown in a synthetic medium in the presence of tritiated amino acids. When analyzed in sodium dodecyl sulfate-7% polyacrylamide gels, the fresh purified preparation contained α, β, γ subunits (referred as the intrinsic subunits) and two other polypeptides (designated as X and component of relative mobility 1.0) whose status as subunits remains to be established. This overall polypeptide composition was similar to that of the F₁-ATPase isolated from the same strain grown in complex medium (J. Carreira, J. M. Andreu, M. Nieto, and E. Muñoz., 1976 Mol. Cell. Biochem.10, 67–76). The distribution of ³H-labeled amino acids into purified F₁-ATPase and its constituent polypeptides under different stages of growth was used to investigate the biosynthetic relationship between the different polypeptides. The incorporation of amino acids into purified BF₁ factor was slower than that of cytoplasmic and other membrane proteins. In isotope-dilution and chase experiments, F₁-ATPase showed one of the slowest rates of decay of the incorporated label. These results point out that F₁-ATPase of M. lysodeikticus undergoes slower turnover than the overall cytoplasmic and membrane proteins. Pulse and chase experiments allowed us to conclude that the α, β, γ subunits and the components of relative mobility 1.0 are independent with differences in their turnover and therefore do not bear any apparent relation as precursors-products. The two major subunits represent seemingly the “core” of ATPase, the β subunit behaving like the most stable component. On the other hand, the γ subunit appears to be synthesized independently from this α + β complex.     

The identification of dimethylphenylarsine as a microbial metabolite using a simple method of chemofocusing

Candida humicola acts on benzenearsonic acid to produce dimethylphenylarsine, which was identified by mass spectroscopy following the chemofocusing of the volatile metabolite onto a mercuric chloride impregnated filter. The same technique established that trimethylarsine is the volatile metabolic product obtained from C. humicola treated with 4-NH₂-2-OHC₆H₃AsO(OH)₂ and (CH₃)₃AsO. Arsanilic acid, 4-NH₂C₆H₄AsO(OH)₂, is not metabolized to a volatile arsine.     

An examination of the rate assay to determine the active site normality of papain

Data are presented which demonstrate that the α-N-benzoyl-l-argine ethyl ester rate assay procedure, based on a burst titration with N-benzyloxy-carbonyl-l-tyrosine p-nitrophenyl ester as previously desribed (1), is an accurate and reliable method for determining the normality of papain in solution.     

A new approach to the characterization of the B and A forms of DNA by I.R. spectroscopy

The RNA conformational changes of B, A and C forms are reflected in the infrared absorption spectra in the region of 800 cm^?1 to 900 cm^?1 and allow one to investigate unoriented samples. The transition to the A form is characterized by the appearence of bands at about 870 cm^?1 and at 813 cm^?1 whereas the B and the C forms exhibit a band at 837 cm^?1, these bands undoubtedly arise from phosphate diester stretching vibrations and yield information about backbone conformation. The presence of these infrared bands provides a criterion for testing the simultaneous presence of two coexisting forms of DNA. It represents a useful method for structural studies of nucleic acid complexes such as protein-DNA for which it is difficult to obtain orientation.     

Cycloheximide: An adrenergic agent

Cycloheximide, a widely used inhibitor of protein synthesis, stimulates glycogenolysis, gluconeogenesis and ureogenesis in isolated rat hepatocytes. The effects of cycloheximide were compared to those of norepinephrine. Both agents, cycloheximide and norepinephrine, produced slight increases in the levels of cyclic AMP (30% increases) which were blocked by propranolol. Interestingly, it was found that the metabolic actions of norepinephrine and cycloheximide (stimulation of glycogenolysis, gluconeogenesis and ureogenesis) were only slightly diminished by the β adrenergic antagonist propranolol but abolished by the selective α₁ adrenergic antagonist prazosin. The ability of cycloheximide to inhibit protein synthesis was not affected by either prazosin or propranolol. It is concluded that the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by cycloheximide in rat hepatocytes, is an effect of the antibiotic independent of its ability to inhibit protein synthesis and that is mediated through activation of α₁ adrenoceptors. The adrenergic activity of cycloheximide should be considered when this drug is used as an inhibitor of protein synthesis.     

The determination of the vitamin D metabolites on a single plasma sample: changes during parturition in dairy cows.

Methods have been developed for the precise measurement of the major known vitamin D metabolites in a single sample of cow plasma (~5 ml). The procedure involves initial extraction with methylene chloride-methanol followed by chromatography on Sephadex LH-20. 25-Hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ were determined using high-pressure liquid chromatography and comparing ultraviolet absorption peak height with absorption peak heights of standards. The dihydroxylated metabolites were further purified and resolved by high-pressure liquid chromatography and determined by radioligand binding assays. The assays were employed to measure the total vitamin D metabolite levels in the plasma of paretic and normal dairy cows at parturition. Parturition had no effect on 25-hydroxyvitamin D levels in either group of cows (paretic, 37–44 ng/ml; normal, 35–38 ng/ml). However, normal cows did show lower mean 25-hydroxyvitamin D levels at every sampling period with the lowest levels in both groups occurring at 7 days postpartum. Plasma 25, 26-dihydroxyvitamin D was higher in paretic animals prepartum and at parturition (0.7–1.0 ng/ml) when compared to nonparetic animals (0.4–0.45 ng/ml). Similar levels (0.6 ng/ml) were observed in both groups postpartum. Cows developing parturient paresis showed a significant (P < 0.05) elevation of 1,25-dihydroxyvitamin D at parturition with a maximum level of 350 pg/ml attained at 1 day postpartum compared to prepartum levels of 60 pg/ml. Normal animals also showed a rise in plasma levels of 1,25-dihydroxyvitamin D with a maximum level of 185 pg/ml observed at 1 day postpartum. Plasma 24,25-dihydroxyvitamin D was initially higher in paretic cows (1.9 ng/ml) with a significant (P < 0.05) drop to 1.05 ng/ml occurring at parturition. This level was maintained for 7 days postpartum. The levels of this steroid were maintained at 1.3–1.4 ng/ml in the normal cows throughout the entire sampling period.     

Temporal changes in embryonic nerve cell recognition: correlate with cholinergic development in aggregate cultures

Chick embryo retina and optic tectum cells can be dissociated into single cells and then reaggregated in suspension cultures to give highly organized and differentiated aggregates. These aggregates show a degree of cholinergic differentiation that is characteristic of each cell type; the low activity of choline acetyltransferase in the optic tectum aggregates probably reflects the condition of natural deafferentation inherent in the culture situation. It is possible, in this respect, to study the retina-tectum interaction in vitro by preparing coaggregates including both types of cells. When coaggregates are prepared from tectum and retina cells of the same developmental age, the activity of choline acetyltransferase measured in the coaggregates is consistently higher than would be expected from the simple addition of the activities of the component cells, pointing to some kind of metabolic synergism between retinal and tectal cells. As for acetylcholinesterase, this synergism occurs only under special circumstances, and it is generally less marked. No synergism was observed when retina and tectum cells of different developmental age were coaggregated, suggesting the existence of a temporal control of neuronal interaction specificity. On the other hand, the synergism is only observed between neuronal systems that are known to establish synaptic connections during normal in vivo development: No interaction could be detected when either retinal or tectal cells were combined with telencephalon, cerebellum, or liver cells. Experimental evidence is presented suggesting that the retina-tectum interaction depends on intimate cell-cell contact, and it is not mediated by freely diffusible molecules. Neurotransmission-related metabolic studies in coaggregates seem to offer a promising tool to study recognition-interaction phenomena in groups of neurons establishing synaptic links during development.     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.