Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes

Oocytes induced to undergo meiotic maturation by progesterone possess a cytoplasmic activity that causes germinal vesicle breakdown (GVBD). The cytoplasmic factor postulated to be responsible for this activity is designated as the maturation promoting factor (MPF). The activity of MPF was assayed by injecting cytoplasm into fully-grown oocytes to induce GVBD. It was found that maturing oocyte cytoplasm possesses MPF activity before GVBD begins. Treatment of progesterone stimulated oocytes with cycloheximide, either applied externally or injected, inhibited the appearance of MPF in the cytoplasm as well as GVBD when the inhibitor treatment was initiated before the cytoplasm exhibited MPF activity. In contrast, the same treatment did not inhibit GVBD when it was applied to oocytes after the cytoplasm possessed MPF activity. Furthermore, cycloheximide treatment of recipient oocytes did not inhibit the induction of GVBD by injected cytoplasm containing MPF. Cytoplasm of oocytes injected with MPF subsequently possessed MPF activity as high as that of the original donor cytoplasm in spite of its extensive dilution. This suggests that amplification of MPF took place in the recipient. Cycloheximide treatment did not inhibit the amplification of MPF. It was concluded that cycloheximide inhibits only the initial phase of induction of MPF activity, but neither its amplification nor its action on the nucleus that causes GVBD. From these results, a hypothesis concerning the cytoplasmic mechanism for the induction of GVBD has been proposed.     

Induction of meiotic maturation in mouse oocytes by adenosine analogs

In this study we have examined the meiosis-inducing influence of adenosine analogs in mouse oocytes. When a varied group of nucleosides and nucleotides were tested on overnight cultures of hypoxanthine-arrested, cumulus cell-enclosed oocytes (CEO), halogenated adenosine nucleosides, but not native adenosine, exhibited a significant meiosis-inducing capability. When tested under a variety of conditions, meiotic induction by 8-bromo-adenosine (8-Br-Ado) and a second adenosine analog, methylmercaptopurine riboside (MMPR), was especially potent in denuded oocytes (DO) compared to CEO and was not dependent on the type of inhibitor chosen to maintain meiotic arrest. Germinal vesicle breakdown (GVB) was stimulated with rapid kinetics and was preceded by an increase in AMP-activated protein kinase (AMPK) activity. Moreover, compound C, an inhibitor of AMPK, blocked the meiosis-inducing activities of both adenosine analogs. When tested for an effect on meiotic progression to metaphase II (MII) in spontaneously maturing CEO, 8-Br-Ado and the AMPK activator, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), increased the percentage of MII-stage oocytes, but MMPR decreased this number. Adenosine and inhibitors of de novo purine synthesis had no effect on the completion of maturation, while compound C suppressed this process. These results support the proposition that oocyte AMPK mediates the positive influence of AICAR and 8-Br-Ado on both the initiation and completion of meiotic maturation. The role of AMPK in MMPR action is less clear.     

Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) prevents ornithine decarboxylase inhibition and apoptosis in L1210 leukemic cells exposed to TGF-β1

Previous studies have shown that growth suppression and apoptosis of leukemic cells exposed to TGF-β₁ is associated with the inhibition of ornithine decarboxylase (ODC) — the key enzyme of polyamine pathway. The aim of the present study was to evaluate the influence of 12-O-tetradecanoylphorbol 13-acetate (TPA) — a potent ODC inducer on antiproliferative and apoptotic effects of TGF-β₁ in L1210 leukemic cells. Cells were incubated in 2%FCS/RPMI1640 medium, supplemented with TGF-β₁ (2 ng/ml), TPA (100 ng/ml) or -difluoromethyl-ornithine (DFMO) (5 mM). Cell proliferation, apoptosis and necrosis were evaluated using [methyl-³H] thymidine, electron microscopy, electrophoresis of DNA and trypan blue exclusion. Expression and activity of ODC were determinated by RT-PCR and measurement of ¹⁴CO₂ release from L-1-¹⁴C ornithine, respectively. TGF-β₁ inhibited proliferation and induced apoptotic and necrotic cell death in L1210 leukemic cells. The above effects were associated with the inhibition of ODC expression and activity, measured 2 and 4 hr after TGF-β₁ administration, respectively. The presence of DFMO, an irreversible inhibitor of ODC, led to apoptotic fragmentation of DNA, similar to that observed in TGF-β₁-treated cultures. Administration of TPA simultaneously with TGF-β₁ significantly reduced antiproliferative, apoptotic and necrotic effects of TGF-β₁, and prevented its inhibitory action on ODC expression and activity. It is concluded that: down-regulation of ODC expression may be one of the early events associated with TGF-β₁-evoked suppression of growth and apoptosis; ODC is involved in the mechanism of protective action of TPA on TGF-β₁-related growth inhibition of L1210 leukemic cells.     

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate)

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.     

Induction and inhibition of meiotic maturation of follicle-enclosed porcine oocytes in vitro

Isolated porcine Graafian follicles which were explanted in vitro and maintained in organ culture were used as a test-system for the meiosis-inducing action of PMSG and hCG. The addition of either PMSG or hCG alone (10 or 20 IU/ml, respectively) to the culture medium was not effective, whereas the simultaneous administration of these hormones ($\text{15}\text{15}\text{IU/ml}$) induced resumption of meiosis in 90.3% ($\text{37}\text{41}$). The same hormone concentrations were used in a second series of experiments in which the inhibition and induction of meiosis of isolated oocytes were tested by transferring them into host follicles. In host follicles containing up to 12 foreign eggs, which were cultured in control media, meiosis was prevented in 86.0% of all oocytes ($\text{104}\text{121}$). By adding PMSG (15 IU/ml) simultaneously with hCG (15 IU/ml) to the medium, meiosis was induced in 95.0% of all oocytes ($\text{133}\text{140}$).The assumption is made that the signal initiating resumption of meiosis of the isolated and transferred oocytes is mediated by the follicular fluid, since intimate contact with the membrana granulosa of the host follicle was prevented by using a roller technique.     

Analysis of zeins' ER retention in Xenopus oocytes

Zeins, maize storage proteins, are retained in the endoplasmic reticulum (ER) during the intracellular protein targeting process. Hydrophobic interaction has been postulated as the driving force of zeins' aggregation and retention in the ER. Recently, a class of zein (the 27K zein) has been proposed to facilitate zeins' ER retention by anchoring to the ER membrane. This study investigated the significance of the two proposed mechanisms toward zeins' ER retention using Xenopus oocyte. Following injection of the total or 27K zein mRNA, zein's movement within the ER was analyzed based upon the extent of diffusion to the non-injected oocyte half. This study indicates that the total zeins freely move within the lumen of the ER, thus, suggesting that the intermolecular aggregation, leading to insolubility and exclusion from the ER lumenal fluid, may not be essential for zeins' ER retention. This study also suggests that the 27K zein may not facilitate zeins' ER retention by virtue of an anchor to the ER membrane based on its free movement in the ER. Free movement of the total and 27K zeins, under conditions where zein aggregates should form, necessitates a reevaluation of the mechanisms responsible for zein polypeptides' ER retention and protein body formation.     

Polyadenylation of histone mRNA in Xenopus oocytes and embryos

Oogenesis of amphibians is an atypical situation in which histone mRNA is polyadenylated. The poly(A) tract on H4 mRNA has been examined by Sl nuclease analysis. Throughout oogenesis the poly(A) tract is very short, and nonexistent on some mRNA molecules. The poly(A) tract is completely removed during maturation of the oocyte, and is absent in embryos and cultured cells.     

Regulation of transglutaminase 1 gene expression by 12-O-tetradecanoylphorbol-13-acetate, dexamethasone, and retinoic acid in cultured human keratinocytes

Transglutaminase 1 (TG1) is an enzyme that is expressed at the late stage of terminal differentiation of keratinocytes and catalyzes the ε-(γ-glutamyl)lysine cross-linking reaction to form a highly insoluble cell envelope. To elucidate the mechanism of TG1 gene expression in keratinocytes, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), dexamethasone, 1,25-dihydroxyvitamin D₃, and retinoic acid on the levels of TG1 mRNA in cultured normal human epidermal keratinocytes (NHEK). Treatment of NHEK with TPA, Up to 10 nM, markedly increased the levels of TG1 mRNA in a dose-dependent manner. The effect by treatment with 1 nM TPA reached a peak after 16 h of incubation (20-fold above the basal level). In contrast, phorbol had no effect on TG1 gene expression. The induction of TG1 mRNA expression by TPA was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine. Dexamethasone at a concentration of 1 μM also increased the TG1 mRNA levels, but the maximum induction was observed (3-fold above the basal level) after 72 h of incubation. The effect of dexamethasone was not suppressed by H-7. Moreover, 1 μM of retinoic acid completely inhibited the induction of TG1 mRNA by both TPA and dexamethasone. 1,25-Dihydroxyvitamin D₃ showed no effect on the TG1 mRNA levels. From these results, we suggest that the expression of TG1 gene may be upregulated by protein kinase C and glucocorticoid receptor systems and down-regulated by the retinoic acid receptor system.     

β-Adrenoceptor antagonists reinitiate meiotic maturation in Clarias batrachus oocytes

Effects of β-adrenoceptor antagonists propranolol and alprenolol in the oocyte maturation of the catfish (Clarias batrachus) were investigated under in vitro. Cyclic AMP (cAMP) levels were also measured in the control, propranolol and phosphodiesterase (PDE) inhibitor treated oocytes. When full-grown folliculated oocytes were cultured in vitro in the presence of different concentrations of propranolol or alprenolol, both the substances induced germinal vesicle breakdown (GVBD) in a dose-dependent manner. The maturational effect of alprenolol at the concentration of 1.0 mM was similar to that of the 1.5 mM dose of propranolol inducing more than 88% GVBD. In the time course study, when the oocytes were treated with 1.5 mM propranolol or with 1.0 mM alprenolol for various times, both the antagonists induced more than 80% GVBD after 4 h of incubations and this induction gradually increased with the increased duration of treatments. On the other hand, 1.5 mM propranolol treatment caused a significant decrease in oocyte cAMP which was maintained upto the duration of the study (36 h). When the oocytes were first stimulated by 1.5 mM propranolol or 1.0 mM alprenolol for 4 h and then treated with various doses of cAMP or PDE inhibitors (IBMX and theophylline), all these substances effectively blocked β-adrenoceptor antagonist-induced GVBD. Both these PDE inhibitors promoted the accumulation of cAMP in the oocytes. These results provide the first example of an existence of a cAMP-mediated mechanism of action of β-adrenoceptor antagonists in the induction of oocyte maturation in fish.     

Effects of 12-O-tetradecanylphorbol 13-acetate (TPA) on a clonal human teratoma-derived embryonal carcinoma cell line

The response of a human embryonal carcinoma cell line LICR LON HT39/7 to 12-O-tetradecanylphorbol 13-acetate (TPA) has been studied. Cells treated with 5 ng/ml of TPA undergo marked morphological changes, becoming flattened with nuclear enlargement and developing a grainy and often vacuolated cytoplasm. Parallel changes in the cell surface phenotype of the treated cells also occur. These include the appearance of membrane fibronectin, the embryonic antigen SSEA-1, and a glycoprotein antigen recognised by a monoclonal antibody. There is also increased expression of histocompatibility antigens. Other membrane molecules, such as peanut agglutinin receptor(s) and a 200 000 membrane glycoprotein appear to be removed from the membrane following TPA treatment. The high levels of alkaline phosphatase normally present in LICR LON HT39/7 are also reduced by TPA. Changes in the ultrastructure of the cells have also been observed, such as increases in nuclear complexity and in the number of intermediate filaments in the treated cells. This latter observation has been confirmed by immunofluorescent staining of the cells for prekeratins, which show an extensive network following the addition of TPA, but not before. 2-Dimensional gel electrophoresis of the proteins synthesized by LICR LON HT39/7 before and after addition of TPA has shown that there are a number of alterations in the proteins synthesised by the treated cells. Furthermore, immunoprecipitation of the culture supernatants from these cells has shown that TPA induces the synthesis and secretion of fibronectin. The alterations in the phenotype of LICR LON HT39/7 induced by TPA are irreversible and the altered cells, whilst they stop dividing, can be maintained for at least three weeks in culture. The analogue of TPA 12-O-tetradecanylphorbol 13-myristate does not produce the effects described above.     

[H]thymidine incorporation in bovine lymphocytes treated with the tumor promoter, 12-O-tetradecanoyl phorobol-13-acetate

Treatment of bovine lymph node lymphocytes with the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) leads to depressed [³H]thymidine incorporation in response to phytohemagglutinin (PHA). Radioautographic and morphological analyses showed that depression was at the level of blast-cell formation. Isotope-dilution experiments, and the use of [³H]deoxycytidine to label DNA indicated that the inhibition was not due to a block in thymidine transport in the treated cells. These experiments, as well as a bioassay designed to measure thymidine in the culture medium, showed that the apparent inhibition of [³H]thymidine incorporation and DNA synthesis was not the result of production of cold thymidine in the cultures. The results taken together support the idea that most TPA-treated cells are inhibited from responding to the mitogenic lectins. Those cells which do respond appear to form blast cells and synthesize DNA at the same rate as do untreated cells.     

The localization of the nuclear protein pool in Xenopus oocytes

It has been demonstrated previously that nuclear proteins in Xenopus oocytes are synthesized in the cytoplasm and maintained in a cellular pool. The present study was performed to determine if any portion of this pool is associated specifically with the nuclear envelope. This was accomplished by first micro-injecting oocytes with [³H]leucine; at various times after injection, nuclear envelope and nucleoplasmic fractions were run on SDS-polyacrylamide gels. In this way labeled polypeptides available in the envelope fraction could be compared to polypeptides which were subsequently incorporated into the nucleoplasm. No evidence was obtained that the nuclear protein pool is associated with the envelope.     

Maturing Xenopus oocytes induce chromosome condensation in somatic plant nuclei

Isolated nuclei of plant (Daucus carota) protoplasts were injected into immature and maturing Xenopus oocytes. In the first series the transplanted nuclei continued to synthesize RNA for at least 18 h, whereas in the latter series the nuclear membrane was disrupted and the chromatin was induced to condense prematurely within 30 min. The results of this study suggest that fundamental biological processes like chromosome condensation during mitosis and meiosis —which are no different in animal and plant kingdom from a morphological point of view—are also very similar with respect to the underlying regulatory control.     

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA. This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility, Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, MD 20205.     

Hyperpolarization-activated Cl current elicited by pituitary adenylate cyclase activating polypeptide in Xenopus oocytes

We examined the electrophysiological effect of pituitary adenylate cyclase activating polypeptide (PACAP) in isolated Xenopus laevis oocytes in vitro. In conventional two-electrode voltage clamp experiments, PACAP (1–10 μM) activated an inward rectifier current at membrane potentials more negative than −60 mV without causing any significant change in currents at potentials more positive than −60 mV both in the follicle-enclosed oocyte and in the defolliculated oocyte. This current reversed at −22.5 mV, close to the theoretical value of Cl⁻ equilibrium potential and the reversal potential of this current was shifted positively by reducing [Cl⁻]_o. This current was blocked by Cl⁻ channel blocker SITS and Ba²⁺. Furthermore, VIP and adenylate cyclase activator forskolin did not elicit the currents. In conclusion, PACAP elicited the hyperpolarization-activated Cl⁻ current in Xenopus laevis oocytes. This current may modulate the membrane potential of the oocyte, thereby affecting the oocyte physiology.     

Integrated microsystem for non-invasive electrophysiological measurements on Xenopus oocytes

We propose a new non-invasive integrated microsystem for electrophysiological measurements on Xenopus laevis oocytes. Xenopus oocyte is a well-known expression system for various kinds of ion channels, that are potential tools in drug screening. In the traditional “Two Electrode Voltage Clamp” (TEVC) method, delicate micromanipulation is required to impale an oocyte with two microelectrodes. In our system, a non-invasive electrical access to the cytoplasm is provided by permeabilizing the cell membrane with an ionophore (e.g. nystatin). Unlike the classical patch-clamp or “macropatch” techniques, this method does not require removal of the vitelline membrane. Cell handling is significantly simplified, resulting in more robust recordings with increased throughput. Moreover, because only part of the oocyte surface is exposed to reagents, the required volume of reagent solutions could be reduced by an order of magnitude compared to the TEVC method. The fabrication process for this disposable microchip, based on poly-dimethylsiloxane (PDMS) molding and glass/PDMS bonding, is cost-efficient and simple. We tested this new microdevice by recording currents in oocytes expressing the human Epithelial Sodium Channel (hENaC) for membrane potentials between −100 and +50 mV. We recorded benzamil-sensitive currents with a large signal-to-noise ratio and we also obtained a benzamil concentration–inhibition curve displaying an inhibition constant IC₅₀ of about 50 nM, comparable to previously published values obtained with the TEVC technique.     

Intracellular transport of vitellogenin in Xenopus oocytes: An autoradiographic study

The role of primordial yolk platelets (PYPs) in the transport of the yolk precursor vitellogenin to the yolk platelets in Xenopus laevis oocytes has been demonstrated by electron microscopic autoradiography. Within 20 min after exposure of the oocyte to ³H-labeled-vitellogenin, silver grains are associated with small PYPs which are formed by the fusion of endosomes. At 40 min after incorporation of ³H-labeled vitellogenin, autoradiographic silver grains are associated with larger PYPs and with the superficial layer of yolk platelets. Thus, the results demonstrate that PYPs are an intermediate in the transport of vitellogenin from endosomes to yolk platelets. These observations are consonant with the general hypothesis that vitellogenin first associates (binds?) with the plasma membrane, then is incorporated by endocytosis into endosomes which fuse to form PYPs, and finally the contents of the PYPs are eventually deposited into yolk platelets.