Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

Functional expression of urea channels in amphibian oocytes injected with frog urinary bladder mRNA.

In amphibian urinary bladder epithelium, vasopressin increases passive urea permeability, concomitant with the appearance of a facilitated urea transport. Amphibian oocytes from Xenopus laevis and Rana esculenta were microinjected with total or fractionated poly(A+) RNA isolated from frog urinary bladder epithelial cells. After several (3-5) days at 18 degrees C, the urea flux was assayed by measuring the uptake and efflux of [14C]urea in water-injected and mRNA-injected oocytes. A 2 to 3-fold increase of urea transport was detected in oocytes injected either with total mRNA or with a 6-10 kilobase mRNA fraction, when compared with water-injected oocytes. This expression of urea channels was inhibited by 0.1 mM phloretin (50% inhibition) and 0.1 mM nitrophenylthiourea (up to 70% inhibition). On the contrary, no expression was detected in brain mRNA-injected oocytes. These results show the specific functional expression of the phloretin- and NPTU-sensitive urea channel (or carrier) from frog urinary bladder epithelial cells, providing an approach for the expression cloning of these urea channels.     

Transplantation of insect giant chromosome nuclei into amphibian oocytes

Polytene salivary gland nuclei of Chironomus pallidivittatus were transplanted into oocytes of Xenopus laevis which were then cultured in vitro for 18 h. The giant chromosomes and nucleoli as well as the entire nuclei enlarged considerably in volume during this time. The polyteny and specific chromomere pattern of the chromosomes were maintained, and the puffing of the salivary gland-specific Balbiani rings was not noticeably changed. — Polytene nuclei from differentiated insect cells transplanted into Xenopus oocytes thus appear suited for exposing giant chromosomes in vivo to purified factors such as regulatory molecules. This paper is dedicated to Professor Wolfgang Beermann on the occasion of his 60th birthday     

Expression of active alpha-3 subunit of rat brain Na+,K+-ATPase from the messenger RNA injected into Xenopus oocytes

Cloned cDNA encoding the so-far uncharacterized alpha-3 subunit of rat brain Na+,K+-ATPase (Hara et al. (1987) J. Biochem. 102, 43-58, Shull et al. (1986) Biochemistry 25, 8125-8132) was incorporated into a vector carrying the SP6 promoter. The mRNA produced in vitro was injected into Xenopus oocytes with the mRNA encoding the Na+,K+-ATPase beta subunit of Torpedo electroplax. Increased Na+,K+-ATPase activity in the oocyte membrane was observed. This newly expressed activity was inhibited by ouabain (Ki = 1.5 x 10(-7) M), suggesting that the alpha-3 subunit of rat brain Na+,K+-ATPase is a highly ouabain-sensitive catalytic subunit.     

Translation of homologous RNA injected into the ciliate Stylonychia mytilus.

Evidence is presented that RNA isolated from $\text{Stylonychia}$ becomes translated when injected back into $\text{Stylonychia}$ cells and that the RNA, synthesized about 1 hour after interaction of $\text{Stylonychia}$ with Con A contains sequences specifically concerned with the first regeneration steps after Con A damage. The use of microinjection combined with a homologous system is discussed.     

A study of the effects of trypan blue injected into amphibian zygotes

Summary Trypan blue was injected (0.001–0.002 mg/egg) into zygotes ofRana. The trypan blue-injected series showed much greater mortality than the controls. A probable suppression of gastrulation was seen. 29.7% of the dye-injected and 50.3% of the control embryos developed beyond stage 14. Abnormalities seemed to appear only after neurulation began. Microcephaly, trunk and tail abnormalities occurred though in a small number. Sections of a few embryos revealed disorganized brain and degeneration of the neural tissue. There were no cases of mesodermalization of the notochord.The results are discussed from the view point that trypan blue alters the physical state of proteins in the cytoplasm of the egg.     

Nuclear transfer into mouse oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).     

In vivo synthesis and turnover of cytoplasmic ribosomal RNA by stage 6 oocytes of Xenopus laevis.

Cytoplasmic ribosomal RNA (rRNA) synthesis was detected in white-banded stage 6 oocytes taken from female Xenopus laevis which were injected with [³H]guanosine 7 days previously. The specific radioactivity of the rRNA in oocytes collected from injected females by weekly laparotomies displays first-order exponential decay. Calculated values for the half-life of rRNA ranged from 9.1–30.9 days in experiments on four animals. The concept of ribosomes in large ovarian oocytes of amphibians as an absolutely stable, long-term storage product appears incorrect.