A comparison of the distribution of muscle type in the tadpole tails of Xenopus laevis and Rana temporaria: an histological and ultrastructural study

The types and the distribution of muscle fibres were analysed and compared in the tails of Xenopus laevis and Rana temporaria tadpoles. The filter feeding tadpoles of X. laevis were found to have both white muscle fibres adjacent to the notochord used for normal locomotory swimming and a superficial layer of small red fibres. The red fibres are probably used for the continuous flickering movement of the tail associated with the maintenance of the mid-water filter feeding position. R. temporaria, a grazing detritus feeding tadpole, was found to have only white muscle fibres used for normal locomotory swimming. Smaller superficial fibres were not red fibres but were thought to be immature white fibres.     

The nuclei of follicular cells do not control oocyte maturation in amphibians induced by gonadotropic hormones in vitro]

The inhibitory effect of actinomycin D (5 micrograms/ml) on maturation of the follicle-enclosed oocytes of Rana temporaria and Xenopus laevis induced in vitro by pituitary suspension and human chorionic gonadotropin, respectively, is not observed at low concentrations of the hormones. These data suggest that nuclei of the follicle cells are not involved in the control of the pituitary-dependent oocyte maturation in amphibians.     

A method for hormonal induction of sperm release in anurans (eight species) and in vitro fertilization in Lepidobatrachus species

Injections of synthetic human gonadotropin releasing hormone (GnRH) into the dorsal pelvic area were used in an attempt to stimulate sperm release in isolated males of eight anuran species including Xenopus laevis, Rana pipiens and Lepidobatrachus laevis . Sperm were obtained within 1–5 h post injection either by mechanical stimulation or by cloacal lavage. Sperm suspensions varied from 8 μL to 7 mL and the cell densities ranged from 4 × 10⁵ to 4 × 10⁷ sperm/mL. The sperm obtained from seven species using GnRH-induced release were viable based on light microscopic observations of motility. In addition, sperm preparations fertilized eggs in vitro and produced normal tadpoles in the case of L. laevis and L. llanensis . This hormonal method of anuran sperm collection will provide a convenient non-injurious way to obtain anuran sperm for basic studies of reproduction and development.     

The cytostatic effect of the cytoplasm of mature, non-activated and cleaving eggs of Rana temporaria, Acipenser stellatus and Xenopus laevis

Corroborated here is the fact, earlier established by Chulitskaya and Felgengauer (1977), that the cytoplasm of mature non-activated eggs of Rana temporaria and Acipenser stellatus, unlike that of Rana pipiens, exerts no cytostatic effect on the nuclei of the cleaving embryo, but acquires such a capacity after being treated with EGTA. EGTA treatment imparts cytostatic properties also to the cytoplasm of cleaving embryos. Revealed is the dependence of the cytostatic effect and death of injected embryos on the dose of EGTA introduced into the egg. No mitotic figures have been detected in embryos with a cytostatic effect. Upon reciprocal transplantation of the cytoplasm between Rana temporaria and Xenopus laevis, only the latter's cytoplasm possessed a cytostatic effect, while the arrest at a metaphase was found only in a few arrested blastomeres.     

The natural stimulus for spawning in Xenopus laevis (Amphibia)

I have already reported (Savage, 1965), preliminary experiments in which "natural spawning", that is, spawning by frogs kept under as natural conditions as aquaria allow, and by frogs not injected or manipulated in any other way, had been induced by a variety of external stimuli, including unialgal cultures of various kinds.
These experiments were planned and executed using as a guide, at every possible point, the statistical investigations of Rana temporaria obtained entirely from field data (Savage, 1935, 1961). The reasons for using Xenopus laevis and not R. temporaria were purely practical. R. temporaria breeds but once a year, and has other disadvantages, but X. laevis is a good laboratory animal.
Since conclusions drawn from mathematical relations in one species have led without important deviation to the expected experimental results in another, it is unlikely that what I now have to report is peculiar to these two species, selected because of availability in one case and ease of working in another. The conclusions are probably quite general. The earlier conclusions have been extended, active materials have been isolated, and other factors relevant to reproduction have been found.     

Oocyte maturation and activation in the common frog and the clawed toad under the action of divalent cations]

Maturation of Rana temporaria and Xenopus laevis oocytes was induced by solutions containing Mn2+ and Co2+ ions. Completion of oocyte maturation was estimated by the following criteria: (1) appearance of the maturation promoting factor (MPF) in the oocyte cytoplasm and (2) oocyte capacity to activation and formation of male pronuclei from the injected sperm nuclei. X. laevis oocytes matured under the effect of Co2+ ions were shown to contain MPF. Oocytes of both species matured under the effect of either ions could not be activated by pricking with a needle and injected sperm nuclei didn't transform into pronuclei. R. temporaria oocytes matured under the effect of ions in late spring, when natural spawning takes place, showed spontaneous activation.     

Is caerulein amphibian CCK?

The amphibian skin decapeptide caerulein is structurally related to the mammalian peptides gastrin and CCK, suggesting that the peptides might share a common evolutionary history. It has been suggested that caerulein is the amphibian counterpart of gastrin and CCK, and that the Amphibia do not possess authentic gastric and CCK. High Performance Liquid Chromatography (HPLC) in conjunction with radioimmunoassay using a caerulein-specific antiserum and C-terminal CCK antisera, was used to characterize CCK-and caerulein-like peptides in amphibian brain and gut. In the brain of Xenopus laevis, two CCK-like peptides were present, one of which was indistinguishable by HPLC from mammalian CCK8. No decapeptide caerulein was detected in the brain of Xenopus laevis or Rana temporaria. In the stomach of Xenopus and in the intestine of both species studied, CCK-like and caerulein-like peptides were present. The results indicate therefore that the Amphibia possess CCK8-like rather than caerulein-like peptides in brain. In contrast, stomach and intestine contain both CCK-like and caerulein-like peptides, but the latter are however distinguishable from the decapeptide found in skin.     

Tail muscle hydrolases during metamorphosis of the anuran tadpole. I. Histochemical studies

Naphthylamidase, beta-glucuronidase and trimetaphosphatase have been studied histochemically in the tail muscles of Rana catesbeiana, Rana japonica and Xenopus laevis tadpoles. During the early stages of growth, tadpole tail muscles showed very low activity of these hydrolases, whereas during the prometamorphic and metamorphic stages, they exhibited a marked increase in enzyme activity. This was localized in the subepidermal and subnotochordal mesenchymous tissue and in the myoseptum, i.e. between the muscle bundles.     

Ammonia as a pheromone in anuran tadpoles

In laboratory experiments, conspecific excretions and ammonia solutions evoked avoidance reactions in tadpoles of three anuran species, Bufo bufo, Rana temporaria, and Rana arvalis. A differential sensitivity of ammonia chemoreception was determined for two anuran species. For Bufo bufo tadpoles, these characteristics at ammonia background concentration of 0.2 mg/l lied in the range 150 % < dI/I < 500 %, and for background of 0.4 mg/l the value lied in the range 400 % < dI/I < 500 %. For Rana temporaria tadpoles, differential threshold against ammonia background concentration of 0.15 mg/l was close to 200 % and against background ammonia concentration of 1.1 mg/l was close to 100 %. These results suggest that such sensitivity of both anurans is sufficient for using ammonia in intra- and interspecies communication.     

Early steroid metabolism in Xenopus laevis, Rana temporaria and Triturus vulgaris embryos.

Embryos of Xenopus laevis, Rana temporaria and Triturus vulgaris exposed to radioactive pregnenolone have been found to convert it to progesterone. Incubations with radioactive progesterone showed that it was actively metabolized by oocytes and embryos. In Xenopus incubations progesterone was converted to 5alpha-pregnane-3,20-dione, 17alpha-hydroxy-4-pregnen-3-one, 4-androstene-3,17-dione and 17alpha-20alpha-dihydroxy-4-pregne-3-one, indicating 5alpha-reductase, 17alpha-hydroxylase, 19-20-desmolase and 20alpha-hydroxylase activities. In oocytes of Triturus and Rana no evidence of 19-20-desmolase was found. In Rana oocytes were also not evidence of 17alpha-hydroxylase activity. All identified activities except 20alpha-hydroxylase were common to embryos of all three species. It is suggested that the steroid enzyme activities present in the embryos are not solely derived from the oocytes but synthentized during early development. Possible meaning of this kind of metabolism during differentiation remains open.     

The main adenosine triphosphate-binding component of amphibian oocytes is ferritin.

After fractionation of mitochondrion-free extracts of Xenopus laevis and Rana temporaria oocytes in sucrose gradients, a distinct peak of adenosine triphosphate (ATP)/guanosine triphosphate (GTP)-binding activity in the 50-70 S range has been detected. This substance has a boyant density in Cs2SO4 of 1.45 g/cm3. The nucleotide-binding substance has been purified to apparent homogenety. By means of electron microscopy, sodium dodecyl sulfate-electrophoresis and other methods it has been identified as ferritin.     

Early Steroid Metabolism in Xenopus laevis, Rana temporaria and Triturus vulgaris Embryos

Embryos of Xenopus laevis , Rana temporaria and Triturus vulgaris exposed to radioactive pregnenolone have been found to convert it to progesterone. Incubations with radioactive progesterone showed that it was actively metabolized by oocytes and embryos.
In Xenopus incubations progesterone was converted to 5α-pregnane-3,20-dione, 17α-hydroxy-4pregnen-3-one, 4-androstene-3,17-dione and 17α,20α:-dihydroxy-4-pregnen-3-one, indicating 5α-reductase, 17α-hydroxylase, 19–20-desmolase and 20α-hydroxylase activities. In oocytes of Triturus and Rana no evidence of 19–20-desmolase was found. In Rana oocytes were also not evidence of 17α-hydroxylase activity. All identified activities except 20α-hydroxylase were common to embryos of all three species.
It is suggested that the steroid enzyme activities present in the embryos are not solely derived from the oocytes but synthetized during early development. Possible meaning of this kind of metabolism during differentiation remains open.     

The effect of the composition of the medium on the concentration of free calcium ions in the cells of the follicular wall in the common frog and in the clawed toad]

The concentration of intracellular free calcium ions in the follicle wall cells and in the follicle cells of Rana temporaria in Ringer solution is 150 +/- 10 and in the follicle wall cells of Xenopus laevis, 220 +/- 10 nM. In a chloride-free saline, its concentration in the same cells is 2.5-3 times that in Ringer solution. Voltage-dependent Ca(2+)-channel blockers diltiazem and verapamil (100 microM) reduce the level of intracellular free calcium ions in R. temporaria follicle wall cells cultivated in a chloride-free saline to 170 +/- 20 nM, which practically does not differ from the level in Ringer solution. Inhibitors (100 microM) decrease the rate of "spontaneous" maturation of R. temporaria follicle-enclosed oocytes both in chloride-free and Ringer solutions. It was concluded that an increased level of intracellular free calcium ions in the follicle cells, among other factors, may determine the stimulating effect of the medium (Ringer or chloride-free solution) on "spontaneous" maturation of follicle-enclosed amphibian oocytes. Voltage-dependent calcium channels appear to be involved in Ca2+ influx into the cells.     

Maturation of Rana temporaria and Acipenser stellatus oocytes induced by the cytoplasm of embryos at different cell cycle phases and at different stages of cleavage and blastulation

Experiments involving injections of cytoplasm from the cleaving embryos of Rana temporaria and Acipenser stellatus into fully grown oocytes of the same species and of Xenopus laevis, show that at all mitotic phases, and throughout the period of synchronous cleavage divisions, the cytoplasm is characterized by high activity of the germinal vesicle breakdown factor. This activity decreases during nuclear division desynchronization and drops sharply after the mitotic index fall upon blastulation. Aside from germinal vesicle breakdown in the oocytes, the A. stellatus embryos' cytoplasm also induces development of a cortical reaction capacity.     

Genomic potential of erythroid and leukocytic cells of Rana pipiens analyzed by nuclear transfer into diplotene and maturing oocytes

In order to determine whether differentiated somatic cells maintain genetic totipotency, nuclear transplantations from several differentiated somatic cell types into eggs and oocytes were performed previously in Rana pipiens and Xenopus laevis. The formation of postneurula embryos and tadpoles under the direction of the test nuclei demonstrated their genetic multipotency. In addition, Rana erythrocyte nuclei transplanted to oocytes directed more extensive tadpole development than those injected into eggs. We have extended our studies of the genomic potential of differentiated somatic nuclei from the peripheral blood of Rana pipiens. First, we show that the developmental potential of erythrocyte nuclei injected into oocytes at first meiotic metaphase was greater than those injected into diplotene oocytes. Second, we demonstrate that erythroblast and leukocyte nuclei transplanted to oocytes at first meiotic metaphase promoted more advanced tadpole development than those previously injected into Xenopus eggs. Third, erythrocyte nuclei were more successful in promoting advanced tadpole development compared with erythroblast and leukocyte nuclei. The results show that differentiated somatic nuclei transferred to the cytoplasm of oocytes at first meiotic metaphase display enhanced genomic and developmental potential over those transplanted to diplotene oocytes and eggs, at least for the three nuclear cell types tested from the peripheral blood.     

Immunochemical markers of embryonic lens differentiation in Rana temporaria. II. Immunohistochemical analysis of the manifestation and localization of individual classes of lens proteins]

Individual lens proteins were studied during development of Rana temporaria. Antisera to alpha-, beta-crystallins of chicks and gamma-crystallins of Rana ridibunda were used as immunochemical markers. Besides the main crystallins, a new antigen was found in the R. temporaria lens tentatively called alphabeta-crystallin. It appears to be characteristic only for the amphibian lens. Using the indirect method of fluorescent antibodies, it was shown that all the antigens under study appeared in the lens of the R. temporaria tadpoles within 1--2 days (at 20 degrees). The crystallins are found initially only in the developing lens fibers and later in the lens epithelium. It was established that the lens epithelium contained gamma-crystallins which appeared somewhat earlier than alpha- and beta-crystallins, but simultaneously with alphabeta-crystallin.     

The epidermis and its degeneration in the larval tail and adult body of Rana temporaria and Xenopus laevis (Amphibia: Anura)

The tail epidermis of the larva and the body epidermis of adults of Rana temporaria and Xenopus laevis are described in terms of electron microscopy.
The activity of lysosomes (determined by the localization of acid phosphatase) in relation to autolysis and the process of cellular cornification, is considered during the periods of climactic disappearance of the larval tail and skin sloughing of adults. The results obtained generally correspond for both genera.
Larval tail epidermal cells completely disappear at metamorphic climax; those of the adult, which are shed, are replaced throughout life after each periodic sloughing. Nevertheless the mechanisms of their epidermal cell loss are comparable, though the level of lysosomal activity in larval tail epidermal cells is higher than in the adult body epidermis. This higher activity of lysosomal enzymes may facilitate the heavy necrosis which ensues in the larval tail at metamorphic climax.     

Effects of high gravity on amphibian development.

In order to clarify the possible effects of high gravity environments on eggs and developing embryos, Rana rugosa and Xenopus laevis fertilized eggs and early embryos were raised in 2 G, 5 G, 7 G and 10 G up to the hatched tadpole stage. The results showed that: (1) High gravity significantly retarded the development of eggs and embryos beginning treatment before the blastula stage and induced various abnormalities, including two heads and microcephally suggesting that high gravity is apt to disrupt the animal-vegital axis. On the other hand, embryos beginning treatment after the gastrula stage showed a striking increase in the number of normal-appearing feeding tadpoles. (2) Autopsy revealed that brains, notochords and muscles were reduced in development and differentiation for embryos and tadpoles developed in high gravity. (3) It seems likely that the system for hydrogen peroxide detoxification develops abnormally in high gravity-treated embryos and tadpoles, which probably results in oxidative stress, leading to considerable cell damage.     

Isolation and characterization of a distantly related member of the beta-gamma crystallin super gene family from Xenopus

In an attempt to isolate gamma crystallin genes from Xenopus genomic library, we have obtained seventeen clones which strongly hybridized to the gamma 1 cDNA probe from Rana temporaria. Of these four clones are characterized. A restriction map was derived by using single and multiple digests with a variety of enzymes. Hybridization selection of lens mRNAs and in vitro translation produced a protein of 26,000 daltons. These data suggest that the above clones may contain at least part of the gamma or beta crystallin sequences.