Hypothalamic neurosecretion and metamorphosis in Xenopus laevis

Summary Normal and propylthiouracil (PTU) treated Xenopus laevis tadpoles were fixed during all stages of metamorphosis and sagittal sections were stained with aldehyde fuchsin (AF) or pseudoisocyanine (PIC). Whereas AF positive neurosecretory material could only be demonstrated in the preoptic nucleus from late prometamorphosis, increasing amounts of PIC positive material were found in cells of the dorsal part of the preoptic region from early premetamorphosis. The development of these cells correlated with that of the thyrotropic cells and the thyroids. Likewise, signs of hyperactivity in thyroids and thyrotropic cells of PTU treated larvae were accompanied by a depletion of the dorsal PIC positive cells. In the ventral preoptic region PIC positive cells developed from late prometamorphosis in control larvae, but failed to do so in PTU treated animals. It is argued that the differentiation of the PIC positive cells is largely dependent on thyroid hormones; that the dorsal PIC positive cells may produce a thyrotropin releasing factor; and that the function of these dorsal cells is inhibited by thyroid hormones.The authors thank Dr. P. G. W. J. van Oordt for his active interest and helpful advices, Miss Tineke Aafjes for technical assistance and Mr. H. van Kooten for making the photographs.     

Hypothalamic neurosecretion and metamorphosis inXenopus laevis

Summary The frontal brain was removed from premetamorphic larvae ofXenopus laevis. Part of the animals was kept in a 0.01% propylthiouracil (PTU) solution for 21, 30 or 90 days, together with unoperated controls. Others were reared in tap water for the same length of time. The hypothalamus, the pituitary and the thyroids were studied in sagittal sections stained with aldehyde fuchsin or pseudoisocyanine (PIC).Operated tadpoles lacking the telencephalon only, could not be distinguished from unoperated controls. Extirpation of the preoptic region of the hypothalamus lowered the activity of the thyrotropic (TSH) cells and of the thyroids, and blocked metamorphosis. In animals with intact PIC-positive cells in the dorsal preoptic region, PTU induced a degranulation of these cells, stimulated the TSH cells and caused a strong hypertrophy of the thyroids. It is concluded that these neurosecretory cells in the dorsal preoptic region produce a thyrotropin-releasing factor (TRF), indispensable for the endocrine regulation of metamorphosis, and that the thyroid hormones have a negative feed-back influence upon the TRF cells.In part of the animals lacking the rostral hypothalamus, new PIC-positive cells were observed to develop immediately behind the preoptic region. In these animals PTU was somewhat less effective than in the animals with an intact dorsal preoptic region. This means either that the newly differentiated PIC-positive cells have a TRF function or that the thyroids can exert a direct negative feed-back upon the TSH cells. The latter possibility is supported by the fact that even in the absence of any neurosecretory cell PTU stimulated the thyroids.Blocking of thyroid hormone production by PTU inhibited the differentiation of the hypothalamus, of the median eminence and of the portal vessels. It may be deducted that during metamorphosis the general morphogenetic effect of the thyroid hormones stimulates the differentiation of the structures necessary for the augmentation of the TSH activity.The author thanks Prof. Dr.P.G.W.J. van Oordt for his active interest and helpful advices, MissTineke Aafjes for technical assistance and Mr.H. van Kooten for making the diagrams and photographs.     

Hypothalamic control of the pars intermedia in Xenopus laevis tadpoles

Summary In Xenopus laevis tadpoles the relation between a paired nucleus of bio-amine producing neurons in the caudal hypothalamus and the pars intermedia of the hypophysis was studied.Treatment of the animals (stage 49 to 50 of Nieuwkoop and Faber's normal table) with reserpine caused aggregation of the skin melanophores within one hour, followed by redispersion five to six hours after the beginning of the experiment. This was at exactly the same time as the bio-amines in the caudal hypothalamus disappeared. However, the drug was ineffective if the nuclei had been removed. This indicates that reserpine acts via these nuclei and does not influence the skin melanophores directly.It was concluded that the initial aggregation of the melanophores may be the result of a reduced extrusion of MSH from the pars intermedia, caused by an increased output of a MIF by the bio-amine producing nuclei. The redispersion was explained by assuming that the bio-amines were depleted and the nuclei stopped with the extrusion of the MIF. This does not mean that the production of a MIF is the only function of the paired bio-amine producing nucleus in the caudal hypothalamus.The author thanks Prof. Dr. P. G. W. J. van Oordt for his helpful comments and criticism. Mr. J. H. I. J. M. ten Berge and Mr. E. W. A. Kamperdijk provided great assistance during the course of the experiments. Mr. H. van Kooten made the diagram and the photograph.     

Corticosteroid binding in plasma of Xenopus laevis. Modifications during metamorphosis and growth

The binding of corticosteroids has been studied by equilibrium dialysis at 4 degrees C and electrophoresis on polyacrylamide gel. Aldosterone was not bound specifically. Large amounts of corticosterone (B) were bound. A high affinity (Ka = 2.8 X 10(8) M-1) low capacity (70.1 nM) component was found in tadpoles. Its affinity and its electrophoretic mobility were not significantly modified during metamorphosis and growth until adult. It differs from mammalian CBG with respect to affinity, electrophoretic mobility and specificity. During growth, the capacity of the high affinity component increased significantly (173 nM in adults) and a low affinity (Ka = 2.0 X 10(5) M-1) high capacity (18.26 microM) component was detected. This last component has the same electrophoretic mobility as bovine serum albumin. These modifications can be related to the large increase in the level of plasma proteins (especially albumins). The concentrations of bound and free B deduced from our data indicate that in tadpoles the increase in the total concentration in the climax is not a good reflection of modifications of these two fractions since the change of free B is larger than that of bound B. This information is an important consideration in interpreting the physiological role of interrenal secretion during metamorphosis.     

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis have been studied. Throughout the larval period to stage 60, the connective tissue consists of a few immature fibroblasts surrounded by a sparse extracellular matrix: few collagen fibrils are visible except close to the thin basal lamina. At the beginning of the transition from larval to adult epithelial form around stage 60, extensive changes are observed in connective tissue. The cells become more numerous and different types appear as the collagen fibrils increase in number and density. Through gaps in the thickened and extensively folded basal lamina, frequent contacts between epithelial and connective tissue cells are established. Thereafter, with the progression of fold formation, the connective tissue cells become oriented according to their position relative to the fold structure. The basal lamina beneath the adult epithelium becomes thin after stage 62, while that beneath the larval epithelium remains thick. Upon the completion of metamorphosis, the connective tissue consists mainly of typical fibroblasts with definite orientation and numerous collagen fibrils. These observations indicate that developmental changes in the connective tissue, especially in the region close to the epithelium, are closely related spatiotemporarily to the transition from the larval to the adult epithelial form. This suggests that tissue interactions between the connective tissue and the epithelium play important roles in controlling the epithelial degeneration, proliferation, and differentiation during metamorphic climax.     

Urea excretion rates and waste nitrogen concentrations during metamorphosis of Xenopus laevis Daudin

• 1.1. Serum urea, ammonia concentrations in the blood and excretion were measured in tadpoles of different stages and juveniles of Xenopus laevis.
• 2.2. The urea excretion rate was determined with the help of injected ¹⁴C-urea.
• 3.3. Urea concentrations are higher during metamorphic climax and at the end of metamorphosis than during prometamorphosis.
• 4.4. Blood ammonia levels remain rather constant throughout metamorphosis.
• 5.5. Coincidentally, the relative amount of urea in the blood increases.
• 6.6. The ¹⁴C-urea excretion rates slow down from very high values (48%/hr) at the beginning of prometamorphosis to low rates (5%/hr) in newly metamorphosed animals.
• 7.7. This means that during metamorphosis not only is the possibility of urea production established. but there is a capacity to retard and store urea to some extent.

     

Influence of 50-Hz electromagnetic field on anurian (Xenopus laevis) metamorphosis

In this study, we show the effect of a 1-mT magnetic field AC at 50 Hz on Xenopus laevis tadpole populations. In the course of a 65-day exposure to the field, tadpole survival showed a small, but significant, decrease (p < 0.0004), together with a striking parallel 6-day shift in tadpole maturation frequency and a significant impairment of their metamorphosis. Particularly, metamorphosis was successful for 85% of individuals in the unirradiated tadpole population and for 45% of individuals in the irradiated tadpole population, respectively.     

Changes in corticosterone and aldosterone concentrations in various tissues of Xenopus laevis tadpoles during the metamorphosis

During the metamorphosis of Xenopus laevis tadpoles, tissue concentrations of corticosterone and aldosterone did not change significantly in forelegs and hindlegs; they increased in tail, liver, skin and intestine. The rise of corticosteroid concentrations appeared in tissues which were deeply transformed during the first part of the climax, when plasma levels of corticosteroids also increased. Highest tissue levels, attained at the mid-climax, were maintained at least until the end of metamorphosis although plasma concentrations of two steroids were then abruptly fallen. Tissues able to retain corticosteroids reacted as Vertebrate "target tissues" and transformations which took place in them could be dependent, at least partially, on corticosteroids.     

Protamine polymorphism in Xenopus laevis laevis

Protamines from individual frogs of the subspecies Xenopus laevis laevis were compared by electrophoresis on polyacrylamide gels containing acetic acid, urea, and Triton X-100 to determine if the expression of protamine genes differs among individuals. Two electrophoretic bands, SP2a and SP2b, appeared to be expressed as allelic variants. Of 33 frogs, 19 expressed only SP2a, 11 expressed both SP2a and SP2b, and three expressed only SP2b. Electrophoretic analysis of partial V8 protease digests could not distinguish the peptides released from SP2a and SP2b. Differences in sperm development between individuals were not detected by light or electron microscopy. The results suggest that protamine polymorphism can exist among individuals of a species without an apparent effect on sperm development or sperm function.     

Morphologic changes of the basal lamina in the small intestine of Xenopus laevis during metamorphosis

Further investigations of the epithelial and mesothelial basal lamina of the duodenum of Xenopus laevis during metamorphosis were performed by means of scanning electron microscopy (SEM) and histochemical techniques using polyethyleneimine (PEI) to demonstrate anionic sites as well as light- and transmission-electron-microscopic methods involving morphometric analysis. The basal lamina of the duodenal epithelial cells was smooth, and it was occasionally curved along the processes of the epithelial cells (stages 56-59). The basal lamina became thicker by folding, and the thickness of the folded basal lamina exceeded 1 micron (stages 60-62). Subsequently, the folded basal lamina disappeared gradually and became almost smooth again and consisted of only one layer (stages 63-66). After removing the epithelium by boric acid, SEM revealed that the small ridges of the basal lamina protruded like a mesh-work into the luminal side, and the luminal surface of the basal lamina became smooth at later stages of the metamorphic climax. The electron-dense granules of PEI-positive material were localized at both sides of the lamina densa at regular intervals (80-100 nm). The basal lamina of the mesothelial cells was almost smooth at stages 56-59 and started to show occasional slight folding. This folding became continuous and deeper (stages 60-62). The folded mesothelial basal lamina disappeared except for the cell-associated basal lamina and became smooth again at later stages of the metamorphic climax (stages 63-66). These morphologic changes of the basal lamina observed in the epithelium and mesothelium may be induced by common factors. We suggest that physical changes in the small intestine involving the shortening and narrowing should be a main factor to cause these changes in the basal lamina. Furthermore, morphometric analysis proposed that the basal lamina becomes more complex by adding newly synthesized basal lamina material, especially in the epithelium.     

Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/ NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-l/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are express     

Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.