Degeneration of the Remaining Pronephros of Rana temporaria after Unilateral Pronephrectomy

If a pronephric duct of a Triturus larva is blocked it degenerates behind the lesion (Fox, 1957). If in Bufo vulgaris japonicus tadpoles the nephrostomial tubules are extirpated, so that there is no continuity between the remainder of the pronephric tubules and the coelom, the pronephric tubules themselves will degenerate (Shimasaki, 1930). Furthermore, pronephric tubules of a young Axolotl larva grafted into the lateral flank of a similar host, when not in functional continuity with the coelom, are stunted and collapsed (Fox, 1960).

Functional connexion of the pronephros and its duct with the body cavity is thus essential to maintain a normal expanded tubular condition.

Nevertheless, normal pronephric degeneration of Rana, which commences at the beginning of metamorphosis, precedes functional discontinuity, for the pronephric duct has a continuous lumen along its length and the nephrostomial tubules are open to the coelom for a considerable time after the onset of pronephros degeneration (Fox, 1962).

In urodele and anuran larvae, extirpation of the pronephros of one side results in hypertrophy of its remaining partner (Howland, 1916, 1921; Fox, 1956; Swingle, 1919; Cambar, 1948); and because an animal with only one pronephros survives and in general appears normal in form and behaviour, it may be presumed that the remaining pronephros does the work normally performed by two—at least during early development—and is thus hyperfunctional.

Since pronephric functional discontinuity and therefore hypofunction is the result and not the cause of normal pronephric degeneration, it may be questioned whether pronephric hyperfunction hastens or inhibits the degeneration process.

Investigations were made on Rana temporaria and the stages described are equivalent to those for R. dalmatina (Cambar & Marrot, 1954). Hatched specimens of R. temporaria at stages 25-26 (approximately)—about 8 days after spawning—were anaesthetized in 1:1000 MS 222 (tricain methonsulphonate in distilled water), and the pronephric blastema on the left side was extirpated (Fox, 1956, 1960). Specimens were maintained for 4 days on a gelatine base in glass crystallizing dishes (8 to each dish), each containing about 80 ml. of fullstrength Holtfreter solution and 0.2 per cent, w/v sodium sulphadiazine, and then for 1 week in one-tenth strength Holtfreter solution and the same concentration of sulphadiazine. Thenceforth all specimens were transferred to tapwater and grown in large aquaria tanks, and maximally fed on boiled lettuce. Controls were similarly treated but merely wounded in the region over the pronephric blastema. The temperature varied between 16° and 20° C.

In R. temporaria the first signs of pronephric degeneration are recognizable at about stage 49 (Fox, 1962). Groups of specimens were therefore chosen at random and killed from about stage 47, till metamorphosis had been completed at stage 54. In controls the time taken from spawning till metamorphosis was about 10 to 12 weeks, and in general the population was a homogeneous one. The time till the completion of metamorphosis in the unilaterally pronephrec-tomized specimens ranged from 12 to 20 weeks, and there was considerable variability among them in size and rate of development.

It seems likely, therefore, that unilateral pronephrectomy inhibits general growth rate in anuran larvae as it does in urodeles (Howland, 1921; Fox, 1960). Because of the difference in rate of development of unilaterally pronephrecto-mized and of control specimens, comparisons of the patterns of degeneration of their pronephroi were made in terms of distinct larval stages, and age as such was not considered in the analysis.

One control and one operated specimen, of each of stages 49-54 inclusive, were killed in Bouin’s solution and studied from transverse sections.

The pattern of degeneration of paired pronephroi in controls of R. temporaria (especially from stage 50 onwards) is similar to and confirms a previous description (Fox, 1962).

A single remaining pronephros of a unilaterally pronephrectomized larva at stage 49 has already begun to degenerate in the typical manner. Some of the tubules possess a small lumen; others are solid heavily stained strands, but in the anterior region of the pronephros a number of sections reveal a few swollen tubules, which possess expanded lumina. These are absent from normal animals. In stages 50 and 51 some of the degenerate tubules still possess a vestigial lumen, though a few expanded tubules, either in front or at the rear, contain degenerate tissue within them. Tubules with swollen lumina are still recognizable among the highly degenerate strands of the remaining pronephros of stage 52 and of stage 53 but not in stage 54, for the pronephric vestige consists of a small mass of degenerate, slightly translucent or partially pigmented strands, invested by lymphocytes and in appearance identical with paired control pronephroi.

Open nephrostomes and ciliated nephrostomial tubules are present in remaining pronephroi of operated specimens up to stage 53, but in those of stage 54 no nephrostomial units were distinguished. In each of stages 49, 50, and 52 after unilateral pronephrectomy the ‘intermediate duct’ between the pro- and mesonephros is tubular, though a lumen was not recognizable in stage 51. This duct is practically solid along its length in stage 53 and completely so in stage 54, but a lumen is found near the front of the mesonephros in both previous stages. The ‘intermediate duct’ of the extirpated pronephros consists of a solid strand in all stages, except in stage 53 when part of it could not be recognized.

Comparison between glomeri in control and in unilaterally pronephrectomized animals shows them to be generally similar in appearance and development. Twelve glomeri of the 6 controls and 11 glomeri of 6 operated specimens were compared (in stage 49 the glomus associated with the extirpated pronephros was missing, presumably because its blastema together with that of the pronephros was removed during the operation).

Glomeri of the two groups were not found to regress to the same extent, within the range studied, as in a previous analysis of normal degeneration (Fox, 1962). Degeneration of the glomus must therefore be considered a more variable phenomenon than pronephros degeneration.

During normal pronephros degeneration in R. temporaria there is a progressive reduction in the size of various components of the pronephric system (Fox, 1962). From these previous data further calculation showed that there is a high positive correlation between, on the one hand, the antero-posterior length of the pronephros, and, on the other hand, the pronephric nuclear population (r = 0.8665), the total volume of cells of the pronephros (r = 0.8700), and the overall pronephros volume (total volume of the tubule cells and the lumina together) (r = 0.8872). Similarly, high correlations were found between measurements of the antero-posterior length of the glomus and its volume (r = 0.9677) and the external surface area of the glomus (r = 0.9933). Measurements of the progressively reduced lengths of the pronephroi and of the glomeri were thus used to express the quantitative pattern of degeneration in control and operated animals, within the range studied, for ifthese measurements oflength show no significant differences between the two groups, then in view of the qualitative similarity of their pronephric systems and because of the correlations described, it is likely that there would be no significant differences between their total cell volumes.

Tests of significance of the means of the measurements of pronephros and of glomus lengths, by an analysis of covariance, and of the slopes of the regression lines of these components related to either staged controls or unilaterally pronephrectomized animals, showed that they do not differ (P < 0.05), and the same was found to be the case between glomeri of either side in the operated specimens (Table 1, Text-figs. 1-3).

Thus in animals at similar stages of development it is reasonable to conclude that degeneration of the pronephric system proceeds at a similar rate in both groups.

Furthermore, in the same stages of R. temporaria (49-54) there are no significant differences in mean length, or in the slopes of the regression lines, of these measurements of the pronephros and of the glomus either in control or in unilaterally pronephrectomized animals on the one hand, and, on the other, in degenerating pronephric systems of animals previously investigated (Fox, 1962); except in the case of the mean glomus length, which was found to be significantly smaller in the latter than in the 6 controls and the 5 operated specimens (calculation obtained by a method of adjusted means) of this present work, by 27 per cent, and 23 per cent, respectively (Table 1, Text-figs. 1, 2).

In terms of arbitrarily determined larval stages of R. temporaria the pattern of degeneration of the remaining pronephros, after unilateral pronephrectomy, is for the most part qualitatively and quantitatively—judged by measurements of its length—similar to that of control paired pronephroi, except that a few of the tubules are seen in cross-section to have an expanded lumen, situated among the degenerate strands, in contrast to the pronephroi of controls where no swollen tubules are recognized. The presence of swollen tubules in a degenerating single pronephros of operated specimens is no doubt a consequence of its earlier hypertrophied condition.

The fact that open inlets and outlets to the pronephric tubules exist even after pronephric tissue degeneration has proceeded some way invalidates the view that normal degeneration is elicited by cessation of function. Indeed, the opposite is more likely the case, and that cessation of pronephros functional activity is a consequence of organ degeneration.

In operated animals the glomus, which had belonged to the extirpated pronephros, did not differ in size or appearance from the contralateral glomus of the remaining pronephros, a result which supported earlier conclusions reached for T. cristatus larvae (Fox, 1956). In contrast Machemer (1925) and Maschkow-zeff (1934) considered that a glomus is ultimately smaller on the side where pronephrectomy is performed, a condition which probably obtains as a result of accidental ablation of some of the blastema of the glomus. In amphibian larvae extirpation of the pronephros alone, at early stages, thus does not influence the development of the glomus.

Unilateral pronephrectomy at early stages inhibits general growth and thus lengthens the time before metamorphosis. The single remaining pronephroi of operated animals are therefore older than their controls. There is thus no finite and unalterable pronephros life-span after which time it will begin to degenerate, but rather that the phenomenon of degeneration is bound up with other metamorphic processes.

1. On a pratiqué la pronéphrectomie unilatérale de têtards de R. temporaria, à des stades approximativement équivalents aux st. 25-26 de R. dalmatina (Cambar et Marrot, 1954), et on a comparé le sort du pronéphros unique restant avec celui de systèmes témoins pairs, pendant la phase de dégénérescence pronéphrétique, qui a heu aux stades 49 à 54.

2. Pour des stades larvaires comparables, le pronéphros restant des individus opérés ne diffère pas, en ce qui concerne sa dégénérescence, des pronéphros témoins, ni qualitativement ni quantitativement (ceci exprimé en longueur du pronéphros ou du glomus), excepté qu’il contient quelques tubules gonflés, résidu de l’hypertrophie tubulaire compensatrice résultant de la pronéphrectomie unilatérale.

3. Après pronéphrectomie unilatérale, des glomérules intacts in situ, qui appartenaient au pronéphros extirpé, sont également identiques aux glomérules contralatéraux du pronéphros restant de l’autre côté, et aux glomérules pairs des témoins.

4. L’hypo-ou l’hyperfonctionnenement n’affecte pas la dégénérescence normale du pronéphros et il est vraisemblable que le phénomène est hé à des processus métamorphiques indépendants de ce fonctionnement.

Professors M. Abercrombie and D. R. Newth read the manuscript and kindly gave advice.