The Development of the Pronephros in theCommon Perch (Perca fluviatilis L.).

By

W. F. H. Stroer,Assistant at the Zoological Laboratory of the University of Amsterdam.

With Plate 33 and 4 Text-figs.

THIS investigation into a series of Perca fluviatilis startedsome time ago.

The material consisted of a number of specimens living inan aquarium, of which some were fixed every day in Bouin'sfluid. The first lot were fixed on April 7. This stage agrees withEhrenbaum's descriptions of fish which had reached the ageof twelve days. The whole embryo lay spread on the vitellus,only the head slightly lifted.

Several specimens were cut at 5 or 10/A, most of them trans-versely or horizontally. They were stained with Ehrlich's(E.h.) or Heidenhain's haematoxylin (H.h.) and eosin.

While the treatment of the larvae gave no trouble at all, theyoung embryos, on the other hand, caused great difficulty. Thelarge quantity of vitellus made it impossible to cut the egg at5 fj, in the ordinary way. To avoid this trouble I cut away theyolk, which can be easily done when with a small metal spatulaa circular cut is made round the embryo through the outer,extra-embryonal layers surrounding the vitellus. It is then quiteeasy to lift the embryo from the yolk with two needles, like a lidfrom a box. Swaen and Brachet followed another method, forthey had at their disposal living embryos, whereas the afore-mentioned material was fixed.

INTRODUCTION AND HISTORY.

The following is only a concise survey of the history of thedevelopment of the pronephros. In this history we may dis-tinguish three groups of investigations or periods:

First, a morphological period, that is to say a period in which

558 W. F. H. STEOER

only the development of the form was described. Eosenberg'swork dates from this period (see Felix, 1897).

The second group embraces the controversy about thearchinephros theory and the theory of Kuckert, that is to say thequestion whether 'pronephros, mesonephros, and metanephrosare simply successive portions of a single, elongated, ancestral,excretory organ possessing a duct and segmentally arranged,serially homologous, tubules' (Graham Kerr on the archinephrostheory), or whether the mesonephric tubules are only 'einezweite vervollkommnete Generation' of the pronephric tubules(Riickert, 1888). The first investigators of this period did notpay much attention to the state of development, so that Semon(1892) described an old stage of theGymnophione Ichthyophisas a typical proof of the correctness of the theory of Eiickert(1888). Some time later, however, Brauer (1902) made a parti-cularly careful and complete investigation of the developmentand especially the appearance of the renal organs of anotherGymnophione Hypogeophis. And nowadays the developmentof Hypogeophis is considered to be one of the best argumentsin favour of the archinephros theory. To Kerr (1919) none of thearguments of the antagonists of this theory is ' so convincingas the very clear evidence afforded by Hypogeoph i s ' .

Felix (1897) gave a good account of the development in theTeleostei; but it was to the magnificent researches of Swaenand Brachet (1899 and 1902) that we owe our knowledge of thissubject. They gave an extensive description of the first appear-ance of the pronephros and came to the conclusion that the modeof development of pronephros and archinephric duct wereprincipally the same. So they decided that the latter is only alarge ' chambre pronephre"tique'.

Arguments in favour of the archinephros theory were broughtforward by the study of Audige (1910) on the renal organs ofadult fishes.

Haller (1908) studied the development of pro- and meso-nephros in Teleostei; and, using certain special considerations,he tried to form a connexion between phylogenesis and function.1

1 Howard and Smith (1930) tried the same for the kidneys of all verte-brates.

PEONEPHEOS IN PEECA 559

He concluded that pro- and meta-nephros are only differentia-tions of the mesonephros. In the last decennium no interestwas shown in the question of the archinephros theory. OnlyMaschkowzeff (1926), in his study on Acipenser, mentionedthe question.

The more precise study of the histological and micro-anatomical state of the adult renal system is characteristic of thethird period.

The paper of Audige is one of the most important studies ofthis period; the histological research connected with experimentsled him to a consideration of the function. And we find anumber of observers, partly before Audige and partly after him,studying the histological structure. I refer to the discussion ofNoll (1924), and mention the modern investigators Verne (1922),Feyel (1928), and Graham Edwards (1928, 1929).

From the above-mentioned review it follows that of late yearsthe study of the development, especially the further develop-ment, of the pronephros of the Teleostei has been neglected, withthe exception of a few observations of Verne (1922).

The following investigation is an effort to fill this gap. Onone hand a description of the development of the tubule, onthe other hand the development of the histological structureof the pronephros, is given.

OBSEBVATIONS.

For the sake of clearness I prefer to divide the followingdescriptions into three groups. The first two groups are con-fined to embryos, the third group to larvae which have leftthe capsules.

I. Let me begin with a brief, separate discussion on the earlieststage that was at my disposal (fixed, April 8). In this embryothe alimentary canal was completely isolated from what re-mained of the yolk. The rudiments of the pronephros form abulging inward of the median edge of the lateral mesoderm(fig. 1, PI. 33). Though the lateral mesoderm is still solid, thenephrocoele is distinctly developed. The archinephric duct isfolded off at full length (fig. 1 a, PI. 33).

560 W. F. H. STROER

The state of development agrees with the descriptions ofSwaen and Brachet (1899 and 1902).

II. Stages 1-6, fixed, April 9-14. The pronephros consistsof a pronephric chamber in which the aorta forms a glomerulus.At an early stage the glomerulus fills the whole cavity of thepronephric chamber (fig. 2, PI. 33), and the cavity does notappear until some time later. The pronephric tubule com-municates with the chamber by means of a funnel. In this

cavity

PST

m? = part a; \*t*t\ = part b; Q = part c.

TEXT-FIG. 1.

Reconstruction of the pronephros of Stage 3 (transversely cut)(150x157).

funnel cilia are present, which are themselves not very distinct.However, we find large basal-granules (H.h.), but they beginat some distance only from the place where the epithelium of thechamber passes into the columnar-epithelium (fig. 3, PL 33).Audige described this in an older stage, but I could observe itin Stage 2.

The pronephric tubule bends frontally in the earliest stages assoon as it originates from the chamber, and forms an anteriorloop, after which it takes a caudal course. At a later stage itfirst bends caudally and forms a posterior loop. During thestay of the embryo in the capsule the differences in form arenot important (cf. Text-figs. 1, 2, and 3).

The histological structure in the succeeding stages is notstrikingly different. The wall of the parts of the tubule hasoriginally a uniform structure. We notice a typical columnar-

PRONEPHEOS IN PERCA 561

epithelium with large nuclei. These .show a chromatin networkwith a large nucleolus (H.h.). Sometimes there are even twonucleoli. The plasma has a somewhat bluish appearance;however it does not absorb the stain everywhere equally strongly.Frequently lighter stained streaks show themselves from thebases of the cells (fig. 4, PI. 33). These are decidedly not fixationproducts; it is possible that later on they will form the spots

aortacavity

TEXT-FIG. 2.

Reconstruction of the pronephros of Stage 6 (horizontally cut).

through which the lymph-cells can creep. In agreement withthis theory is the fact that both occur mostly in the part of thetubule stretching towards the archinephric duct.

In this duct the appearance is mainly the same as that above-mentioned, only the nucleolus is generally not very distinct.

At later stages we find differences between the parts of thetubule.

Especially in Stage 6 we notice a larger cavity in the ascendingpart of the tubule, while the cells are higher, which is chieflycaused by the quantity of plasma. The nuclei are situated nearthe base of the cells (fig. 5, PI. 33). The descending part of thetubule forms a transition to the typical archinephric duct,which has a very small cavity.

Brush-borde r .—In the younger stages we notice a brush-

562 W. F. H. STROEB

border in the anterior loop. At later stages, however, it is moredistinct (especially with E.h.).

P s e u d o l y m p h o i d tissue.—Originally only situatedbehind the pronephric chamber, it expands at later stages andfinally fills up the space between the right and left tubule.

C a r d i n a l vein.—Ventrally to the tubules, partly situatedbetween the parts of the anterior loop, we observe the cardinalvein (figs. 2 and 3, PI. 33), which accompanies the archinephricduct in its further course.

III. Stages 7-13, fixed, April 15-21. These specimens are

aorta

TEXT-FIG. 3.

Reconstruction of the pronephros of Stage 9 (horizontally cut).

larvae, that is to say they have left the capsule. The demandsof the new surroundings are demonstrated in the developmentof the pronephros, that is to say in the development of the form,but also in the differentiation of the histological structure.

The tubule becomes much elongated and coiled in the sameway as this occurs in Polypterus described by Kerr (1919).

Now we may distinguish two kinds of coils: (1) the primaryloops, that is to say the anterior and posterior loop; (2) thesecondary coils. These vary topographically as well as in size.In later stages they may also occur in the archinephric duct(cf. Text-figs. 3 and 4). We believe that the secondary coils

PRONEPHROS IN PERCA 568

are only the result of the functional demands to the tubule, thatis to say a surface-increase of the latter, but that the primaryloops are allied with a constitutional factor. (We must observe

sorts

TEXT-FIG. 4.

Keconstruction of the pronephros of Lophius piscatorius (length:4 mm., horizontally cut).

that the latter are different in P e r c a and L o p h i u s ; cf.Text-figs. 2, 3, and 4.)

From an histological point of view the tubule consists ofthree parts: (a) the beginning of the posterior loop, which isonly a continuation of the funnel; (6) the anterior loop; (c) thepart that forms a transition between (b) and the archinephricduct.

564 W. F. H. STEOBR

(a) Audige (1910) gave a description of this part (le collet) fora larva of Bar bus (p. 433, &c). I have little to add to this;besides, there is little or no difference from my descriptionsof Stages 1-6, only I noticed here sometimes a slightly stainedspace round the nuclei.

At the posterior loop fissures penetrate the tubule betweenthe cells and may even reach the cavity. Haller (1908) describedthe same for the renal tubules of the adult fishes. Often henoticed lymph-cells creeping through the fissures. PersonallyI cannot affirm this, but it is a matter of fact that these fissureswere most distinct in older stages in which the pseudolymphoidtissue surrounded the tubule. Yet I must admit the possibilitythat we have to do here with artifacts.

(b) This loop is the most interesting part of the tubule.Stage 7. In animals which have just left the capsule nothing

special is to be noticed. The protoplasm shows more affinityto haematoxylin than in (a), while the situation of the nucleiis less regular (fig. 6, PI. 38). Characteristic are the lighterstained spots that lie in the plasma. These are the rudimentsof the vacuoles. Further, we notice some granules (only distinctwith H.h.). They often lie against the nuclei or quite near them.

The cavity in (b) is twice or three times as big as in (a). Thisholds good for the ascending part; the part taking a caudalcourse has a less-developed cavity.

Here the cells are not so high, while we find distinct vacuoles.In one or two in the wall of the tubule I could detect a lymph-cell.

Stages 8 and 9. In older stages we find a stronger developmentof the lighter stained patches and vacuoles in the whole anteriorloop. For this reason the protoplasm has been repelled to thebase of the cells. It has a bluish appearance and is filled upwith granules. These 'Korner', as Noll (1924) calls them ingeneral, are on the one hand dispersed irregularly round thenuclei at the bases of the cells, and on the other hand they formrows stretching from tip to base in the cells.

These granules are mitochondria; that is to say they first formthe rudiments of the 'reseaux protoplasmiques' described byAudige (1910), Policard and Mawas (1906), &c. The rows of

PRONEPHROS IN PERCA 565

mitochondria are the rudiments of the 'Stabchen' of Heiden-hain (1874). Benda (1903) described the same for the kidneyof the embryo of a mouse (rnetanephros ?) and the kidney (?)of a larva of E a n a . He did not find both systems of mito-chondria in one cell.

In spite of the negation of Benda (1903) I am able to confirmthe observations of Audige that the rows of mitochondriaterminate in the brush-border. I must admit the possibilitythat we have to do with the strips of the cementing substance('Kittleiste').

In the ascending part of the anterior loop of the tubule theprotoplasm of every cell has been repelled by a vacuole and showsa triangular shape (fig. 8 a, PI. 33); but in the descending partit has a more rectangular shape; here the vacuoles are lessdeveloped (fig. 8 b, PI. 33).

Adjacent to the above-mentioned granules we find one or twograins lying in the vacuoles. These are of course 'grainsurinaires' or granules of excretion.

Stages 11-13. These larvae do not show important differ-ences compared with the above-mentioned stages.

Larger nuclei with a distinct nucleolus are conspicuous; theprotoplasm has a granular appearance. The number of themitochondria is diminished, but there are more granules ofexcretion (fig. 9, PI. 33).

The presence of a tunica is noteworthy, for I could not detectit in younger stages.

Brush-border.—Although it is present in animals ofStages 7, 8, and 9, it is not always distinct. Whether this is theeffect of the bursting of the vacuoles in the cavity of the tubule(cf. Eegaud et Policard, 1902 a and b; Audige, 1910), or anartifact, is difficult to settle.

In older stages the border is better developed and forms abright, distinctly striated border with regularly dispersed basal-granules. So we understand that we have to do here with aciliated border. This agrees with the observations of Nussbaum(cited by Felix, 1904).

(c) Conspicuous is the lack of granules and vacuoles in thispart. Besides, I could not detect a brush-border round the less-

566 W. F. H. STROEB

developed cavity. Practically there are no differences in thehistological structure in the successive stages.

The transition between (b) and (c) is quite sudden.P s e u d o l y m p h o i d tissue.—We have little to add to

the description of Stages 1-6. Now it lies even between thedifferent parts of the tubules. Many cells are in division.

PHYSIOLOGY.

We may conclude from the preceding descriptions that part(b) of the larval pronephric tubule has an excretory function.

I have, moreover, made some observations which are notpreviously mentioned.

In one specimen (Stage 8, transversely cut) I could not ob-serve a communication between pronephric chamber and tubule,neither on the left nor on the right side. The lack of this com-munication on the right side only I observed in an animal ofStage 9 (horizontally cut). Both animals were free swimming.

Our conclusion is that the part played by the pronephricchamber with glomerulus, which we may compare with thecapsule of Bowman (cf. Haller, 1908), in the excretory functionis not very important, that is to say that these observations arein favour of the theory of Bowman-Heidenhain. The aglomeru-lar kidneys of the Lophobranch fishes support these views.Besides, Verne (1922) did not observe here a back-resorption.

Maschkowzeff (1926) admits in Ac ipense r an excretoryfunction of the archinephric duct, while it is accompanied bythe cardinal vein.1 The histological structure in P e r c a tellsin every way against this view.

DISCUSSION.

Audige (1910) has distinguished in the mesonephric tubuleof the adult Teleostei the following parts: ' 1 , le glomerule deMalpighi avec la capsule de Bowman; 2, le collet; 3, le tubecontourne; 4, le canal collecteur; 5, l'uretere.'

A comparison between his descriptions of 2, 3, and 4 and the

1 This is not only accepted by Boveri (cf. Maschkowzeff, 1926), but alsoby Wheeler (1899).

PRONEPHROS IN PERCA 567

preceding descriptions of the parts (a, b, and c) of the larvalpronephric tubule shows a great similarity.

We conclude that the pronephros agrees with one meso-nephric tubule of the adult fishes.

These investigations have not produced any evidence inconnexion with the archinephros theory.

But as Swaen and Brachet (1899) concluded that the archi-nephric duct was only a large pronephric chamber, I mustobserve that the lack of excretory function is not in favour ofthis theory.

Of course it is not allowed to use physiological arguments indiscussing the problem of homology; but the fact that thearchinephric duct has no excretory function, in spite of thecardinal vein running along it, makes it clear that other factors,that is to say factors dependent on the constitution of theanimals, are present here.

SUMMARY.

1. A description of the development of the external form ofthe pronephric tubule of Perca f luviatilis is given.

2. In two animals a communication between pronephricchamber and tubule was not found.

3. A description of the histological structure of the three partsof the pronephric tubule is given.

4. Three parts in the larval tubule are distinguished whichagree with the parts distinguished by Audige in the meso-nephric tubule.

I wish here to acknowledge my indebtedness to the Directorof our Institute, Professor J. E. W. Ihle, for the kind and helpfulinterest he has shown in my observations.

LITERATURE CITED.

1. Audige, J.—"Contributions a 1'etudedesreinsdes poissons teleosteens'','Arch. Zool. Exp.', torn, iv, 1910.

2. Benda, C.—"Die Mitochondria des Nierenepithels", 'Verh. Anat.Ges.', 1903.

3. Brauer, A.—"Beitrage zur Kenntnis der Entwicklung und Anatomieder Gymnophionen: Die Entwicklung der Excretionsorgane",'Zool. Jahrb. Anat.', Bd. 16, 1902.

568 W. F. H. STROBR

4. Ehrenbaum, E.—'Eier und Larven von Fischen des nordischenPlanktons', 1905-9.

5. Felix, W.—"Beitrage zur Entwioklungagesohichte der Salmoniden",'Anat. Hefte', 1897.

6. "Die Entwicklung der Ham- und Geschlechtsorgane", O.Hertwig, 'Handb. der Entw.gesch. der Wirbeltiere', Bd. 3, torn, i,1904-6.

7. Feyel, P.—'Arch. Anat. mier.', 24, 4, 1928.8. Graham Edwards, J.—"Studies on aglomerular and glomerular

kidne37s: 1. Anatomical", 'Amer. Journ. Anat.', v. 42, 1928.9. • "Studies on aglomerular and glomerular kidneys: 3. Cytological"

'Anat. Rec.', v. 44, 1929.10. Haller, B.—"Znr Phylogenese des Nierenorganes (Holonephros) der

Knochenfische", 'Jen. Zeitsch. Nat.', Bd. 43, 1908.11. Howard and Smith.—"The glomerular development of the vertebrate

kidney in relation to habitat", 'Biol. Bulletin', lix, no. 2, 1930.12. Kerr, J. Graham.—'Text-book of Embryology', vol. ii, 1919.13. Maschkowzeff, A.—"Zur Phylogenie des Urogenitalsystems der Wir-

beltiere auf Grund der Entwicklung des Mesoderms, des Pronephros,der Analofinung und der Abdominalporen bei Acipenser stellatus,T. I und T. II", 'Zool. Jahrb. Anat.', Bd. 48, 1926.

14. Noll, A.—"Die Excretion: Wirbeltiere", 'Handb. d. vergl. Physiol.',Bd. II, 2 H., 1924, S. 780 a.s.o.

15. Polioard et Mawas.—"Le canalicule urinaire des teleosteens",' Bibliographie Anat.', torn, xv, 1906.

16. Regaud et Policard.—"Etude sur le tube urinifeTe de la lamproie",'C. R. de l'Assoc. d. Anat.', 1902.

17. "Notes histologiques sur la secretion renale, iii", 'C. R. desseances de la Soc. de Biol.', 1902.

18. Riickert, J.—'Arch. f. Anat. u. Entw.gesch.', 1888.19. Semon, R.—"Studien iiber den Bauplan des Urogenitalsystems der

Wirbeltiere", 'Jen. Zeitach. Nat.', Bd. 66, 1892.20. Swaen et Brachet.—"Etudes sur les premieres phases du developpe-

ment des organes derives du mesoblaste chez les poissons t616ost6ens ",prem. partie, 'Arch. d. Biol.', torn, xvi, 1899; deux, partie, 'Arch,d. Biol.', torn, xviii, 1902.

21. Verne, J.—"Contribution a 1'etude des reins aglomerulaires; 1'appareilr6nal des poissons lophobranehes", 'Arch. Anat. micr.', torn. 18,1922.

22. Wheeler, W. M.—"The development of the urogenital organs of theLamprey", 'Zool. Jahrb. Anat.', Bd. 13, 1899.

Quart. Joiirn. Micr. Sci. Vol. 75, X. S., PL 33

L m ascending part' f tubi/Lk

8b

grains ofexcretion

10

PRONEPHROS IN PERCA 569

EXPLANATION OF PLATE 33.

ABBREVIATIONS ON PLATE AND TEXT-FIGURES.

a.d., archinephric duct; &.&., brush-border; b.gr., basal-granules; c.str.,strips of cementing substance; gl., glomerulus; l.m., lateral mesoderm;TO., mitochondria; my., myotome; n., notochord; n.n., nuclei; n.sl., nephro-stome; p.ch., pronephric chamber; p.n., pronephros; ps.l., pseudolymphoidtissue; s.c, spinal cord; v,, vacuole; v.c, cardinal vein.

Fig. 1.—Transverse section of Pe rca (fixed, April 8) through therudiment of the pronephros (E.h.).

Fig. la.—Idem, through the archinephric duct (E.h.).Fig. 2.—Transverse section of Stage 1 (fixed, April 9) through the

pronephric chamber before the nephrostome (H.h.).Fig. 3.—Transverse section of Stage 2 through the nephrostome (H.h.).Fig. 3 a.—The right nephrostome of fig. 3 (H.h.).Fig. 4.—Transverse section through the archinephric duct (Stage 1)

(H.h.).Fig. 5.—Transverse section through the ascending part of the tubule

of Stage 6 (E.h.).Fig. 6.—Horizontal section through the nephrostome of Stage 7 (H.h.).Fig. 6 a.—Horizontal section through the descending part of the tubule

(Stage 7) (H.h.).Fig. 7.—Sagittal section through the posterior loop of the tubule

(Stage 8) (H.h.).Fig. 8 a.—Horizontal section through the descending part (Stage 9)

Fig. 8 6.—Horizontal section through the transition between parts 6 and c.Fig. 8 c.—Horizontal section through the network of mitochondria and

strips of cementing substance (H.h.).Fig. 9.—Horizontal section through the descending part of the tubule

of Stage 13 (H.h.).Fig. 10.—Horizontal section through the archinephric duct of Stage 9