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The chromaffin and interrenal cells of Acanthobrama terrae-sanctae (Cyprinidae, Teleostei)
WNHt41.
AND
COMPARATIVE
The
14, 542-550 (1970)
ENDOCRINOLOGY
Chromaffin
and
Interrenal
fame-sancfae
Cells
(Cyprinidae,
of Acanthobrama Teleostei)
Z. YARON Department
of
Zoology,
Tel
Aviv
University,
Tel
Aviv,
trael’
Received October 20, 1969 The interrenal and the chromaffin cells of Acanthobrama tewaesanctas are situated around the veins passing through the head kidney, 3,f3- and H/3-hydroxysteroid dehydrogenases were demonstrated in the interrenal cells. Using Wood’s method, norepinephrine was detected in the chromaffin cells of the head kidney. Reserpine treatment caused the depletion of stained material from the chromaffin cells. Dexamethasone treatment caused degenerative changes in the interrenal cells. Accordingly, this steroid may be used for experimental induction of interrenal hypofunction. Annual changes in histological features of the interrenal cells were studied. The highest mean diameter of cells and nuclei was found to coincide with vitellogenic activity in the ovary. The possible interactions between the ovary and the interrenal cells are discussed.
Acanthobrama terrae-sanctae,
a “sardine-
like” eyprinid from Lake Tiberias, during the winter (Sivan, 1967;
spawns Yaron, 1969), but in contrast to other winter breeders, such as the salmonids, its osmotic environment does not change considerably throughout the year. The relations between the interrenal function and reproduction of teIeost fishes has been studied mainly in salmonids (Chester Jones, Phillips, and Holmes, 1959; Robertson et al., 1961; Honma
and Tamura,
19633). It was obvious,
that Acanthobrama terraetherefore, sunctae would serve well as a different subject for studying the interrelations between reproduction and interrenal function. The aims of the present study were to locate the interrenal cells and study their seasonal
changes
in relation
with
gonadal
activity. MATERIALS AND METHODS Monthly samples of 50 live adult &h each were collected from ring-net fishing catches of fishermen in Lake Tiberias. Fish were brought alive to the ’ Present address: Department of PhysiologyAnatomy, University of California, Berkeley, California 94720.
shore and some were fixed in Allen’s fluid (Gurr, 1962) after incision of the abdomen. Others were transported alive to the laboratory and kept there for experimental work in tanks provided with running tap water. Histology
The right head kidneys were removed from the fixed specimens and longitudinal sections S,u thick, containing the posterior cardinal vein, were stained by the Azan method (Gurr, 1962). Measwements
The mean cell diameter was calculated from the number of interrenal cells crossed by the line of an eyepiece micrometer in sections of the head kidney containing interrenal cells only. The number of cells was divided by the total length of the line and the quotient was recorded as the mean for the given specimen. At least 10 sections from each fish were measured. The nuclear diameter was measure,d by a micrometric eyepiece (10 X) in 100 cells of each fish using an 100 X oil immersion objective. The relative abundance of interrenal cells was estimated by determining the proportion of interrenal cells underlying the line of the micrometric eyepiece viewed horizontally on sections at the magnification of 400X. Successive fields were changed at random. All sections of the head kidneys were measured.
FISH
CHROMAFFIN
AND
Histochemistry Interrenal
Cells. Activity
of BP-hydroxysteroid dehydrogenase was demonstrated in the interrenal cells by the method of Rubin et al. (1963) using frozen or freeze-dried paraffin-embedded tissue (Barka and Anderson, 1965~). bhromafiin Cells. These cells were identified using the method of Wood (1963).
Dexametkasone
Treatment
Males were kept in outdoor tanks for 2 weeks prior to the experiment. Three &roups of 10 fish each were placed in well-aerated, ‘20~liter aquaria, with flowing tap water and were fed with Tubifex. The experiment started on January 4 and ended on January 25, 1967, Water temperatures varied from 14”-16” during the day and between 9”-12” during the night. Fish were lightly anaesthetized wiath MS-222 (Sandoz) before injection of the drugs in order to facilitate handling. One group was injected every other day for 21 days with 10 pg dexamethasone (9a-fluoro-16ametl~yl-l1~,17a,21-trihydroxypregna-l,4-cliene-3,2Odione) in 0.1 ml of solution. The injections were given in dorsal myotomes anterior to the dorsa.1 fin. The dexamethasone (DXM) was dissolved in ethanol and mixed with amphibian Ringer to give a final concentration of 5% ethanol. Each fish received a tot.al dose of 100 pg DXM. The second &~oup received 0.1 ml injections of the solvent, and a third group was left untreated. On the 22nd day B& were anaesthetized with MS-222 and fixed in Allelk% &id. REXJLTS
General
The
Morphology
head
kidneys of Acanthobrama form the thick anterior ends of each mesonephros. They are covered by part of the peritoneum containing numerous melanophores. The head kidneys begin at the level of the fifth rib and extend anteriorly underneath the Weberian ossicles and ventrally t,o the pericardium. The Sateroposterior end of the head kidney reaches the swim bladder and the lateroanterior end touches the fifth ceratobranchid bone. terrae-sanctae
Bkood Supply The drainage dcanthobramnu.
metric;
of the
mesonephroi of is asymcardinal vein is un-
terrae-sanctae
the posterior
INTERRENAL
CELLS
643
paired and passes along the right mesonephros. Short transverse venules dra& the left kidney into the right cardinal vein, This is also the case for the head kidneyrs, i.e., the posterior cardinal vein passes through. the right head kidney and is absent in the left. An isthmus of lymphoid tissue passes transversally and bridges the dorsal ends of both head kidneys. The anterior cardinal vein enter% the rostra1 end of each head kidney and: at the ventral end of each, Cuvier’s duct drains into the sinus venosus (Figs. 1 and 2;. Thus, at the right head kidney> there is 8 junction of three main veins (Cuvier’s duet, anterior and posterior cardinal veins) ) whereas in the left only the first two are found. Large arteries supplying fhe head kidneys were not encountered. Nistology
The main mass of the head kid.ney is composed of lymph&d tissue, while endocrine components constitute only about 10% of the whole mass. Interrenal cells form one or more layers along t.he main blood vessels. Small clumps of chmmaEn cells are scattered among the interrenal cells, usually outside the inner lager of interrenal cells. A sympathetic gangljlon is found in the right head kidney (Fig, 3) I Interrenal C&s. The interrenlal cells are polygonal with a diameter of 6-~O+K.. In the autumn and spring they are somewhat shrunken and resemble a coiumriar epitbelium surrounding the cardinal veins. After fixation in AUen’s Auid and ham staining, the cytoplasm is homogeneous light grayish-violet or contains fine basophilie granuies. In a few casesprominent, fuchsinophiie granules were observed. Vacuoles, va.riable in size and distribution, are present.; in active eelis with large diameters, vacuoles are numerous, giving the cyI.oplasm a spongy appearance. The nuclear diameter varies from 3.2-4.9 ha; t’he nucleus cMain.s deeply stained dense chromatin and a. very prominent fuchsinophile nucleolus (Fig. 4) I Steroidogenic Activitz~. The identificat8ion of the interrenal cells is based on the hi&ochemical demonstration of 3/J-hydrnxysteroid dehydrogenase (S/Z?-HSDI and ‘IIF-
544
Z.
YARDN
FISH
CHROMAFFIN
AND
hvdroxysteroid dehydrogenase (ll/?-HSD) . DIformazan granules, representing the activity sites of these enzymes, are seen in the cytoplasm but not in the nucleus. ChromafEn cells do not show such granules (Figs. 5 and 6). Control sections incubated without the suitable substrates did not react, Chromafin Cells. The chromaffin cells with an average diameter of 16~~ are larger than the interrenal cells. The cell has a few projections, sometimes reaching the wall of the vein. The cytoplasm has little affinity for st,ains of Azan method and is almost transparent; only fine ,granules can be distinguished. In the oval nucleus, which is stained pink, there is a coarse net of chromatin and one or more, peripherally situat,ed, nucleoli (Fig. 4). Cytoplasmic inclusions are visualized using the method of Wood (1963) for epinephrine and norepinephrine. The yellow content indicates norepinephrine. Administration of reserpine (4 mg per 1OOg body weight) to the ,fish 24 hr prior to fixation caused the depletion of the stainable inclusions (Figs. 7 and $1. Dexamethasone Treatment
The interrenal, cells of males treated with dexamethasone (ea. 5 ,ug per g body weight) showed signs of degeneration. Figure 9 shows that the mean diameter of the interrenal cells decreased after dexame&asone treatment (p < 0.025 and p < 0.005, as compared with treated and untreated controls, respectively) I Nuclear diameter also decreased as compared with treated (p < 0.05) and untreated (p < 0.001’1 controls. The interrenal cells in the head kidneys of dexamethasone-treated fish
INTERRENAL
545
CELLS
appeared to be less abundant, than in control groups, but the diflerenw is significant (p > 0.2) I Seasonal Variations in the ~~te~e~a~ Cells During the summer, the me nuclear diameters are rather h wards October cell and nuclear diameters fall abruptly and interrenal cells become relatively ra,re in the head kidney (‘Fig. 10) p From October to December, ~o~l~o~ita~~ with the increase of ovarian weight (Si-m+ 1967) the interrenal cell and nuclear diameters increase and cells are more abundant, During the following winter months there is a gradual decrease in the diameter of interrenal cells and nuclei with minimal values occurring in May. DISCUSsION Distribution
of Interrenal
Cells in the
Kidney
The close association of the interrenal cells wit’h the asymmetrical cardinal veins results in the inequal d~stribut~oll of these cells ; the right head kidney containing more cells than the left one. A similar asymmetry was found in Betta splendsns by ‘G’an Overbeeke (1960)) who attributed ix Lo the difference in the size of the right and Jef”r, posterior cardinal veins. Medullury Cells Histochemical identification of catechcalamines using the ehromaffin reaction bss been performed in several teleost fishes and reviewed by Van Overbeeke (~~6~~ I who could not state conclusively whether the chromaffin or phaeochrome reaction could demonstrate the presence of adrenaline irb _I__
___I___
-- ..-..--.-
1. Sagittal section through the right head kidney of 14-mm fingerling. Connection between the cardinal veins and Cuvier’s duct. Stain: Azan, green filter. Scale 100 p. dc-Cuvier’s duct; h-liver; hk---right head kidney; ph-pharynx; sv-sinus venosus; vca-anterior cardinal vein; vcp-posterior cardinal vein; vt-ventricle. FIG. 2. Sagit,tal section of left head kidney of the same specimen. IConnection between the anierior cardinal vein and the Cuvier’s duct. Staining, filter and scale as in Fig. 1. de-Cuviefs duct; h-liver; hk---left, head kidney; ph-pharynx; vca-anterior cardinal vein. FIG. 3. Ganglion within the head-kidney of an adult. Stain: Azan; yellow filter; &ale 100 P. cr--chsomaf’& cells; g-ganglion; ir-interrenal cells; nv-nerve; vcp-posterior cardinal vein. FIG. 4. Tnterrenal (ir) and chromaffin (cr) cells in a fish caught in February. Stain: Azan; green filter. Scale 20 pd FIG.
546
FISH
CHROMAFFIN
AND
INTERRENAL
547
CELLS
DXM SIY UN CELL DIAMETER
NUCLEUS
DIAMETER
RELATIVE
ABUNDANCE
FIG. 9. ERect of dexamethasone treatment on cell diameter, nucleus diameter and relative abundance of interrenal tissue in the head kidney of Acunthobramz terra+sanctae males. DXM-dexamethasone-treated fish; SLV-solvent-injected fish; UNT-untreated fish. (Vertical 1ines-S.E.)
kleost fishes or whether other catechols are involved in this reaction. The method of WTood (1963) enables the differentiation between adrenaline and noradrenaline-containing cells in the hamster. When applied to the head kidney of Acunthobrama, only one cell type was noticed. Therefore, we are not in a position to state, according to the tinctorial features only, which of the two cateeholamines described in teleosts is present in these cells. Nevertheless, the total vacuolization and the disappearance of stainable material from the chromaffin cells 24 hr after reserpine treatment probably indicates that norepinephrine is the stained material in the chromaffin cells when Wood’s method is applied. Similarly, Olivereau (1963) showed that reserpine treatment in the eel (Anguilla anguilla) caused the depletion of norepinephrine from the chromaffin cells. The existence of two different types of
medullary cetk in mammals was questioned by Picard and Vitry (1964) ) who stated that ““cell ‘dimorphism appears to result not from ultrastructural but rather from chemical differences v r I and express a different functiona behavior of a single cell type-” As far as tinctorial evidence can be relied on, only one type of chromaEin cells is present in the head kidney of Aca~~~~~b~~m,~. The Interrenal Cells
The histology
celis ?n to that of other fish (Van Overbeeke, 1960; Mahon et CLI.~1962). However, no direct comparison can be made because of the changes in size and cytological details of the interrenal cells of A~~~t~~b~arn~ throughout the year; only in a few cases was the description of these cells in previous papers foilowed by notes on other glands, especially the gonads (Stanworth,
Acanthobrama
of the interrenal,
terraa-sanctae is similar
-I_----
FIG. 5. Interrenal cells (ir) showing activity of 3pdydroxysteroid dehydrogenase. Substrate: Dehydroepiandrosterone. Freeze-dried, paraffin-embedded tissue. Green filter. Scale 20 p, cr-chromafin cells; v-venule. FIG. 6. 11 fl-hydroxysteroid dehydrogenase in interrenal cells (ir). Frozen section. Substrate: A4 Androstene-l&%ol-3,17-dione. Green filter. Scale 40 p. FIG. 7. Chromaffin cells. Stain: Wood; blue filter; scale 15 JL Abbreviations as in Fig. 5. FIG. 8. Chromaffin cells 24 hr after reserpine injection. Stain: Wood; bIue filter; scale 15 pp.Nate the disappearance of stained material from the chromaffin cells as compared with E‘ig. 7. Abbreviations as in Fig. 5.
548
Z. YARON
vided that suitable controls are used, may serve as a better parameter for evaluation of steroid synthesis in the interrenal cells, using small amounts of tissue. The obtained results are in good correlation with biochemical findings (Baillie, Ferguson, and McK. Hart, 1966). Nevertheless, the application of enzyme histochemistry for identification of steroidogenic sites in tissues is still limited to oxidative enzymes (dehydrogenases) only. Moreover, negative histochemical results are not necessarily conclusive and may be due to diffusion of the enzymes from the cells. The comparison between the histochemical preparations and routine histological sections allowed the proper use of fixed tissues for the detection of seasonal changes in the interrenal cells of the fish under study. However, the assumption that hypertrophied and hyperplastic interrenal tissue ,with large nuclei indicates elevated production and secretion of corticosteroids is indirect and based on correlations found in teleosts between the level of 17-OH steroids and the histological et al., 0’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 1 picture of the tissue (Robertson 1961; Hane et al., 1966). JJASONDJFMAM Further evidence that the histologic apFIG. 10. Seasonal variations in the (A) ovary pearance of the interrenals reflects the weight (‘% of body weight), data from Sivan (1967); of the cells is obtained (B) mean diameter of interrenal cells; IC) mean secretory activity the pituitary-interdiameter of nuclei; and (D) relative abundance of from studies involving renal axis in a variety of fish. For example, interrenal cells in the head kidney of Acanthobrama hypophysectomy or cortisol acetate injecterrae-sanctae, males and females. (Vertical linestion causes atrophy of interrenal cells in S.E.) Salvo and Tilapia (Donaldson and Mc1953; Hane et al., 1966; Honma and Bride, 1967; Basu, Nandi, and Bern, 1965), while metopirone treatment causes them to Tamura, 1963). Various histochemical methods have b,een hypertrophy in Poecilia and AnpiZZa (Ball and OIivereau, 1966). The present results applied to the piscine interrenals: lipid of dexamethasone treatment indicate that stains with Sudan black B, phospholipids and Schultz reaction for cholesterol (Chav- the principles of interrenal control by the and the pituitary gland in in, 1966; Mahon, Hoar, and Tabata, 1962), hypothalamus Acanthobrama terrae-sundae are basically but their specificity has been questioned similar to those found in mammals and in (Barka and Anderson, 1965). Moreover, the interpretation of the results is equivocal. the Salmon (Dallman and Yates, 1968; Thus, for instance, the presence of choles- Fagerlund and McBride, 1969). Since DXM terol within the interrenal cells could be treatment apparently depresses the level of explained as aggregation of precursors for activity of interrenal tissue, this drug may be utilized for experimental induction of increased synthesis of steroid hormones, interrenal hypofunction in teleosts where and at, the same time, one may interpret the phenomenon as accumulation of pre- surgical ablation is rather difficult (Chester cursors due to decrease in steroid synthesis. Jones et al., 1964; Butler et al., 1969). Robertson et al. (1961) found a rise of The use of enzyme histochemistry, pro-
FISH
CHRQMAFXIN
AND
x7-QH steroid level associated with hypertrophy of the interrenal cells in the plasma of maturing Salrno and Oncorhynchus. Hypert,rophy of interrenal cells during gonadal development in fish has been reported also by Stanworth (1953), Honma (1Q60), and Honma and Tamura (1963). Tt has been proposed that adrenal hyperplasm, indicating hyperfunction of the tisof sue, is due to ‘“severe derangement metabolism owing to development of the gonad” rather than to the “‘energy demand of upstream migration>’ (Honma and Tamura, 1963) e Svislotsky (1960) found that in Acan~~o~ranaa terrcwsanctae there is a reduction in weight of the liver and alimentary canal during the development of the gonads, especially in females. This raises the possibility that the liver provides materials for yolk formation. However, information concerning the effects of corticosteroids on the synthesis of yolk proteins in fish is still limited. Accordingly, it is speculative to state that gonad development and accumulation of yolk within the oocytes is supplied by mobilization of material from tissues which are affected by cortical hormones. awever, it is noteworthy in this regard that the hypertrophy and hyperplasia of the interrenal cells (Fig, 2) occur at the same time as the surge of vitellogenesis in the ovary. SCKNOWLEDGMENTS 1 thank Professor H. Steinita of the Hebrew University, Jerusalem, for his encouragement and advice, Mrs. L. Dekel for assistance, and Professor E. Kochva and Professor P. Kraicer of the TelAviv University, and Professor Charles S. Nicoll of the University of California at Berkeley for reading the manuscript. REFERENCES A. H., FERGTJSON, M. M., AND McK. HART, D. (1966). “Developments in Steroid Histochemistry.” Academic Press, London and New York.
BAILLIE,
BALL,
J.
X.,
AND
QL~VEIWAU,
MADELEINE.
(1966).
Identification of ACTH cells in the pituitary of two teleosts, Poecilia lati?-Jinna and Alzguilla anguilla e correlated changes in the interrena and in the pars distalis resulting from administration of metopirone (SU 4885). Gen. Comp. EncEocrino k. 6, 5-18 ~
INTERRENAL
BARKA,
CELLS
T.,
AND
ANDEBSON,
P, J.
(1968.
““&to-
chemistry, Theory, Practice, and Ribliographgr,“’ Harper & Row (Hoeber), New York. BAGU,
J., IYINDI,
J., AND BERN,
H. A.
<1966).
homolog of the pitnita~-adrenocort~~a~ the teleost fish TiEapia mossambica. Zoos?, 159, 347-356.
%a
atis in S. Esp.
BUTLER: D. G., CLARKE, W. C., DONALDSON, i?i. %!kp AND LANGFORD, R. w. (1969.9). &w&‘iCa~ Xke?X%~-
ectomy of a teleost fish (Anguilla ros&ato LeSueur) : effect on plasma cortisol and tiSSUe electrolyte and carbohydrate concentrations.. Gen. Camp. Endoctinol. 12, 503-514, CHAYIN, W. (1966). Adrenal histochemist*ry oh” some freshwater and marine teleosts. Gen. Con8.n. Endocl-inoZ. 6, 183-194. CHESTEEJONES, I.,. PHUIPS, J. G., .~?n PPOLMEB. w.
N,
(1959).
Comparative
physislogy
of
the
adrenal cortex. In “Comparative Endocrinology” (A. Gorbman, ed.), pp. 582-612. Wiley, ‘New York. CHESTER-JONES,
I., HENDERSON,
I. W.. AND Mosn,~,
W. (1964) ~ Methods for the adrenalectomy of the European eel (Anguilla angdh L.), J. &azdocrinol.3Q, DALLMAN-;,
155-156. MAW F., AND YATES,
F. E. (1968). Anatomical and functional mapping of central nervous input and feedback pathways of adrenocortical system. Mem. Sot. EndocrinoE. 17,X&-72.
DONALDSON,
E. M., AND MCBRIDE,
J. X. (%iX;
I The
effects of hypophysectomy in the rainbow trout Salmo gczirdnerii (Rich.) with special reference to the pituitary-interrenal axis. Gel. Camp, Endocrinol. 9, 93-161. FAGERWND,
U. H.
M.,
AND MCBRIDE,
J. R,
(
Suppression by dexamethasone of interren tivity in adult sockeye salmon (~~~~o~~~~c~~~ nerka) eGen. Camp. &&ooctinol. 12, 651-957, GURR, E. (1962). “Staining, Practical and Theoretical.” Wilhams and WiIkins, Baltimore. HANE, S., ROBERTBON, 0. H., WEXLER, KRUPP, M. A. (1966), Adrenocortical response to stress and ACTH in Pacific salmon (Oncorhqthus tshawytscha) and steelhead trout (Salvo saidmiil at successive stages in the sex-c& cycle. l!hdowinology 48, 791-800. HONXA, Y ~ (1960) ~ Studies on the endocrine glands of the salmonoid fish, Ayu, Plecoglossus a&t&.&& Temmimk & Sehlegel III. Changes in the r&eml co&al tissue during the life-span oi the fish. Annot. Zool. Jap. 33, 234-246. HoNM.~,
Y.,
AND TAMURA,
E.
(1963)
I Studies
on
the endocrine glands of the salmonoid f&h, the Ayu, Plecoglossus altivelti Temminck $ S&&gel. V. Seasonal changes in the endocrines of the land-focked form, the Koayu. Zoeb&a 44 2532.
550
2. YARON
adrenal tissues of the gold6sh. Can. J. 2001. 40, 449-464. OLIVEREAV, M. (1963). Action de la r&erpine sur l’hypophyse, l’interr&al et les cellules chromaffines de 1’Anguille (Arzguilla anguilZa L.) 6. R. Sot, Viol. 157, 1357-1360. hXRD, D., ET VITRY, G. (1964). Cytophysiologie de la cellule m&dullosurrknalienne. Arch. Biologic 75, 453496. ROBERTSON, 0. H., KRUPP, M. A, FAVOWR, C. B., HANE, S., AND (1961). Hyperadrenocorticism in
S. F,, B. C. spawning migratory and nonmigratory rainbow trout (Xalmo gairdnerii) ; comparison with Pacific salmon (genus Oneorhynchus) . Gen. Comp. Endocrinol. 1, 473484. RUBIN, B. L., DEANE, H. W., HAMILTON, J. A., AND DRIKS, E. C. (1963). Changes in A5-3phydroxysteroid dehydrogenase activity in the ovaries of maturing rats. Endocrinology 72, 924-930. SWAN, P. (1967). Seasonal changes in the gonads of Acunthobruma terrae-sanctae H. Steinitz from THOMAS, WEXLER,
Lake Tiberias. Sea Fish. Res. Stat. Haifa, Bull. 44, 22-41. STANWORTN, P. (1953). A study of reproduction in the three-spined stickleback Gasterosteus aculeatus L. with some reference to endocrine control. Thesis, Faculty Pure Science, Sheffield University. SVISLOTSKY, P. (1960). Seasonal changes in the gonads of Acanthobrama terraesanctae from Lake Tiberias. MSc. Thesis (In Hebrew), The Hebrew University, Jerusalem. VAN OVERBEEKE, A. P. (1960). “Histological Studies on the Interrend and the Phaeochromic Tissue in Teleostei.” Vrije Universiteit, Amsterdam. Van Munster’s Drukkerijen, Amsterdam. WOOD, J. G. (1963.). Identification of and observations on epinephrine and norepinephrine containing cells in the adrenal medulla. Amer. J. Anat. 112, 285-303. YARON, Z. (1969). Correlation between spawning, water temperature and thyroid activity in Acanthobrama terrae-sanctae (Cyprinidae) of Lake Tiberias. Gen. Comp. Endocrinol. 12, 604-608.
AND
COMPARATIVE
The
14, 542-550 (1970)
ENDOCRINOLOGY
Chromaffin
and
Interrenal
fame-sancfae
Cells
(Cyprinidae,
of Acanthobrama Teleostei)
Z. YARON Department
of
Zoology,
Tel
Aviv
University,
Tel
Aviv,
trael’
Received October 20, 1969 The interrenal and the chromaffin cells of Acanthobrama tewaesanctas are situated around the veins passing through the head kidney, 3,f3- and H/3-hydroxysteroid dehydrogenases were demonstrated in the interrenal cells. Using Wood’s method, norepinephrine was detected in the chromaffin cells of the head kidney. Reserpine treatment caused the depletion of stained material from the chromaffin cells. Dexamethasone treatment caused degenerative changes in the interrenal cells. Accordingly, this steroid may be used for experimental induction of interrenal hypofunction. Annual changes in histological features of the interrenal cells were studied. The highest mean diameter of cells and nuclei was found to coincide with vitellogenic activity in the ovary. The possible interactions between the ovary and the interrenal cells are discussed.
Acanthobrama terrae-sanctae,
a “sardine-
like” eyprinid from Lake Tiberias, during the winter (Sivan, 1967;
spawns Yaron, 1969), but in contrast to other winter breeders, such as the salmonids, its osmotic environment does not change considerably throughout the year. The relations between the interrenal function and reproduction of teIeost fishes has been studied mainly in salmonids (Chester Jones, Phillips, and Holmes, 1959; Robertson et al., 1961; Honma
and Tamura,
19633). It was obvious,
that Acanthobrama terraetherefore, sunctae would serve well as a different subject for studying the interrelations between reproduction and interrenal function. The aims of the present study were to locate the interrenal cells and study their seasonal
changes
in relation
with
gonadal
activity. MATERIALS AND METHODS Monthly samples of 50 live adult &h each were collected from ring-net fishing catches of fishermen in Lake Tiberias. Fish were brought alive to the ’ Present address: Department of PhysiologyAnatomy, University of California, Berkeley, California 94720.
shore and some were fixed in Allen’s fluid (Gurr, 1962) after incision of the abdomen. Others were transported alive to the laboratory and kept there for experimental work in tanks provided with running tap water. Histology
The right head kidneys were removed from the fixed specimens and longitudinal sections S,u thick, containing the posterior cardinal vein, were stained by the Azan method (Gurr, 1962). Measwements
The mean cell diameter was calculated from the number of interrenal cells crossed by the line of an eyepiece micrometer in sections of the head kidney containing interrenal cells only. The number of cells was divided by the total length of the line and the quotient was recorded as the mean for the given specimen. At least 10 sections from each fish were measured. The nuclear diameter was measure,d by a micrometric eyepiece (10 X) in 100 cells of each fish using an 100 X oil immersion objective. The relative abundance of interrenal cells was estimated by determining the proportion of interrenal cells underlying the line of the micrometric eyepiece viewed horizontally on sections at the magnification of 400X. Successive fields were changed at random. All sections of the head kidneys were measured.
FISH
CHROMAFFIN
AND
Histochemistry Interrenal
Cells. Activity
of BP-hydroxysteroid dehydrogenase was demonstrated in the interrenal cells by the method of Rubin et al. (1963) using frozen or freeze-dried paraffin-embedded tissue (Barka and Anderson, 1965~). bhromafiin Cells. These cells were identified using the method of Wood (1963).
Dexametkasone
Treatment
Males were kept in outdoor tanks for 2 weeks prior to the experiment. Three &roups of 10 fish each were placed in well-aerated, ‘20~liter aquaria, with flowing tap water and were fed with Tubifex. The experiment started on January 4 and ended on January 25, 1967, Water temperatures varied from 14”-16” during the day and between 9”-12” during the night. Fish were lightly anaesthetized wiath MS-222 (Sandoz) before injection of the drugs in order to facilitate handling. One group was injected every other day for 21 days with 10 pg dexamethasone (9a-fluoro-16ametl~yl-l1~,17a,21-trihydroxypregna-l,4-cliene-3,2Odione) in 0.1 ml of solution. The injections were given in dorsal myotomes anterior to the dorsa.1 fin. The dexamethasone (DXM) was dissolved in ethanol and mixed with amphibian Ringer to give a final concentration of 5% ethanol. Each fish received a tot.al dose of 100 pg DXM. The second &~oup received 0.1 ml injections of the solvent, and a third group was left untreated. On the 22nd day B& were anaesthetized with MS-222 and fixed in Allelk% &id. REXJLTS
General
The
Morphology
head
kidneys of Acanthobrama form the thick anterior ends of each mesonephros. They are covered by part of the peritoneum containing numerous melanophores. The head kidneys begin at the level of the fifth rib and extend anteriorly underneath the Weberian ossicles and ventrally t,o the pericardium. The Sateroposterior end of the head kidney reaches the swim bladder and the lateroanterior end touches the fifth ceratobranchid bone. terrae-sanctae
Bkood Supply The drainage dcanthobramnu.
metric;
of the
mesonephroi of is asymcardinal vein is un-
terrae-sanctae
the posterior
INTERRENAL
CELLS
643
paired and passes along the right mesonephros. Short transverse venules dra& the left kidney into the right cardinal vein, This is also the case for the head kidneyrs, i.e., the posterior cardinal vein passes through. the right head kidney and is absent in the left. An isthmus of lymphoid tissue passes transversally and bridges the dorsal ends of both head kidneys. The anterior cardinal vein enter% the rostra1 end of each head kidney and: at the ventral end of each, Cuvier’s duct drains into the sinus venosus (Figs. 1 and 2;. Thus, at the right head kidney> there is 8 junction of three main veins (Cuvier’s duet, anterior and posterior cardinal veins) ) whereas in the left only the first two are found. Large arteries supplying fhe head kidneys were not encountered. Nistology
The main mass of the head kid.ney is composed of lymph&d tissue, while endocrine components constitute only about 10% of the whole mass. Interrenal cells form one or more layers along t.he main blood vessels. Small clumps of chmmaEn cells are scattered among the interrenal cells, usually outside the inner lager of interrenal cells. A sympathetic gangljlon is found in the right head kidney (Fig, 3) I Interrenal C&s. The interrenlal cells are polygonal with a diameter of 6-~O+K.. In the autumn and spring they are somewhat shrunken and resemble a coiumriar epitbelium surrounding the cardinal veins. After fixation in AUen’s Auid and ham staining, the cytoplasm is homogeneous light grayish-violet or contains fine basophilie granuies. In a few casesprominent, fuchsinophiie granules were observed. Vacuoles, va.riable in size and distribution, are present.; in active eelis with large diameters, vacuoles are numerous, giving the cyI.oplasm a spongy appearance. The nuclear diameter varies from 3.2-4.9 ha; t’he nucleus cMain.s deeply stained dense chromatin and a. very prominent fuchsinophile nucleolus (Fig. 4) I Steroidogenic Activitz~. The identificat8ion of the interrenal cells is based on the hi&ochemical demonstration of 3/J-hydrnxysteroid dehydrogenase (S/Z?-HSDI and ‘IIF-
544
Z.
YARDN
FISH
CHROMAFFIN
AND
hvdroxysteroid dehydrogenase (ll/?-HSD) . DIformazan granules, representing the activity sites of these enzymes, are seen in the cytoplasm but not in the nucleus. ChromafEn cells do not show such granules (Figs. 5 and 6). Control sections incubated without the suitable substrates did not react, Chromafin Cells. The chromaffin cells with an average diameter of 16~~ are larger than the interrenal cells. The cell has a few projections, sometimes reaching the wall of the vein. The cytoplasm has little affinity for st,ains of Azan method and is almost transparent; only fine ,granules can be distinguished. In the oval nucleus, which is stained pink, there is a coarse net of chromatin and one or more, peripherally situat,ed, nucleoli (Fig. 4). Cytoplasmic inclusions are visualized using the method of Wood (1963) for epinephrine and norepinephrine. The yellow content indicates norepinephrine. Administration of reserpine (4 mg per 1OOg body weight) to the ,fish 24 hr prior to fixation caused the depletion of the stainable inclusions (Figs. 7 and $1. Dexamethasone Treatment
The interrenal, cells of males treated with dexamethasone (ea. 5 ,ug per g body weight) showed signs of degeneration. Figure 9 shows that the mean diameter of the interrenal cells decreased after dexame&asone treatment (p < 0.025 and p < 0.005, as compared with treated and untreated controls, respectively) I Nuclear diameter also decreased as compared with treated (p < 0.05) and untreated (p < 0.001’1 controls. The interrenal cells in the head kidneys of dexamethasone-treated fish
INTERRENAL
545
CELLS
appeared to be less abundant, than in control groups, but the diflerenw is significant (p > 0.2) I Seasonal Variations in the ~~te~e~a~ Cells During the summer, the me nuclear diameters are rather h wards October cell and nuclear diameters fall abruptly and interrenal cells become relatively ra,re in the head kidney (‘Fig. 10) p From October to December, ~o~l~o~ita~~ with the increase of ovarian weight (Si-m+ 1967) the interrenal cell and nuclear diameters increase and cells are more abundant, During the following winter months there is a gradual decrease in the diameter of interrenal cells and nuclei with minimal values occurring in May. DISCUSsION Distribution
of Interrenal
Cells in the
Kidney
The close association of the interrenal cells wit’h the asymmetrical cardinal veins results in the inequal d~stribut~oll of these cells ; the right head kidney containing more cells than the left one. A similar asymmetry was found in Betta splendsns by ‘G’an Overbeeke (1960)) who attributed ix Lo the difference in the size of the right and Jef”r, posterior cardinal veins. Medullury Cells Histochemical identification of catechcalamines using the ehromaffin reaction bss been performed in several teleost fishes and reviewed by Van Overbeeke (~~6~~ I who could not state conclusively whether the chromaffin or phaeochrome reaction could demonstrate the presence of adrenaline irb _I__
___I___
-- ..-..--.-
1. Sagittal section through the right head kidney of 14-mm fingerling. Connection between the cardinal veins and Cuvier’s duct. Stain: Azan, green filter. Scale 100 p. dc-Cuvier’s duct; h-liver; hk---right head kidney; ph-pharynx; sv-sinus venosus; vca-anterior cardinal vein; vcp-posterior cardinal vein; vt-ventricle. FIG. 2. Sagit,tal section of left head kidney of the same specimen. IConnection between the anierior cardinal vein and the Cuvier’s duct. Staining, filter and scale as in Fig. 1. de-Cuviefs duct; h-liver; hk---left, head kidney; ph-pharynx; vca-anterior cardinal vein. FIG. 3. Ganglion within the head-kidney of an adult. Stain: Azan; yellow filter; &ale 100 P. cr--chsomaf’& cells; g-ganglion; ir-interrenal cells; nv-nerve; vcp-posterior cardinal vein. FIG. 4. Tnterrenal (ir) and chromaffin (cr) cells in a fish caught in February. Stain: Azan; green filter. Scale 20 pd FIG.
546
FISH
CHROMAFFIN
AND
INTERRENAL
547
CELLS
DXM SIY UN CELL DIAMETER
NUCLEUS
DIAMETER
RELATIVE
ABUNDANCE
FIG. 9. ERect of dexamethasone treatment on cell diameter, nucleus diameter and relative abundance of interrenal tissue in the head kidney of Acunthobramz terra+sanctae males. DXM-dexamethasone-treated fish; SLV-solvent-injected fish; UNT-untreated fish. (Vertical 1ines-S.E.)
kleost fishes or whether other catechols are involved in this reaction. The method of WTood (1963) enables the differentiation between adrenaline and noradrenaline-containing cells in the hamster. When applied to the head kidney of Acunthobrama, only one cell type was noticed. Therefore, we are not in a position to state, according to the tinctorial features only, which of the two cateeholamines described in teleosts is present in these cells. Nevertheless, the total vacuolization and the disappearance of stainable material from the chromaffin cells 24 hr after reserpine treatment probably indicates that norepinephrine is the stained material in the chromaffin cells when Wood’s method is applied. Similarly, Olivereau (1963) showed that reserpine treatment in the eel (Anguilla anguilla) caused the depletion of norepinephrine from the chromaffin cells. The existence of two different types of
medullary cetk in mammals was questioned by Picard and Vitry (1964) ) who stated that ““cell ‘dimorphism appears to result not from ultrastructural but rather from chemical differences v r I and express a different functiona behavior of a single cell type-” As far as tinctorial evidence can be relied on, only one type of chromaEin cells is present in the head kidney of Aca~~~~~b~~m,~. The Interrenal Cells
The histology
celis ?n to that of other fish (Van Overbeeke, 1960; Mahon et CLI.~1962). However, no direct comparison can be made because of the changes in size and cytological details of the interrenal cells of A~~~t~~b~arn~ throughout the year; only in a few cases was the description of these cells in previous papers foilowed by notes on other glands, especially the gonads (Stanworth,
Acanthobrama
of the interrenal,
terraa-sanctae is similar
-I_----
FIG. 5. Interrenal cells (ir) showing activity of 3pdydroxysteroid dehydrogenase. Substrate: Dehydroepiandrosterone. Freeze-dried, paraffin-embedded tissue. Green filter. Scale 20 p, cr-chromafin cells; v-venule. FIG. 6. 11 fl-hydroxysteroid dehydrogenase in interrenal cells (ir). Frozen section. Substrate: A4 Androstene-l&%ol-3,17-dione. Green filter. Scale 40 p. FIG. 7. Chromaffin cells. Stain: Wood; blue filter; scale 15 JL Abbreviations as in Fig. 5. FIG. 8. Chromaffin cells 24 hr after reserpine injection. Stain: Wood; bIue filter; scale 15 pp.Nate the disappearance of stained material from the chromaffin cells as compared with E‘ig. 7. Abbreviations as in Fig. 5.
548
Z. YARON
vided that suitable controls are used, may serve as a better parameter for evaluation of steroid synthesis in the interrenal cells, using small amounts of tissue. The obtained results are in good correlation with biochemical findings (Baillie, Ferguson, and McK. Hart, 1966). Nevertheless, the application of enzyme histochemistry for identification of steroidogenic sites in tissues is still limited to oxidative enzymes (dehydrogenases) only. Moreover, negative histochemical results are not necessarily conclusive and may be due to diffusion of the enzymes from the cells. The comparison between the histochemical preparations and routine histological sections allowed the proper use of fixed tissues for the detection of seasonal changes in the interrenal cells of the fish under study. However, the assumption that hypertrophied and hyperplastic interrenal tissue ,with large nuclei indicates elevated production and secretion of corticosteroids is indirect and based on correlations found in teleosts between the level of 17-OH steroids and the histological et al., 0’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 1 picture of the tissue (Robertson 1961; Hane et al., 1966). JJASONDJFMAM Further evidence that the histologic apFIG. 10. Seasonal variations in the (A) ovary pearance of the interrenals reflects the weight (‘% of body weight), data from Sivan (1967); of the cells is obtained (B) mean diameter of interrenal cells; IC) mean secretory activity the pituitary-interdiameter of nuclei; and (D) relative abundance of from studies involving renal axis in a variety of fish. For example, interrenal cells in the head kidney of Acanthobrama hypophysectomy or cortisol acetate injecterrae-sanctae, males and females. (Vertical linestion causes atrophy of interrenal cells in S.E.) Salvo and Tilapia (Donaldson and Mc1953; Hane et al., 1966; Honma and Bride, 1967; Basu, Nandi, and Bern, 1965), while metopirone treatment causes them to Tamura, 1963). Various histochemical methods have b,een hypertrophy in Poecilia and AnpiZZa (Ball and OIivereau, 1966). The present results applied to the piscine interrenals: lipid of dexamethasone treatment indicate that stains with Sudan black B, phospholipids and Schultz reaction for cholesterol (Chav- the principles of interrenal control by the and the pituitary gland in in, 1966; Mahon, Hoar, and Tabata, 1962), hypothalamus Acanthobrama terrae-sundae are basically but their specificity has been questioned similar to those found in mammals and in (Barka and Anderson, 1965). Moreover, the interpretation of the results is equivocal. the Salmon (Dallman and Yates, 1968; Thus, for instance, the presence of choles- Fagerlund and McBride, 1969). Since DXM terol within the interrenal cells could be treatment apparently depresses the level of explained as aggregation of precursors for activity of interrenal tissue, this drug may be utilized for experimental induction of increased synthesis of steroid hormones, interrenal hypofunction in teleosts where and at, the same time, one may interpret the phenomenon as accumulation of pre- surgical ablation is rather difficult (Chester cursors due to decrease in steroid synthesis. Jones et al., 1964; Butler et al., 1969). Robertson et al. (1961) found a rise of The use of enzyme histochemistry, pro-
FISH
CHRQMAFXIN
AND
x7-QH steroid level associated with hypertrophy of the interrenal cells in the plasma of maturing Salrno and Oncorhynchus. Hypert,rophy of interrenal cells during gonadal development in fish has been reported also by Stanworth (1953), Honma (1Q60), and Honma and Tamura (1963). Tt has been proposed that adrenal hyperplasm, indicating hyperfunction of the tisof sue, is due to ‘“severe derangement metabolism owing to development of the gonad” rather than to the “‘energy demand of upstream migration>’ (Honma and Tamura, 1963) e Svislotsky (1960) found that in Acan~~o~ranaa terrcwsanctae there is a reduction in weight of the liver and alimentary canal during the development of the gonads, especially in females. This raises the possibility that the liver provides materials for yolk formation. However, information concerning the effects of corticosteroids on the synthesis of yolk proteins in fish is still limited. Accordingly, it is speculative to state that gonad development and accumulation of yolk within the oocytes is supplied by mobilization of material from tissues which are affected by cortical hormones. awever, it is noteworthy in this regard that the hypertrophy and hyperplasia of the interrenal cells (Fig, 2) occur at the same time as the surge of vitellogenesis in the ovary. SCKNOWLEDGMENTS 1 thank Professor H. Steinita of the Hebrew University, Jerusalem, for his encouragement and advice, Mrs. L. Dekel for assistance, and Professor E. Kochva and Professor P. Kraicer of the TelAviv University, and Professor Charles S. Nicoll of the University of California at Berkeley for reading the manuscript. REFERENCES A. H., FERGTJSON, M. M., AND McK. HART, D. (1966). “Developments in Steroid Histochemistry.” Academic Press, London and New York.
BAILLIE,
BALL,
J.
X.,
AND
QL~VEIWAU,
MADELEINE.
(1966).
Identification of ACTH cells in the pituitary of two teleosts, Poecilia lati?-Jinna and Alzguilla anguilla e correlated changes in the interrena and in the pars distalis resulting from administration of metopirone (SU 4885). Gen. Comp. EncEocrino k. 6, 5-18 ~
INTERRENAL
BARKA,
CELLS
T.,
AND
ANDEBSON,
P, J.
(1968.
““&to-
chemistry, Theory, Practice, and Ribliographgr,“’ Harper & Row (Hoeber), New York. BAGU,
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homolog of the pitnita~-adrenocort~~a~ the teleost fish TiEapia mossambica. Zoos?, 159, 347-356.
%a
atis in S. Esp.
BUTLER: D. G., CLARKE, W. C., DONALDSON, i?i. %!kp AND LANGFORD, R. w. (1969.9). &w&‘iCa~ Xke?X%~-
ectomy of a teleost fish (Anguilla ros&ato LeSueur) : effect on plasma cortisol and tiSSUe electrolyte and carbohydrate concentrations.. Gen. Camp. Endoctinol. 12, 503-514, CHAYIN, W. (1966). Adrenal histochemist*ry oh” some freshwater and marine teleosts. Gen. Con8.n. Endocl-inoZ. 6, 183-194. CHESTEEJONES, I.,. PHUIPS, J. G., .~?n PPOLMEB. w.
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I., HENDERSON,
I. W.. AND Mosn,~,
W. (1964) ~ Methods for the adrenalectomy of the European eel (Anguilla angdh L.), J. &azdocrinol.3Q, DALLMAN-;,
155-156. MAW F., AND YATES,
F. E. (1968). Anatomical and functional mapping of central nervous input and feedback pathways of adrenocortical system. Mem. Sot. EndocrinoE. 17,X&-72.
DONALDSON,
E. M., AND MCBRIDE,
J. X. (%iX;
I The
effects of hypophysectomy in the rainbow trout Salmo gczirdnerii (Rich.) with special reference to the pituitary-interrenal axis. Gel. Camp, Endocrinol. 9, 93-161. FAGERWND,
U. H.
M.,
AND MCBRIDE,
J. R,
(
Suppression by dexamethasone of interren tivity in adult sockeye salmon (~~~~o~~~~c~~~ nerka) eGen. Camp. &&ooctinol. 12, 651-957, GURR, E. (1962). “Staining, Practical and Theoretical.” Wilhams and WiIkins, Baltimore. HANE, S., ROBERTBON, 0. H., WEXLER, KRUPP, M. A. (1966), Adrenocortical response to stress and ACTH in Pacific salmon (Oncorhqthus tshawytscha) and steelhead trout (Salvo saidmiil at successive stages in the sex-c& cycle. l!hdowinology 48, 791-800. HONXA, Y ~ (1960) ~ Studies on the endocrine glands of the salmonoid fish, Ayu, Plecoglossus a&t&.&& Temmimk & Sehlegel III. Changes in the r&eml co&al tissue during the life-span oi the fish. Annot. Zool. Jap. 33, 234-246. HoNM.~,
Y.,
AND TAMURA,
E.
(1963)
I Studies
on
the endocrine glands of the salmonoid f&h, the Ayu, Plecoglossus altivelti Temminck $ S&&gel. V. Seasonal changes in the endocrines of the land-focked form, the Koayu. Zoeb&a 44 2532.
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adrenal tissues of the gold6sh. Can. J. 2001. 40, 449-464. OLIVEREAV, M. (1963). Action de la r&erpine sur l’hypophyse, l’interr&al et les cellules chromaffines de 1’Anguille (Arzguilla anguilZa L.) 6. R. Sot, Viol. 157, 1357-1360. hXRD, D., ET VITRY, G. (1964). Cytophysiologie de la cellule m&dullosurrknalienne. Arch. Biologic 75, 453496. ROBERTSON, 0. H., KRUPP, M. A, FAVOWR, C. B., HANE, S., AND (1961). Hyperadrenocorticism in
S. F,, B. C. spawning migratory and nonmigratory rainbow trout (Xalmo gairdnerii) ; comparison with Pacific salmon (genus Oneorhynchus) . Gen. Comp. Endocrinol. 1, 473484. RUBIN, B. L., DEANE, H. W., HAMILTON, J. A., AND DRIKS, E. C. (1963). Changes in A5-3phydroxysteroid dehydrogenase activity in the ovaries of maturing rats. Endocrinology 72, 924-930. SWAN, P. (1967). Seasonal changes in the gonads of Acunthobruma terrae-sanctae H. Steinitz from THOMAS, WEXLER,
Lake Tiberias. Sea Fish. Res. Stat. Haifa, Bull. 44, 22-41. STANWORTN, P. (1953). A study of reproduction in the three-spined stickleback Gasterosteus aculeatus L. with some reference to endocrine control. Thesis, Faculty Pure Science, Sheffield University. SVISLOTSKY, P. (1960). Seasonal changes in the gonads of Acanthobrama terraesanctae from Lake Tiberias. MSc. Thesis (In Hebrew), The Hebrew University, Jerusalem. VAN OVERBEEKE, A. P. (1960). “Histological Studies on the Interrend and the Phaeochromic Tissue in Teleostei.” Vrije Universiteit, Amsterdam. Van Munster’s Drukkerijen, Amsterdam. WOOD, J. G. (1963.). Identification of and observations on epinephrine and norepinephrine containing cells in the adrenal medulla. Amer. J. Anat. 112, 285-303. YARON, Z. (1969). Correlation between spawning, water temperature and thyroid activity in Acanthobrama terrae-sanctae (Cyprinidae) of Lake Tiberias. Gen. Comp. Endocrinol. 12, 604-608.