JOURNAL OF MORPHOLOGY 212:65-70 (1992)

Functional Stages in the lnterrenal Cells of Rana perezi (Anura: Ranidae)

RAFAEL ALVAREZ Departamento de Biologia Celular y Anatomia, Uniuersidad de Ledn, 24071 Ledn, Spain

ABSTRACT The electron density of the lipid droplets and mitochondrial matrix of the interrenal cells of Ranaperezi differs during the year. This makes it possible to characterize the different stages of interrenal cell activity. A dropletjmitochondria index, based on their relative size, may provide an indicator of cellular activity.

In lower vertebrates, including amphibi- ans, the adrenal gland is composed of interre- nal and chromaffin tissue, synthesizing corti- costeroids and catecholamines, respectively (Beaumont and Cassier, '82). The interrenal cells possess attributes typical of steroid- producing cells: droplets, mitochondria with tubular cristae, and smooth endoplasmic re- ticulum (Chester-Jones, '87). These cells show seasonal changes in structure which reflect variation in their synthetic activity (Lofst and Bern, '72; Licht et al., '83).

The various functional stages in amphib- ian interrenal tissue have been described by several authors (Lofst and Bern, '72; Fritsch et al., '77; Accordi et al., '81). Characteristics that change in relation to the degree of func- tional activity include electron density of the lipid droplets and mitochondrial matrix and the development of smooth endoplasmic retic- ulum and mitochondria. Fritsch et al. ('77) suggested that changes in mitochondria and lipid droplets might be useful as indicators of the degree of hormonal production.

In the present study, changes in the elec- tron density of lipid droplets and mitochon- drial matrix in interrenal cells of Ranaperezi were determined over a period of one year. In addition, another possible morphological in- dicator of interrenal cell activity was mea- sured: the average diameter of lipid droplets in relation to the average diameter of mito- chondria.

MATERIALS AND METHODS

Forty-eight adult female Rana perezi were collected in September 1983 from their natu- ral habitat near Ledn (Spain). Specimens were maintained for a year under regulated light and temperature conditions (12L/ 12D and 20/22OC) in enclosures, each containing a

receptacle of water which was changed every second day. All frogs were fed live worms, flies, and grasshoppers every three days. Four animals were sacrificed each month over a period of one year. Both kidneys were re- moved immediately, fixed in 2.5% glutaralde- hyde in Sorensen buffer at pH 7.4, postfixed in 1% O,O, in the same buffer, dehydrated in acetone, and embedded in Araldite. Ultrathin sections double-stained with lead citrate and uranyl acetate were examined with a JEOL 100-C electron microscope.

For each month of the study, sections were prepared from the two kidneys in each of the four animals sacrificed. Four grids of sections from each kidney were selected, and four cells from each field were chosen for analysis. Thus 128 cells were observed monthly for assessment of electron density of lipid drop- lets and mitochondria.

The electron density of the lipid droplets was evaluated and assigned values as follows: electron dense (value 11, intermediate (value 2), or electron lucent (value 3). The mitochon- drial matrix was also evaluated and assigned the following values: electron lucent (value l ) , intermediate (value 2), or electron dense (value 3).

Thirty-two cells per animal were examined monthly and eight lipid droplets and mito- chondria per cell (256 droplets and mitochon- dria per animal) were measured. Measure- ments of the diameters of the lipid droplets and mitochondria were obtained from photo- graphs by using an IBAS I1 Kontron Image Analysis System. The values ranged from 1 to 3. The index obtained by dividing the aver- age diameter of the lipid droplets by the average diameter of the mitochondria was designated an i value. This index value was

o 1992 WILEY-LISS, INC.

66 R. ALVAREZ

calculated from the data obtained each month.

The average values for each month and their respective standard deviations were cal- culated, represented graphically, and com- pared to each other by application of Stu- dent's "t" test (P < 0.05).

RESULTS

In Rana perezi, the suprarenal gland is located on the ventral surface of the kidney as an elongated structure covered by the same mesothelium that covers the kidney. The cells are arranged in cords and surrounded by large blood sinuses (Fig. 1). The glands are composed of three types of cells which are randomly distributed (Fig. 2). The cytoplasm of two of these types of cells is filled with granules, while the cytoplasm of the third cell type appears spongy and lacks definite boundaries. At the ultrastructural level, the three kinds of cells can be identified un- equivocally. The granulated cells are epineph- rine- and norepinephrine-producing cells comprising the chromaffin tissue, while the remainder of the cells are corticosteroid- synthesizing cells comprising the interrenal tissue. These interrenal cells are elongated with eccentrically located nuclei. Their cyto- plasm contains three elements characteristic of the cells which secrete steroid hormones: smooth endoplasmic reticulum, mitochon- dria with tubular cristae, and lipid droplets (Fig. 3).

Over a 12-month period, the lipid droplets and mitochondrial matrix vary considerably in density and appearance (Fig. 4a-d). As indicated in Table 1, the average density of the lipid droplets is relatively low (2.27-3.00) during summer and autumn (June through November) but higher (1.16-2.00) during winter and spring (December through May) (Figs. 5 , 6). In contrast, the average density of the mitochondrial matrix is relatively high (1.97-2.66) during summer and autumn (June through November) but lower (1.11- 1.66) during winter and spring (December through May) (Figs. 5 , 6). When monthly changes are considered, it is evident that the maximum changes in density of the lipid droplets coincide temporally with those of mitochondria. The greatest decline in density of the lipid droplets occurs between May and June, while the mitochondrial matrix shows its greatest increase in density at this time. Moreover, the greatest increase in density of the lipid droplets occurs between November

and December, while the mitochondrial ma- trix shows its greatest decrease in density at this time.

Changes in i values also display seasonal variation. As shown in Table 1, the values are above 2.00 in material fixed during the period of August through January, but less than 2.00 in material fixed during the period of February through July. The greatest decline per month occurs between January and Feb- ruary, while the greatest increase per month occurs between July and August. Thus, changes in the average sizes of the lipid drop- lets in relation to those of mitochondria ap- pear to be two months out of synchrony with changes occurring in the electron densities of these structures (Fig. 7).

DISCUSSION

Interrenal cells in both Rhacophorus leuco- mystax (Rhacophoridae) and in Xenopus lae- uis (Pipidae) (Accordi and Cianfoni, '81; Ac- cordi et al., '81) show lipid bodies varying in size, mitochondria with tubular cristae, and smooth endoplasmic reticulum which may show variation related to various functional stages of the cell (Hanke, '78; Accordi and Cianfoni, '81; Accordi et al., '81). The present study has shown that the anatomy, struc- ture, and ultrastructure of the interrenal tissue of Rana perezi closely resemble those considered typical of the anuran amphibians by other authors.

The different functional stages of interre- nal cells have been described by many au- thors, including Kemenade ('68), Fritsch et al. ('77) and Accordi et al. ('81) in Rana esculenta, R. temporaria, and Xenopus lae- uis. The stage of greatest functional activity is characterized by the presence of electron- dense lipid droplets and mitochondria with moderately electron-dense matrix. The stage of least activity is characterized by the pres- ence of electron-lucent lipid droplets and elec- tron-dense mitochondrial matrix.

On the other hand, Lofst and Bern ('72) noted that the high accumulation of lipid droplets in the cytoplasm indicates low func- tional activity, while the disappearance of such droplets is a sign of enhanced activity. These authors also observed variability in the development of smooth endoplasmic retic- ulum and mitochondria in several periods throughout the year and inferred that this variability is linked to changes in functional activity of the cells.

In the present work, not only were some of the characteristics mentioned previously by

FUNCTIONAL STAGES IN R. PEREZI INTERRENAL CELLS 67

Fig. 1. Rana perezi. Adrenal gland (A) close to the kidney (K). v, blood vessels. Scale bar = 50 Fm.

Fig. 2. Rana perezi. Adrenal gland showing cells pro- ducing epinephrine (e), norepinephrine (ne), and corticos- teroids (open arrows). Arrowheads, mesothelium. Scale bar = 4 pm.

Fig. 3. Rana perezi. Detail of interrenal cell. Note nucleus (n), mitochondria with tubular cristae (m), smooth endoplasmic reticulum (I), and lipid droplets (d). Scale bar = 0.1 wm.

Fig. 4. Rana perezi. Examples of variation in electron density of lipid droplets (d) and mitochondrial matrix (arrows). Lipid droplets are electron dense in a, electron lucent in b, and intermediate in density in c and d. Mitochondria1 matrix is moderately dense in a, dense in b, and intermediate in density in c and d. Scale bars = 0.1 pm.

Fig. 5. Rana perezi. Interrenal cells in active condi- tion. d, lipid droplets; n, nucleus; arrowheads, mitochon- dria. Scale bar = 1 pm.

Fig. 6. Ranaperezi. Interrenal cells in inactive condi- tion. d, lipid droplets; n, nucleus; arrowheads, mitochon- dria. Scale bar = 1 bm.

FUNCTIONAL STAGES IN R. PEREZ1 INTERRENAL CELLS 69

TABLE 1 . Monthly density values oflipid droplets (0) and mitochondrial matrix (M), and ratios of diameters of lipid droplets and mitochondria (i)

Spring 3 4 5

3.00 f 0.00 2.85 f 0.34 2.85 f 0.34

1.33 f 0.47 2.00 f 0.45

2.50 f 0.50 2.27 f 0.86

1.16 f 0.37 1.50 f 0.70 1.50 Ifr 0.50

1.11 f 0.31' 1.50 -t 0.50

2.50 f 0.50 2.22 f 0.41

1.70 r 0.04

1.59 f 0.04 2.42 f 0.03

'Months a r r numbered from January 11, to Ikwmber (121. .Values are means 5 standard drviations of ohsenations in 128 cells per month. 'Valucs a r r means z standard deviations of ratios ot'diamrters of 1,024 lipid droplrts and mitochondria per month

several authors considered, but also the size ratios of lipid droplets and mitochondria were evaluated as possible indicators of hormonal production. The data suggest relationships between lower dropletst mitochondria ratios and lower activity. I t seems likely that changes in lipid droplets reflect functional activity better than changes in mitochondria, since the droplets are more abundant and variable than mitochondria. Fritsch et al. ('77) considered the sizes of both mitochon- dria and lipid droplets as possible indicators of hormonal production in Xenopus laevis. Further research may reveal the reasons for non-synchrony between the electron density of lipid droplets and mitochondrial matrix, and the dropletst'mitochondria index.

Our findings show that it is possible to use ultrastructural morphology to characterize the various interrenal activity periods throughout the year. An active stage, charac-

terized by interrenal cells with electron- dense droplets and moderately electron-lu- cent mitochondrial matrix, occurs in winter and spring; a stage of reduced activity, char- acterized by cells with electron-lucent drop- lets and electron-dense mitochondrial ma- trix, occurs in summer and au tumn. Furthermore, in spring there is a low drop- letst'mitochondria index, whereas this index is high in autumn.

The seasonal changes in Rana perezi de- scribed here, and any disagreement with the results described by other authors, may pos- sibly be attributed to geographical and cli- matic causes. The fact that the interrenal cells in R. perezi show seasonal variations, in spite of the fact that the animals are kept for a year under constant light and temperature conditions, should be the subject of further research.

Ranaperezi. Values of density of lipid droplets (D) and mitochondrial matrix (M) and ratios of diameters of lipid droplets and mitochondria (i) plotted from data shown in Table 1. Data for the year studied are plotted for two consecutive years in order to emphasize their

cyclic nature. Numbers on the abscissa are months of the year. Values from 1.0 to 3.0 on the ordenate are estimates of electron density for D and M, and of ratios of diameters for z.

70 R. ALVAREZ

ACKNOWLEDGMENTS

Thanks to the anonymous reviewers who assisted me in improving the manuscript.

LITERATURE CITED Accordi, F., and P. Cianfoni (1981a) Histology and ultra-

structure of the adrenal gland of Rhacophorus leuco- mystux (Amphibia, Anura). Boll. Zool. 48t277-288.

Accordi, F., V.P. Galla, and E. Grasi Milano (1981) The adrenal gland of Xenopus laevis (Daudin) (Anura, Pip- idae): Histological and ultrastructural observations. Monitore Zool. Ital. (N.S.). 15163-174.

Beaumont, A,, and P. Cassier (1982) Biologie animale. Les Cordes. Anatomie compare6 des Vertebres. Paris: Bordas.

Chester-Jones, I. (1987) Structure of the adrenal and interrenal glands. In I. Chester-Jones, P.M. Inglenton, and J.G. Phillips (eds): Fundamentals of Comparative Vertebrate Endocrinology. New York: Plenum Press, pp. 95-120.

Fritsch, H.A.R., F.-W. Pehlemann, and H. Faltz (1977) Effect of partial hepatectomy of the interrenal tissues of Xenopus laevis (Daudin). Cell Tissue Res. 179:197- 209.

Hanke, W. (1978). The adrenal cortex of Amphibia. In I. Chester-Jones and I.W. Henderson (eds): General, Com- parative and Clinical Endocrinology of the Adrenal Cortex, Vol. 2. London: Academic Press, pp. 419456.

Kemenade, J.K.M. van (1968) Effect of ACTH and hy- pophysectomy of the interrenal tissue in the common frog, Rana temporaria. 2. Zellforsch. Mikrosk. Anat. 92t549-553.

Licht, P., B.R. McCreery, R. Barnes, and R. Pang (1983) Seasonal and stress related changes in plasma gonadot- ropins, sex steroids and corticosterone in the bullfrog, Rana catesbeiana. Gen. Comp. Endocrinol. 50r124- 145.

Lofst, B., and N.A. Bern (1972) The functional morphol- ogy of steroidogenic tissues. In D.R. Idler (ed): Steroids in Nonmammalian Vertebrates. New York: Academic Press, pp. 37-51.