Renin immunohistochemistry in the adrenal gland of the mouse fetus and neonate.код для вставкиСкачать
THE ANATOMICAL RECORD 227:124-131 (1990) Renin lmmunohistochemistry in the Adrenal Gland of the Mouse Fetus and Neonate YASUHIRO KON, YOSHIHARU HASHIMOTO, HIROSHI KITAGAWA, MAKOTO SUGIMURA, AND KAZUO MURAKAMI Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Hokkaido University, Kita-ku Kita 18-jou Nishi 9-choume Sapporo 060 ( Y . K., Y . H., M . S.); Department of Veterinary Anatomy, Faculty of Agriculture, Tottori University, Tottori 680 (H. K.); and Department of Applied Biochemistry, University of Tsukuba, Zbaraki 305 ( K . M.), Japan ABSTRACT The development of renin-containing cells in fetal and neonatal adrenal glands of the mouse was studied using immunohistochemistry. On days 13-14 of gestation, immunoreactivity for renin was first observed in a few cortical cells of the gland, appearing a s small patchy or granular reaction products in the perikaryon. The mitotic configurations of the cells demonstrating immunoreactivity were noted. On day 16 of gestation, a number of intensively immunoreactive cells were distributed in the aortal side of the cortical zone. On day 18 of gestation, and day 1 postparturition, a small number of potent immunoreactive cells were still found in the cortical area. Immunoreactivity of the cytoplasm was observed in the cells, some showing a n intensive reaction and others possessing numerous tiny granules just below the cell membrane. On days 3, 5, and 7 after birth, no renincontaining cells were found in the adrenal gland. The ratio of the numbers of renin-positive cells in certain areas to the numbers in the entire cortical area was significantly increased on day 16 of gestation, but there was no sexual difference in the ratios. The ratios were decreased subsequently until day 1 after birth. The possible significance of renin synthesis in specific adrenal cells in fetal life is discussed with respect to a n important involvement of angiotensin I1 in the morphogenesis of the adrenal gland of the mouse. Renin, which is synthesized mainly by the juxtaglomerular cells of the kidney, has a triggering role in the control of circulating blood pressure by cleaving angiotensinogen to form angiotensin I (Davis and Freeman, 1976). Recently, renin (or renin-like) activities or renin-containing tissues in the extrarenal regions have been reported in several organs, namely, in the submandibular gland (Cohen e t al., 1972; Tanaka et al., 1980), brain (pineal, anterior, and intermediate pituitary) (Deschepper et al., 1985; Haulica et al., 1975; McKenzie et al., 1985),adrenal gland (Naruse and Inagami, 1982; Naruse et al., 1983; Ryan, 1972), testis (Parmentier et al., 1983)) uterus (Eskildsen, 19721, ovary (Fernandez et al., 1985a,b),placenta (Poisner et al., 1981), and vascular wall (Gould e t al., 1964; Swales et al., 1983). It is generally accepted that these extrarenal renins may function as local, independent reninangiotensin systems in individual organs rather than as a circulating one such as that originating from the kidney (Deschepper and Ganong, 1988). However, there have only been a few morphological identifications of the cell types in individual organs. We have studied the development of the intrarenal and extrarenal renin-angiotensin system ontogenetically using immunohistochemical techniques for enzyme renin (Kon et al., 1984, 1989). Thus, in the present study, we examined the renin-containing cells in fetal and neonatal adrenal glands of mouse from the viewpoint of the development of the renin-angiotensin0 1990 WILEY-LISS, INC aldosterone system, and we discuss here the functional meaning of the regional occurrence of renin in developmental adrenals. MATERIALS AND METHODS Mouse (Balbk strain) fetuses from 11 to 18 days of gestation and neonates, aged 1 to 7 days, were used in this examination. For demonstration of gestational stages, adult male and female mice were placed together overnight, and day 0 of the gestational period was determined with the appearance of a vaginal plug. All animals examined in this study were anesthetized with ether. Whole fetal specimens or dissected adrenal glands were fixed in Bouin’s solution overnight. Tissues were dehydrated by graded ethanol series, embedded in paraffin, and sectioned transversely o r sagittally into 5-km-thick slices through the adrenal glands. The sections were deparaffinized, rehydrated, and processed by routine staining with hematoxylineosin, periodic acid Schiff-hematoxylin, azan-trichrome, or Grimerius’ impregnation, and by a n immunohistochemical method using specific antimouse submandibular gland renin rabbit-serum (Kon et al., 1989).Grimelius’ impregnation was carried out for the detection of the argyrophilic cells, especially the adre- Received March 13, 1989; accepted August 14, 1989. RENIN IN ADRENAL GLAND 125 Fig. 1. Sagittal section of the adrenal gland on day 12 of gestation. The gland is composed of a cluster of undifferentiated cells (arrow). Hematoxylin-eosin, x 80 Fig. 2. Transverse section of the adrenal gland on day 13 of gestation. a: Argyrophilic cells are scattered within the adrenal parenchyme. Adrenal glands are surrounded by arrows. Grimelius’ impregnation. x 80. b: Note a few renin-immunoreactive cells containing small patchy or granular reaction products (arrows). Immunohistochemical method. x 160. c: Renin immunoreactivity in the wall of an intra-adrenal vessel (arrow). Immunohistochemical method. x 160. 126 Y. KON ET AL nal medulla cells. Briefly, this method consisted of the following successive incubation in: 1)0.003% silver nitrate solution for 3 hours a t 60" C; 2) reducing solution containing 1 % hydroquinone and 5% sodium sulfite for 1 minute a t 45" C; and 3) 5% sodium thiosulfate for 2 minutes a t room temperature. The immunostaining sequence for renin comprised the following steps: 1) treatment with 0.1% hydrogen peroxide and with absolute methanol for 30 minutes, respectively; 2) preincubation in 1% normal goat serum for 1 hour a t room temperature; 3) incubation with rabbit antimouse renin antiserum 1:2,000-3,000 for 48 hours a t 4" C; 4) incubation with goat antirabbit immunoglobulin G serum 1 : l O O for 1 hour a t room temperature; 5) incubation with soluble rabbit PAP serum 1:lOO for 1hour at room temperature; and 6) exposure to 3,3'-diaminobenzidine-HC1. Controls for the immunohistochemical staining consisted of normal rabbit serum o r phosphate-buffered saline substituted for the serum of the specific antibody. The sections were counter-stained slightly with hematoxylin. Because renin-antiserum was raised against male mouse submandibular gland renin, morphometrical analysis was employed to determine if the development of immunoreactivity for renin in the male adrenal gland was different from those in female. For morphometrical analysis, the transverse serial sections of adrenal glands obtained from timed animals, namely, on days 14, 16, and 18 of gestation and on day 1 after birth, were used, and the sex of each fetus was determined with observation of the primordial genital organs. In sections impregnated a t each stage with Grimelius' method, the areas of the whole parenchyme or the medulla were divided and, in the neighboring sections immunostained for renin, the renin-positive areas were shown as numerical values. The data of these measurements, obtained by using a computenized planimeter system (Cosmozone-1 system, software by Nikon, Inc.), were represented finally a s a percentage of the renin-positive area against the entire cortical area. RESULTS Days 11 and 12 of Gestation Although there were conglomerations of undifferentiated adrenal cells a t the caudoventral region of the primordial metanephros, no capsule surrounding the adrenal gland was present. This allowed no obvious discrimination of the cortex and the medulla at this stage (Fig. 1). No renin immunoreactivity was demonstrated within the adrenal tissue in this gestational period. A bbreuiations Ad adrenal gland Ao aorta Cap capsule M medulla MT metanephros Day 13 of Gestation The capsule, the cortex, and the medulla were still immature; however, a few argyrophilic cells were found to be sparsely scattered, and some were conglomerated within the parenchyma (Fig. 2a). Immunoreactivity for renin was demonstrated in the parenchymal cells of the adrenal gland in only one fetus of the five examined in this gestational period (Fig. 2b). The reaction products in the cytoplasm of the positive cells appeared a s small patchy or granular forms. The wall of the arteriole flowing into the adrenal tissue also contained a renin immunoreactive cell (Fig. 2c). Day 14 of Gestation A thin, fibrous capsule partly constructed with the mesothelial tissue was found to surround the gland. Angiogenesis was advanced, and many blood capillaries were found to occur irregularly among the parenchymal cells. Because the aggregation of argyrophilic cells started to form numerous small islets centripetally, i t was noted, in varying degrees, that the area of separation of the gland split into a n inner medullary and a n outer cortical zone. In this period, the renin-containing cells were scattered within the gland and distributed numerously in the medial region of the cortical zone near the aorta (Fig. 3a), whereas cortical cells in the lateral regions contained weaker or no immunoreactions. The renincontaining cells occurring in the medullary zone were found neighbouring the small islets of medulla cells. The immunoreactive cells were represented a s large, round-to-oval shapes, where the cytoplasm possessed small dots or rod-shaped immunoreactive inclusions near the nucleus (Fig. 3b). In addition, there were some mitotic cells showing slightly demonstrable immunoreactivity. Day 16 of Gestation The results obtained from serial sections exposed to antirenin serum or impregnated with Grimelius' method showed that renin-containing cells were localized only in the cortical zone (Fig. 4a, b). During this gestational period, a number of intensive immunoreactive cells were distributed in the cortical zone near the aorta, whereas the opposite region contained numerous feebly reacting cells as well a s those found on day 14 of gestation. The outer zone of the cortical tissue just below the capsule was not generally composed of immunoreactive cells. The continuous appearance of immunoreactive cells from the deep cortical zone to this outer zone was rarely encountered, especially in the ventral region of the gland (Fig. 412). Immunoreactivity in the cytoplasm of positive cells was observed to have various morphologies, namely, from showing intense activity thoughout the cytoplasm to demonstrating a number of small-sized granular appearances. Most granular configurations of the reaction products were generally detected in their peripheral cytoplasm. In the deep or intermediate zone of the cortex, the intensely reactive cells were sparsely distributed rather than making cellular aggregations. Furthermore, mitotic cells showing immunoreactivity for renin were still observed throughout the cortical zone (Fig. 4d). Fig. 3. Renin-containing cells of the adrenal gland on day 14 of gestation. a: Many renin-immunoreactive cells are observed in the cortical zone near the aorta. Immunohistochemical method. x 80. b: The cytoplasm of the positive cells contains small dots or rod-shaped immunoreactive inclusions (arrows). Mitotic cell expressing immunoreactivity is observed (arrowhead). Immunohistochemical method. x 310. Fig. 4. The adrenal gland on day 16 of gestation. The serial sections stained with immunohistochemistry for renin (a)and with Grimelius’ impregnation (b). x 80. c: Immunoreactivity located just below the adrenal capsule (arrow). Immunohistochemical method. x 310. d: A mitotic configuration containing renin reaction (arrow).Immunohistochemical method. x 500. 128 Y. KON ET AL. Day 18 of Gestation The parenchymal cells had grown into a more tightly packed arrangement, and the capsule decreased in thickness. In the adrenal cortex stained with hematoxylin-eosin, one or more layers of cells just below the capsule showed a basophilic appearance; however, the cells of deeper zones were demonstrated as larger acidophilic ones, most of which showed a diffuse, weak reaction for renin (Fig. 5a). Ten or more potent immunoreactive cells were found in solitude or in aggregations in the medial region of the adrenal cortex near the aorta per a 5-pm-section. Although immunoreactive cells were also found in the opposite side of the cortex, most were distributed solitarily. Immunoreactive cells decreased their cytoplasmic reactivity in this period; however, numerous granules with marked reactivity were located just below the cell membrane (Fig. 5b), and a few mitotic immunoreactive cells were still observed in the middle cortical zone as well a s those found on day 16 of gestation. Day 1 After Birth Immunoreactive cells still markedly decreased in number and were scattered in the middle cortical region facing the aorta (Fig. 6a). The immunoreactivity of their cytoplasm ranged from intensive reactions to possessing a few tiny granules below the cell membrane and vacuolized figure (Fig. 6b). Days 3, 5, and 7 After Birth No renin-containing cells were found in any areas of the adrenal gland (Fig. 7a, b). The ratios of the reninpositive area to the whole area of the cortex, determined during day 14 of gestation and day 1 after birth in the male and female, respectively, are shown in Figure 8. The positive area in the day-16 of gestation fetus showed a significant increase. The averages were 44.9% in the male and 36.9% in the female, but there was no sexual difference between the two groups. The ratios decreased subsequently until day 1 after birth. DISCUSSION Our data demonstrate the presence of renin-immunoreactivity in cortical cells in the adrenal gland of the mouse, a t least during fetal and neonatal life. Although it has been reported that the adrenal gland is possessed of the enzyme renin in vivo and in vitro, the results for the cells or regions containing renin do not always correspond to each other because of differences in cell distributions among animal species: examples include the adrenal glomerulosa region in the rat (Deschepper et al., 1986; Doi et al., 1984; Nakamaru e t al., 19851, the innercortex of the adrenal gland in the mouse (Naruse e t al., 1984), and the medullary tissue in the bovine (Ryan, 1972). In any case, the fact that many renin-containing cells, some even showing mitotic figures, were demonstrated on day 16 of gestation corresponds with the results in the kidney, where renin-positive cells were also detected numerously in the same prenatal life (Kon et al., 1989). It is suggested that this gestational period may be a n important period in the ontogeny of the system regulating blood pressure. In the present study, immunoreactivity in the cyto- plasm on day 14 was detected a s dots or rod-shaped figures near the nucleus, whereas that on days 16 to 18 showed small granular contours just below the cell membrane. Such movement of immunocytochemical production suggests that the renin observed in adrenal cortical cells is not taken from circulating blood, but it is synthesized in the cytoplasm. This renin, when moved to peripheral positions in the cytoplasm, may be released to the intercellular spaces. Further studies on the ultrastructural detection of renin should be carried out. The present results-showing that the number of renin-containing cells in the glands decreased significantly during perinatal development and that no cells were demonstrable using immunohistochemistry 3 days after birth-may lead to the following hypotheses. First, the adrenal gland may not be the main source of circulating renin, but it does possess a preparatory role in the maintenance of blood pressure. Such a hypothesis has been accepted concerning the significant elevation of adrenal renin activity in experimantal bilateral nephrectomy in mouse and rat (Naruse and Inagami, 1982; Naruse et al., 1984). Second, a n amount of renin too small to be demonstrable with immunohistochemical procedures may perform a n important physiological function in the release of aldosterone (Doi et al., 1983; Vecsei et al., 1978). In the present study, the enzyme renin may be derived from the cortical cells located in fascicular and reticular zones. The idea that renin originally existing in the adrenal gland is involved in the release of aldosterone, generally known to be produced by zona glomerulosa cells via angiotensin series (Vecsei et al., 1978), suggests a possible role in the paracrine function of renin-containing cells. Such a possibility has been suggested previously by some investigators (Deschepper et al., 1986; Naruse et al., 1984). Finally, it is possible that variable masking of antigenic determinants, interference against combinations of antigens and antibodies with unknown proteins, or some antigenic alterations of extrarenal renin occur in the adrenal tissue during the later development of the mouse. Further studies are required to clarify these issues. The other components functioning a s a reninangiotensin system without renin examined in this paper have been reported to exist in the adrenal gland. Fig. 5. Adrenal gland stained with immunohistochemistry on day 18 of gestation. a: A few cells immunoreactive for renin are observed in the middle cortical zone (arrows). Immunohistochemical method. x 120. b: Numerous granules with marked reactivity are located a t the cell membrane (arrow).Immunohistochemical method. x 310. Fig. 6. The adrenal gland on day 1 after birth. a: A few renincontaining cells with intense or feeble reactions are distributed individually in the middle cortical region (arrows). Immunohistochemical method. x 80. b: Higher magnification showing some granules below the cell membrane and a vacuolated figure in the cytoplasm (arrows). Immunohistochemical method. x 310. Fig. 7. Adrenal glands on days 3 (a) and 7 (b) after birth. Immunoreactivity for renin is not found in the gland. Numerous immunoreactive cells are found in blood vessels of the metanephros. Immunohistochemical method. x 40. RENIN IN ADRENAL GLAND Figs. 5-7 129 Y. KON ET AL. 130 m aJ L 5 7 m u (%I .r +J L 0 -r 50 U ? 5 c aJ L Female 40 -0 m L Q W 30 rn aJ L m W > 20 c, -r. v) 0 n .-SS 10 I-r L W + 0 W 0 1 4 t h day 1 6 t h day 18th day 1st day after birth 0, 5 c, S W U Gestational a n d neonatal periods L W a Fig. 8. The ontogenetical changes of the ratio of the renin positive area against the whole cortical area from day 14 of gestation to day 1 of birth in the male and female, respectively. There is no sexual difference between the two groups. For example, angiotensin I, angiotensin 11, angiotensin 111, and angiotensin-converting enzyme have been measured in the rat adrenal gland (Mendelsohn, 1982; Nakamaru et al., 1985), and mRNAs of renin and angiotensinogen have been detected with the Northern blot technique (Campbell and Habener, 1986). These investigations suggest that the extrarenal adrenal renin-angiotensin system provides a local independent regulating function. Our present results may be of some significance in elucidating the ontogenetical development of the extrarenal renin-angiotensin system in the mouse. Because the initial appearance of renin immunoreactivity was observed in adrenal cortical cells accompanied by cell divisions at days 13-14 of gestation, there is a n assumption that the angiotensin series also appear in the gland a t or after the fetal period, as in the present observations (Celio et al., 1985). Angiotensin I1 is known to have various functions in tissues; for instance, in the bovine adrenal gland i t is a potent promoting factor for the mitosis of cortical cells (Gill et al., 1977). Furthermore, angiotensin I1 has also been shown to be a significant stimulating factor for angiogenesis and prostaglandin synthesis in several organs, and the prostaglandin in the adrenal gland has also been reported to stimulate steroidogenesis (Fernandez et al., 1985; Flack et al., 1969). These results suggest that there is a n important involvement of angiotensin I1 produced by renin synthesis in the morphogenesis of the adrenal gland in the fetal stage of the mouse. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (no. 63760233) from the Ministry of Education, Science and Culture, Japan. LITERATURE CITED Campbell, D.J., and J.F. Habener 1986 Angiotensinogen gene is expressed and differentially regulated in multiple tissues of the rat. J . Clin. Invest., 78:31-39. Celio, M.R., P. Groscurth, and T. Inagami 1985 Ontogeny of renin immunoreactive cells in the human kidney. Anat. Embryol., 173: 149-155. Cohen, S., J.M. Taylor, K. Murakami, A.M. Michelakis, and T. Inagami 1972 Isolation and characterization of renin-like enzymes from mouse submaxillary gland. Biochemistry, 11:4286-4292. Davis, J.O., and R.H. Freeman 1976 Mechanisms regulating renin release. Physiol. Rev., 56:l-56. Deschepper, C.F., and W.F. Ganong 1988 Renin and angiotensin in endocrine glands. In: Frontiers in Neuroendocrinology. L. Martini and W.F. Ganong, eds. Raven Press, New York, Vol. 10, pp. 79-98. Deschepper, C.F., J. Bouhnik, F. Cumin, and W.F. 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