Cell Tiss. Res. 194, 269-277 (1978)
Cell and Tissue Research 9 by Springer-Verlag 1978
Electron Microscopic Study of the Response of the Adrenocortical X-Zone in Mice Treated with Sex Steroids* Y. T o m o o k a and T. Yasui** Department of Biology,Faculty of Science,Okayama University, Tsushima Okayama 700, Japan
Summary. In the adrenal cortex of 23-to 27-day-old C3H/Tw female mice, the eosinophilic X-zone became increasingly undetectable after 3 injections of 100 ~tg testosterone propionate (TP). Whorls of smooth endoplasmic reticulum (sER) and peculiar complexes of mitochondria and sER, characteristics of X-zone cell, were no longer present in mice given 7 daily injections of TP. The ordinary mitochondria, although reduced in number, became swollen and actually increased in percent area occupied. They had well-developed tubulovesicular cristae. The lipid droplets increased in size and number after 3 daily TP injections, but decreased after 7 daily injections. Rough endoplasmic reticulum and sER were reduced in area in mice receiving 7 daily injections. The X-zone also became indistinguishable from the zona fasciculata after 7 daily injections of 5~-dihydrotestosterone propionate. Injections of progesterone or estradiol17fl had no effect on the X-zone. Key words: Adrenal cortex - X - z o n e - Zona reticularis - Sex steroids - Electron microscopic morphometry.
The X-zone of the adrenal of mice consists of small eosinophilic cells. In the female, this zone is well developed and occupies the inner part of the cortex, persisting for at least a year. At advanced ages the zone undergoes involution with or without fatty degeneration (Masui and Tamura, 1926; Howard-Miller, 1927; Takewaki, 1937; Miintener and Theiler, 1974). Administration of androgen results in disappearance of the X-zone, whereas the zone does not respond uniformly to exogenous estrogen Y. Tomooka, Department of Zoology and Cancer Research Laboratory, University of California, Berkeley,CA 94720 U.S.A. * Supported by a Grant-in-Aid for ScientificResearch from the Ministryof Education, Scienceand Culture of Japan to Professor Takasugi ** The authors are gratefulto ProfessorH.A. Bern (Universityof California, Berkeley)for his critical reading of the manuscript and to Professor N. Takasugi (Okayama University) for his constant encouragement and guidance Send offprint requests to:
0302-766X/78/0194/0269/$01.80
270
Y. Tomooka and T. Yasui
(cf. Chester Jones, 1957). Holmes and Dickson (1971) studied the effects of various hormones on this zone and concluded that only androgen and progesterone caused its disappearance. Ultrastructural studies of the X-zone revealed that its cells are characterized by the formation of peculiar mitochondrial complexes and a whorled pattern of smooth endoplasmic reticulum (sER) (Ross, 1967; Sato, 1968; Hirokawa and Ishikawa, 1974). During androgen-induced disappearance of the female X-zone, the cells degenerate and disappear, leaving cell remnants and connective tissue surrounding the medulla. The fate of the region after disappearance of the X-zone has long been disputed (Howard-Miller, 1927; Deanesly, 1928; Chester Jones, 1948; Kimura, 1959; Mfintener and Theiler, 1974). The present study was undertaken to examine the morphometric changes in X-zone cells following administration of various sex steroids.
Materials and Methods Treatment Sixty-four female mice of the C3H/Tw strain were used. Thirty-six of them were separated into 3 groups of 12 mice each. Each group of mice was subcutaneously injected with a daily dose of 100 ~tg testosterone propionate (TP), 100 ~tg progesterone (P) or 100 Ixg estradiol-17fl (E z) dissolved in 0.04 ml sesame oil starting at 20 days of age. Twelve other control mice received injections of the 0.04 ml oil vehicle only. Each of these groups was further divided into 3 subgroups of 4 mice which received 1, 3 and 7 daily injections. The injected mice were sacrificed by decapitation 24h after the last injection. Four other groups consisting of 3 mice each were injected with 100 ~tg or 200 lag 5~-dihydrotestosterone (DHT), 200 ~tg testosterone (T) and 20 I~g 5~-dihydrotestosterone propionate (DHTP) in 0.04 ml sesame oil for 7 days starting at 20 days of age and were sacrificed on the day after the last injection. Another group of 4 intact mice was sacrificed at 20 days of age.
Fixation For light microscopy, the left adrenals were fixed in Bouin's solution, embedded in paraffin, sectioned at 7~tm and stained with Delafield's hematoxylin and eosin. The right adrenals were fixed in 3 glutaraldehyde in 0.1 M phosphate buffer (PH 7.4) for 2 h and post-fixed in 1 ~ osmium tetroxide in 0.1 M phosphate buffer for 2 h. The tissues were embedded in Epon 812. The thin sections were stained with uranyl acetate and lead citrate, and observed with a Hitachi HU-11 E electron microscope.
Quantitative Analysis Electron micrographs at a magnification of 3,400 were prepared o f the X-zone cells of 20-day-old intact mice and of the mice given injections of TP, P and oil. In each mouse, measurements were carried out randomly on 25 cells of the innermost cortical tissue adjacent to the connective tissue bordering the medullary region (100 cells per treatment group). The cytoplasmic area and the number and area of mitochondria and lipid droplets were measured in each of these cells. Occasionally a few degenerating cells were seen; these were not included in the measurements. In order to estimate the percent area of the cytoplasm occupied by mitochondria and lipid droplets, the longest and the shortest axes of these organelles and droplets were measured. The areas were calculated as ellipsoids at a magnification of 10,000. The cytoplasmic area was measured with planimeter. There was no significant difference in cytoplasmic area between control and experimental groups. Numbers of mitochondria and lipid droplets were also counted to estimate the average area occupied. The percent area of sER and of rough endoplasmic reticulum (rER) was assessed in about 40 cells of each group (TP, P and oil) following Weibel's method (1969) at a magnification of 23,800. The data were statistically analyzed by the Student t-test.
AdrenalX-Zoneand Sex Steroids
271
Results X-Zone in Intact and Oil-Injected Female Mice (Controls)
In 20- to 27-day-old control mice, the X-zone consisted of eosinophilic cells adjacent to the medullary region. Electron microscopy revealed that the X-zone cells are characterized by peculiar complexes of mitochondria and sER (cf. Sato, 1968; Hirokawa and Ishikawa, 1974). The mitochondrial structure is undeveloped tubulo-vesicular. The lipid droplets are small and spherical with a tendency to be aggregated (Fig. 1). X-Zone in Female Mice Given Injections o f TP
In mice given a single injection of TP, the X-zone cells were decreased in eosin stainability. Electron microscopic observations indicated that the number of lipid droplets per cell increased (P < 0.01) and that the average area of the lipid droplets was double that of the controls (Figs. 6 a, 8 a). The area occupied by lipid droplets reached a maximum of 9.95 % (P
Cell and Tissue Research 9 by Springer-Verlag 1978
Electron Microscopic Study of the Response of the Adrenocortical X-Zone in Mice Treated with Sex Steroids* Y. T o m o o k a and T. Yasui** Department of Biology,Faculty of Science,Okayama University, Tsushima Okayama 700, Japan
Summary. In the adrenal cortex of 23-to 27-day-old C3H/Tw female mice, the eosinophilic X-zone became increasingly undetectable after 3 injections of 100 ~tg testosterone propionate (TP). Whorls of smooth endoplasmic reticulum (sER) and peculiar complexes of mitochondria and sER, characteristics of X-zone cell, were no longer present in mice given 7 daily injections of TP. The ordinary mitochondria, although reduced in number, became swollen and actually increased in percent area occupied. They had well-developed tubulovesicular cristae. The lipid droplets increased in size and number after 3 daily TP injections, but decreased after 7 daily injections. Rough endoplasmic reticulum and sER were reduced in area in mice receiving 7 daily injections. The X-zone also became indistinguishable from the zona fasciculata after 7 daily injections of 5~-dihydrotestosterone propionate. Injections of progesterone or estradiol17fl had no effect on the X-zone. Key words: Adrenal cortex - X - z o n e - Zona reticularis - Sex steroids - Electron microscopic morphometry.
The X-zone of the adrenal of mice consists of small eosinophilic cells. In the female, this zone is well developed and occupies the inner part of the cortex, persisting for at least a year. At advanced ages the zone undergoes involution with or without fatty degeneration (Masui and Tamura, 1926; Howard-Miller, 1927; Takewaki, 1937; Miintener and Theiler, 1974). Administration of androgen results in disappearance of the X-zone, whereas the zone does not respond uniformly to exogenous estrogen Y. Tomooka, Department of Zoology and Cancer Research Laboratory, University of California, Berkeley,CA 94720 U.S.A. * Supported by a Grant-in-Aid for ScientificResearch from the Ministryof Education, Scienceand Culture of Japan to Professor Takasugi ** The authors are gratefulto ProfessorH.A. Bern (Universityof California, Berkeley)for his critical reading of the manuscript and to Professor N. Takasugi (Okayama University) for his constant encouragement and guidance Send offprint requests to:
0302-766X/78/0194/0269/$01.80
270
Y. Tomooka and T. Yasui
(cf. Chester Jones, 1957). Holmes and Dickson (1971) studied the effects of various hormones on this zone and concluded that only androgen and progesterone caused its disappearance. Ultrastructural studies of the X-zone revealed that its cells are characterized by the formation of peculiar mitochondrial complexes and a whorled pattern of smooth endoplasmic reticulum (sER) (Ross, 1967; Sato, 1968; Hirokawa and Ishikawa, 1974). During androgen-induced disappearance of the female X-zone, the cells degenerate and disappear, leaving cell remnants and connective tissue surrounding the medulla. The fate of the region after disappearance of the X-zone has long been disputed (Howard-Miller, 1927; Deanesly, 1928; Chester Jones, 1948; Kimura, 1959; Mfintener and Theiler, 1974). The present study was undertaken to examine the morphometric changes in X-zone cells following administration of various sex steroids.
Materials and Methods Treatment Sixty-four female mice of the C3H/Tw strain were used. Thirty-six of them were separated into 3 groups of 12 mice each. Each group of mice was subcutaneously injected with a daily dose of 100 ~tg testosterone propionate (TP), 100 ~tg progesterone (P) or 100 Ixg estradiol-17fl (E z) dissolved in 0.04 ml sesame oil starting at 20 days of age. Twelve other control mice received injections of the 0.04 ml oil vehicle only. Each of these groups was further divided into 3 subgroups of 4 mice which received 1, 3 and 7 daily injections. The injected mice were sacrificed by decapitation 24h after the last injection. Four other groups consisting of 3 mice each were injected with 100 ~tg or 200 lag 5~-dihydrotestosterone (DHT), 200 ~tg testosterone (T) and 20 I~g 5~-dihydrotestosterone propionate (DHTP) in 0.04 ml sesame oil for 7 days starting at 20 days of age and were sacrificed on the day after the last injection. Another group of 4 intact mice was sacrificed at 20 days of age.
Fixation For light microscopy, the left adrenals were fixed in Bouin's solution, embedded in paraffin, sectioned at 7~tm and stained with Delafield's hematoxylin and eosin. The right adrenals were fixed in 3 glutaraldehyde in 0.1 M phosphate buffer (PH 7.4) for 2 h and post-fixed in 1 ~ osmium tetroxide in 0.1 M phosphate buffer for 2 h. The tissues were embedded in Epon 812. The thin sections were stained with uranyl acetate and lead citrate, and observed with a Hitachi HU-11 E electron microscope.
Quantitative Analysis Electron micrographs at a magnification of 3,400 were prepared o f the X-zone cells of 20-day-old intact mice and of the mice given injections of TP, P and oil. In each mouse, measurements were carried out randomly on 25 cells of the innermost cortical tissue adjacent to the connective tissue bordering the medullary region (100 cells per treatment group). The cytoplasmic area and the number and area of mitochondria and lipid droplets were measured in each of these cells. Occasionally a few degenerating cells were seen; these were not included in the measurements. In order to estimate the percent area of the cytoplasm occupied by mitochondria and lipid droplets, the longest and the shortest axes of these organelles and droplets were measured. The areas were calculated as ellipsoids at a magnification of 10,000. The cytoplasmic area was measured with planimeter. There was no significant difference in cytoplasmic area between control and experimental groups. Numbers of mitochondria and lipid droplets were also counted to estimate the average area occupied. The percent area of sER and of rough endoplasmic reticulum (rER) was assessed in about 40 cells of each group (TP, P and oil) following Weibel's method (1969) at a magnification of 23,800. The data were statistically analyzed by the Student t-test.
AdrenalX-Zoneand Sex Steroids
271
Results X-Zone in Intact and Oil-Injected Female Mice (Controls)
In 20- to 27-day-old control mice, the X-zone consisted of eosinophilic cells adjacent to the medullary region. Electron microscopy revealed that the X-zone cells are characterized by peculiar complexes of mitochondria and sER (cf. Sato, 1968; Hirokawa and Ishikawa, 1974). The mitochondrial structure is undeveloped tubulo-vesicular. The lipid droplets are small and spherical with a tendency to be aggregated (Fig. 1). X-Zone in Female Mice Given Injections o f TP
In mice given a single injection of TP, the X-zone cells were decreased in eosin stainability. Electron microscopic observations indicated that the number of lipid droplets per cell increased (P < 0.01) and that the average area of the lipid droplets was double that of the controls (Figs. 6 a, 8 a). The area occupied by lipid droplets reached a maximum of 9.95 % (P
