Light and electron microscopic evidences of the presence of c-fos-like immunoreactivity in the rat adrenal cortex

The presence and localization of c-fos-like immunoreactivity in the rat adrenal cortex has been demonstrated by immunocytochemical methods at both light and electron microscopic level. C-fos-like immunoreactivity was detected in the zona fasciculata and the zona reticulata, but not in the zona glomerulosa. Ultrastructurally, all products of c-fos-like immunoreaction were localized exclusively in the regions associated with the euchromatin in the nucleus of the immunoreactive cells. Moreover, a higher density of the immunoreactive cells in the adrenal cortex of pregnant rats was found with quantitative immunocytochemistry as compared to the non-pregnant. The characteristic zonation of c-fos-like immunoreactivity in the adrenal cortex suggest that the c-fos protein is involved in the normal function of the glucocorticoid-producing cells of mammalian adrenals. The numerical increase in the immunoreactive cells in pregnant rats implies that basal expression of the c-fos-like protein may vary with the functional state of the cortical cells.     

Functional alteration of dihydropyridine-sensitive Ca(2+) channels in the adrenal glomerulosa of pregnant rats

Our previous work on aldosterone secretion suggested that dihydropyridine-sensitive calcium channels, one type of voltage-dependent calcium channels (VDCC), are functionally impaired in adrenal capsule preparations from the pregnant rat. The aim of this study was to determine whether, during pregnancy, the density and/or activity of these channels is altered in the adrenal zona glomerulosa. These VDCC measured with [(3)H]nitrendipine binding were not different between membrane preparations of nonpregnant and pregnant rats. Western blots were performed using two different antibodies, a polyclonal (PcAb) directed against the alpha(1)-subunit of VDCC and a monoclonal (McAb) that recognizes an intracellular domain of that protein. McAb immunoreactivity showed a significant decrease in preparations from pregnant rats, whereas no difference was observed with PcAb. VDCC activity was estimated by (45)Ca(2+) uptake in isolated adrenal cortex and by intracellular calcium concentration ([Ca(2+)](i)) in adrenal glomerulosa cells with the Ca(2+) probe fura PE3. These measurements revealed that KCl stimulation produced greater Ca(2+) influx in nonpregnant than in pregnant rats. Nifedipine (a blocker of VDCC) inhibited this stimulation only in nonpregnant rats, whereas BAY K 8644 (an activator of VDCC) increased Ca(2+) influx in pregnant rats only. These data suggest that, during pregnancy, the altered regulation of calcium homeostasis in adrenal glomerulosa is linked to a conformational alteration of VDCC.     

促肾上腺皮质激素引起大鼠肾上腺c-fos原癌基因表达的免疫组化研究

采用冰冻切片及免疫组化法观察了注射促肾上腺皮质激素(ACTH,75U/kg)、胰岛素及正常对照大鼠中肾上腺各部分c-fos原癌基因表达产物Fos蛋白的出现和分布特点。结果表明注射ACTH后90min,大鼠肾上腺皮质网状带出现Fos蛋白染色阳性细胞,阳性染色物集中于细胞核,肾上腺皮质束状带仅见少数Fos蛋白染色阳性细胞,肾上腺髓质则未见Fos蛋白染色阳性细胞。与注射ACTH相反,注射胰岛素引起肾上腺髓质出现Fos蛋白染色阳性细胞。注射生理盐水对照组动物肾上腺皮质和髓质均未见Fos蛋白染色阳性细胞。上述结果表明,注射ACTH或胰岛素可以引起大鼠肾上腺不同部位c-fos原癌基因表达。     

Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibers in the adrenal gland of rats

Summary The present immunohistochemical study reveals that a small number of chromaffin cells in the rat adrenal medulla exhibit CGRP-like immunoreactivity. All CGRP-immunoreactive cells were found to be chromaffin cells without noradrenaline fluorescence; from combined immunohistochemistry and fluorescence histochemistry we suggest that these are adrenaline cells. In addition, all CGRP-immunoreactive cells simultaneously exhibited NPY-like immunoreactivity. CGRP-chromaffin cells were characterized by abundant chromaffin granules with round cores in which the immunoreactive material was densely localized. These findings suggest the co-existence of CGRP, NPY and adrenaline within the chromaffin granules in a substantial number of chromaffin cells.Thicker and thinner nerve bundles, which included CGRP-immunoreactive nerve fibers, with or without varicosities, penetrated the adrenal capsule. Most of them passed through the cortex and entered the medulla directly, whereas others were distributed in subcapsular regions and among the cortical cells of the zona glomerulosa. Here the CGRP-fibers were in close contact with cortical cells. A few of the fibers supplying the cortex extended further into the medulla. The CGRP-immunoreactive fibers in the medulla were traced among and within small clusters of chromaffin cells and around ganglion cells. The CGRP-fibers were directly apposed to both CGRP-positive and negative chromaffin cells, as well as to ganglion cells. Immunoreactive fibers, which could not be found close to blood vessels, were characterized by the presence of numerous small clear vesicles mixed with a few large granular vesicles. The immunoreactive material was localized in the large granular vesicles and also in the axoplasm. Since no ganglion cells with CGRP-like immunoreactivity were found in the adrenal gland, the CGRP-fibers are regarded as extrinsic in origin. In double-immunofluorescence staining for CGRP and SP, all the SP-immunoreactive fibers corresponded to CGRP-immunoreactive ones in the adrenal gland. This suggests that CGRP-positive fibers in the adrenal gland may be derived from the spinal ganglia, as has been demonstrated with regard to the SP-nerve fibers.     

Distribution of P2X receptors in the rat adrenal gland

The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7-immunoreactive chromaffin cells exceeding that of P2X5. The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3. Adrenal cortical cells were positively immunostained for P2X4-7. Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5-7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6, followed by P2X7 and then P2X5. The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland.     

Immunohistochemical localization of 3 beta-hydroxysteroid/delta 5-delta 4-isomerase, tyrosine hydroxylase and phenylethanolamine N-methyl transferase in adrenal glands of sheep fetuses throughout gestation and in neonates.

Immunoreactive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) was localized in adrenal glands of sheep fetuses in cortical-type cells, but not in medullary-type cells, from day 43 of gestation to term and in 2-4-day-old neonates. From day 54 of gestation, the formation of distinct zones within the adrenal cortex was apparent and immunoreactive 3 beta-HSD was found in cortical cells in the zona fasciculata and in groups and cords of cortical cells within the developing medulla, with weak positive staining in the zona glomerulosa. At this stage, most medullary cells were positive for immunoreactive tyrosine hydroxylase, and some of these cells with a juxtacortical distribution also stained positively for immunoreactive phenylethanolamine N-methyl transferase (PNMT). Between days 65 and 130, the adrenal medulla increased in size with little change in the width of the cortex. Organization and zonation of immunoreactive 3 beta-HSD staining cells were evident in the zona fasciculata and in groups of cells in the medulla. Between day 130 and term, uniform immunoreactive 3 beta-HSD staining was found throughout the zona fasciculata, and there was also staining in single cells and small clusters of cells throughout the medulla. At this stage, immunoreactive tyrosine hydroxylase was distributed in most cells throughout the medulla, but in two distinct patterns: cells staining intensely for immunoreactive tyrosine hydroxylase in the central region of the medulla, and cells exhibiting weaker staining for immunoreactive tyrosine hydroxylase localized in a juxta-cortical position. These juxta-cortical cells were also positive for immunoreactive PNMT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of follistatin in rat tissues.

We have used immunohistochemistry to localize follistatin/activin-binding protein in adult male and female rats. A polyclonal antibody directed against a follistatin peptide (residues 123-134) was used as a specific immunologic probe. Intense and specific follistatin immunoreactivity was evident in spermatogenic cells of seminiferous tubules in the testis. The predominant staining was in nuclei of spermatocytes and spermatids, but no immune reaction was observed in spermatogonia or spermatozoa. Moderate immunoreactivity was detected in Leydig cells. Sertoli cells were follistatin-negative. Significant immunoreactivity was evident in ovarian granulosa cells. The intensity of the staining changed with follicle development: no immunoreactivity was observed in granulosa cells of primordial to primary follicles, but the cells of secondary to Graafian follicles displayed moderate to strong staining and finally luteal cells of the corpus luteum became negative. The epithelial lining of the oviduct and the smooth muscle of the myometrium of the uterus were intensely immunoreactive. Immunoreactive follistatin staining was present in the pituitary: a group of round-shaped cells were specifically stained. Immunostainable follistatin was visible in the epithelial layers of renal tubules with moderate to strong staining reactivity. Hepatic cells in the liver demonstrated homogeneous immunoreactivity from moderate to strong. The cortex of the adrenal gland, white pulp of the spleen and the brain cortex were also stained weakly but distinctly with the antiserum. In conclusion, immunoreactive follistatin is widespread in rat tissues, suggesting that follistatin/activin-binding protein is a ubiquitous protein, regulating a wide variety of activin actions.     

Substance P-like immunoreactivity in adrenal chromaffin cells and intra-adrenal nerve fibers of rats

Summary The present peroxidase-antiperoxidase immunohistochemical study demonstrated a relatively small number of cells with substance P(SP)-like immunoreactivity in the adrenal medulla of rats. These cells were found alone or in small groups, were polygonal in shape and lacked long cytoplasmic processes. At immunoelectron microscopy, the immunoreactive cells were characterized by abundant granular vesicles, and the immunoreactive material was confined to the round core of the vesicles. Thus, it is suggested that SP co-exists with catecholamines in a population of chromaffin cells of the rat adrenal medulla. In addition a few SP-immunoreactive nerve fibers with varicosities were found in the adrenal medulla of rats. They extended between small clusters of chromaffin cells and had their dotlike terminals around and within the cell clusters. The SP-immunoreactive nerve fibers were characterized by the presence of abundant small clear vesicles mixed with a few large granular vesicles; the immunoreactivity appeared in the latter, but was also perfused throughout the entire axoplasm. The nerve fibers formed synapses on nonimmunoreactive chromaffin cells. Judging from the presence of bundles of SP-immunoreactive nerve fibers penetrating the adrenal capsule and cortex as well as the absence of SP-immunoreactive ganglion cells in the medulla, the intramedullary SP-immunoreactive nerve fibers seem to be extrinsic in origin.     

Substance P-like immunoreactivity in adrenal chromaffin cells and intra-adrenal nerve fibers of rats

The present peroxidase-antiperoxidase immunohistochemical study demonstrated a relatively small number of cells with substance P(SP)-like immunoreactivity in the adrenal medulla of rats. These cells were found alone or in small groups, were polygonal in shape and lacked long cytoplasmic processes. At immunoelectron microscopy, the immunoreactive cells were characterized by abundant granular vesicles, and the immunoreactive material was confined to the round core of the vesicles. Thus, it is suggested that SP co-exists with catecholamines in a population of chromaffin cells of the rat adrenal medulla. In addition a few SP-immunoreactive nerve fibers with varicosities were found in the adrenal medulla of rats. They extended between small clusters of chromaffin cells and had their dot-like terminals around and within the cell clusters. The SP-immunoreactive nerve fibers were characterized by the presence of abundant small clear vesicles mixed with a few large granular vesicles; the immunoreactivity appeared in the latter, but was also perfused throughout the entire axoplasm. The nerve fibers formed synapses on nonimmunoreactive chromaffin cells. Judging from the presence of bundles of SP-immunoreactive nerve fibers penetrating the adrenal capsule and cortex as well as the absence of SP-immunoreactive ganglion cells in the medulla, the intramedullary SP-immunoreactive nerve fibers seem to be extrinsic in origin.     

A study of cell migration in the adrenal cortex of the rat using bromodeoxyuridine

Abstract Bromodeoxyuridine (BrdU) was administered by a single intraperitoneal injection to immature (14 days) male and female and adult male Sprague-Dawley rats. Animals were killed at intervals from 2 hr to 28 days following injection. Labelled cells in the adrenal cortex were identified by an indirect immunoperoxidase technique using a monoclonal antibody to BrdU. At 2 hr, labelling was maximal in the outer zona fasciculata and zona glomerulosa in both prepubertal and adult rats. The numbers of immunopositive cells were greater in the 14 day rats. In both groups, the front of immunopositive cells moved deeper into the cortex with time. These results support the centripetal migration theory of adrenal growth.     

Nestin expression in rat adrenal gland

The constituents of the intermediate filament network of adrenal gland cells have not been deeply investigated in vivo. Adrenocortical cells have been reported to express cytokeratins and vimentin, but the intermediate filament components of the adrenomedullary cells are still unknown. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous and muscle systems. It has been reported to be unable to form filaments by itself and it co-assembles with vimentin. Using immunocytochemical and biochemical approaches, the present study demonstrates that nestin is expressed in situ either in the cortex or in the medulla of adult rat adrenal glands. Nestin-negative cells prevalently form the zona glomerulosa whereas the zona fasciculata and the zona reticularis are mainly nestin-immunoreactive. Nestin-positive cells always express vimentin-like immunoreactivity but several cells apparently expressing only vimentin are detectable too. Nestin is also expressed by adrenomedullary cells that also display a faint vimentin-like immunoreactivity. We hypothesise that the inconstant detection of nestin in adrenocortical cells depends on their different functional moments. Moreover, even though our data do not allow to confirm vimentin in adrenomedullary cells, in situ detection of nestin in the adrenal medulla indirectly supports in vivo expression of vimentin in chromaffin cells.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Relationships between NADPH diaphorase staining and neuronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues

 The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent enzymes – neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450 reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue. However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver, sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea, zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms. Accepted: 2 September 1996     

Protein kinase c-β-like immunoreactivity in the developing and adult rat adrenal gland

Summary Protein kinase c--like immunoreactivity was studied in the adrenal gland of adult rats and at different pre- and postnatal stages of development (E17-P21) with an antibody specific to both the ₂₁ and - subtypes of the kinase. In the adult rat adrenal gland, the immunoreactivity was seen in numerous nerve fibres in the adrenal medulla both in bundles and individually forming occasionally dense networks around chromaffin cell groups. Several protein kinase c--immunoreactive fibres were also observed transversing the adrenal cortex towards the medulla. No remaining immunoreactive fibres two weeks after transection of the splanchnic nerve could be seen; nor was any immunoreactivity observed in the chromaffin cells of the adrenal medulla or in the cortical cells, but some faintly immunoreactive ganglion cells were detected in the adrenal medulla. The amount and distribution of protein kinase c--like immunoreactivity in the fetal and developing adrenals was very similar to that seen in the adrenal glands of adult rats. On the basis of its localization, the -subtype of protein kinase c does not appear to be directly involved in the release of catecholamines from the adrenal medulla, but it might have a role in the regulation of neurotransmitter release from preganglionic cholinergic neurons.     

Immunocytochemical localization of basic fibroblast growth factor in bovine adrenal gland, ovary, and pituitary

We studied the distribution of basic fibroblast growth factor (bFGF) immunoreactivity in bovine adrenal gland, ovary, and pituitary, using a polyclonal anti-bFGF antibody. In the adrenal gland, the inner layers of the capsule, the zona glomerulosa of the cortex, and the chromaffin cells of the adrenal medulla were intensely stained. In the ovary, follicular epithelial cells of growing follicles and granulosa cells of mature follicles showed strong bFGF-like immunoreactivity. Endocrine cells of the pituitary anterior and intermediate lobes displayed a positive immunoreaction. Blood vessels, including endothelial and smooth muscle cells, as well as stromal cells in all three organs studied, were not stained. This distribution pattern of bFGF immunoreactivity is only partially compatible with the established mitogenic role of this protein, and suggests a wider spectrum of bFGF functions.     

Aldosterone biosynthesis and cytochrome P-45011 beta: evidence for two different forms of the enzyme in rats

Whereas cytochrome P-45011 beta has been recently shown to catalyze the two-step conversion of corticosterone to aldosterone in the bovine and porcine adrenal cortex, the identity of the enzyme involved in the two final steps of aldosterone biosynthesis in the rat adrenal cortex is as yet unknown. Mitochondria from capsular adrenals of sodium-deficient, potassium-replete rats converted corticosterone to 18-hydroxycorticosterone and aldosterone at markedly higher rates than mitochondria from capsular adrenals of sodium-replete, potassium-deficient rats. However, the same preparations exhibited no difference in the 11 beta-hydroxylase activity, i.e. the conversion of deoxycorticosterone to corticosterone. Only mitochondria of zona glomerulosa from rats with stimulated aldosterone biosynthesis contained a 49K protein which showed a strong cross-reactivity with a monoclonal antibody raised against purified bovine cytochrome P-45011 beta. By contrast, a crossreactive protein with a molecular weight of 51K was found in mitochondria of zona fasciculata and in mitochondria of zona glomerulosa from rats with a suppressed aldosterone biosynthesis. These findings indicate the existence of two different forms of cytochrome P-45011 beta in the rat adrenal cortex, with only one of them, i.e. the 49K form, being capable of catalyzing the two final steps of aldosterone biosynthesis in situ.     

Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.     

Sex differences in adrenocortical structure and function. V. The effects of postpubertal gonadectomy and gonadal hormone replacement on nuclear-cytoplasmic ratio, morphology and histochemistry of rat adrenal cortex.

Studies were carried out on adult rats of Wistar strain. Six weeks after postpubertal gonadectomy some of the orchiectomized rats were injected with a single dose of testosterone cypionate and ovari-ctomized rats with estradiol cypionate (17 beta-cyclopentyloproprionate esters of testosterone or estradiol). The control rats were sham operated. Investigations were carried out 8 weeks after surgery. Absolute and relative adrenal weight is lower in the male than in the female rat. Orchiectomy increases these weights while testosterone restores them to their normal level. Ovariectomy has no effect on the weight of adrenal, estradiol, however, increases the relative weight of the gland. The adrenal cortex of the adult female is wider than in the adult male rat and in female gland sudanophobic zone is lacking. Orchiectomy leads to the broadening of the adrenal cortex and testosterone replacement has an opposite effect. Ovariectomy has no effect on the structure of adrenal cortex and estradiol replacement resulted in the narrowing of zona glomerulosa. There were no differences in the nuclear-cytoplasmic ratio of zona glomerulosa cells in male and Female rats and neither gonadectomy nor gonadal hormone replacement has no effect on this parameter. The nuclear-cytoplasmic ratio of zona fasciculata cells in the male is markedly higher than in female. Orchiectomy lowers this ratio and testosterone restores it to the normal level. Neither ovariectomy nor estradiol replacement has effect on the nuclear-cytoplasmic ratio of zona fasciculata cells. Similar changes as those in the zona fasciculata were observed in zona reticularis cells. Among the oxidoreductases studied, the most marked sex differences were found in alpha-glycerophosphate dehydrogenase activity. In control female rats an intense reaction for this enzyme is observed in broad band of cells of the zona fasciculata interna, while in male rats only individual cells showed this reaction. In orchiectomized rats the reaction for this enzyme is similar as in control female rats and testosterone restores it to normal. Ovariectomy has no effect on localisation of reaction while after estradiol replacement reaction was more intense. Regarding the remaining oxidoreductases studied, in the adrenal cortex of rats of both sexes no marked differences were observed in localization, however, intensity of reaction depended upon applied experimental conditions. More distinct sex differences were observed in topochemistry of some hydrolases and intensity of reaction depended upon the applied experimental conditions.