Renin in Exocrine Glands of Different Mouse Strains

The renin-angiotensin system (RAS) controls the regulation of blood pressure and water-mineral balance. Recently, renin has been reported to be synthesized and secreted outside the kidney. The present study investigated immunohistochemically the distribution of renin in exocrine glands, i.e. salivary glands and coagulating glands, in male mice of 13 strains. In some strains, renin was detected in the sublingual and submandibular salivary glands, but not in the parotid glands. It was restricted to the striated or granular portions of excretory ducts. In coagulating glands, variable staining patterns were found. These results demonstrate the existence of variations in renin content in mouse strains and offered a selection of mouse strains for investigation of exocrine gland renin.     

Expression of Angiotensinogen in the Uterus induced by Coagulating Gland Renin in Mice

Coagulating gland (CG) renin is one of the components of the local renin-angiotensin system (RAS), which plays important roles in the maintenance of homeostasis in several tissues. Although the existence of local renin has been reported in many tissues, its function is not yet well understood. In the present study, the relationship between CG renin and uterine angiotensinogen was investigated by immunohistochemical and in situ hybridization techniques. In mating experiments with male and female C57BL/6 mice, renin was demonstrated immunohistochemically on the epithelial cells of the uterus on the first day after coitus; however, it was not detected on the second and third days after coitus. Neither immunoreactive cells nor messenger signals for renin were demonstrated on the epithelial cells of the uterus throughout the experiments. On the first day after mating, it was noted that positive signals for angiotensinogen mRNA were expressed on the epithelial cells of the uterus in situ hybridization, but not on the second and third days after coitus. These results suggest the possibility that CG renin is transferred to the uterine epithelium at mating and temporarily activates the expression of angiotensinogen in the uterus. Angiotensin II produced by angiotensinogen may act as an important mediator for vascularization of the first step of pregnancy.     

Expression of Renin in the Rat Liver

Renin is well-known to be a trigger enzyme in the renin-angiotensin system (RAS). In contrast to the classical RAS, the local RAS has recently been noted in several tissues. The local RAS has a function independent from that of the classical RAS, although its physiological principles are not well known. In the present study, we immunohistochemically demonstrated that hepatocytes in the rat express renin. No renin-immunoreactive cells were detected in rat liver at 0 min after death. At 15 min after death, a small number of renin-positive cells was demonstrated in the lamina hepatica, and they increased with time to the end of observation. Immunoreactivity for renin was scarce throughout the cytoplasm, sometimes condensed to below the cell membrane and around the intracellular granules. Histoplanimetrically, the values from 15 min to 120 min after death were significantly different from that at 0 min after death. Hybridohistochemistry revealed no hybrid signals throughout the liver at either 0 min or 30 min, although renin-immunoreactivity was clearly demonstrated in adjacent sections of liver at 30 min after death. In RT-PCR, the radioactivities in kidney and liver at 0 min after death were not different from those at 30 min after death, respectively. These results suggest the existence of hepatic renin in the rat.     

Study of migration of neural crest cells to adrenal medulla by three-dimensional reconstruction

Adrenal medullary cells are derived from the neural crest. To study the formation process of the adrenal medulla in the embryonic period, we visualized chromaffin cells of rat embryos at 13 to 17 days of gestation using anti-tyrosine hydroxylase (TH) antiserum, and created three-dimensional images from serial tissue sections. Between 13 and 15 days of gestation, TH-positive cells (chromaffin cells) migrated from a group of TH-positive cells present dorsal to the adrenal primordium via the medial cranial end of the adrenal primordium into the adrenal primordium. At or after 16 days of gestation, the adrenal capsule was formed except on the ventral aspect of the cranial end of the adrenal gland, from which TH-positive cells penetrated into the adrenal gland. The reconstructed images showed that TH-positive cells were present contiguously from the sympathetic chain ganglia through a group of TH-positive cells ventral to the adrenal gland into the adrenal cortex, and that the group of TH-positive cells ventral to the adrenal gland communicated with the preaortic ganglion present ventral and caudal to the adrenal gland. These results suggest that neural crest cells use the same pathway to migrate to the sympathetic chain ganglia dorsal to the adrenal gland, to the adrenal gland, and to the preaortic ganglion.     

Colocalization of gamma-aminobutyric acid immunoreactivity and acetylcholinesterase activity in nerve fibers of the mouse adrenal gland.

The present immunohistochemical and enzyme histochemical study showed gamma-aminobutyric acid (GABA) immunoreactivity and acetylcholinesterase (AChE) activity in the mouse adrenal gland. Weak GABA immunoreactivity was seen in clusters of chromaffin cells showing noradrenaline fluorescence. This finding suggests that both GABA and noradrenaline may be released from the granules of noradrenaline cells by adequate stimuli. GABA-immunoreactive varicose nerve fibers densely contacted adrenaline cells and large ganglion cells, but they were sparse in the periphery of clusters of noradrenaline cells. AChE activity was strong in a few large ganglion cells and weak in chromaffin cells showing noradrenaline fluorescence, and was found in numerous nerve bundles and fibers of the medulla. AChE-active nerve fibers more densely contacted noradrenaline cells than adrenaline cells. By using double labeling technique, numerous GABA-immunoreactive nerve fibers in the medulla were reactive for AChE in the same sections. These results suggest that both GABA and acetylcholine may be colocalized in the intra-adrenal nerve fibers and may have some secretory effects on the chromaffin cells.     

Immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation

To elucidate the relationship between steroidogenic hormones and developing adrenal glands, we investigated the immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation. Fetal adrenal glands were obtained from three horses at 217, 225 and 235 days of gestation. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Histologically, cortex and medulla cells were clearly observed in the three fetal adrenal gland tissue samples. P450scc and P450c17 were identified in cortex cells close to medulla cells and in some medulla cells in the fetal adrenal glands. P450arom was present in both cortex and medulla cells in the fetal adrenal glands. However, 3betaHSD was not found in any of the equine fetal adrenal gland tissue samples. These results suggest that equine fetal adrenal glands have the ability to synthesize androgen and estrogen, which may play an important physiological role in the development of equine fetal adrenal glands.     

An Immunohistochemical Study on the Embryonic Development of Renin-containing Cells in the Mouse and Pig

The prenatal occurrence and distribution of renin-containing (RC) cells were investigated immunohistochemically in mouse and pig embryos. The RC cells of the mouse embryo were first observed at the 13th day of gestation at the walls of the renal, the mesonephric, the adrenal, the abdominal arteries, the adrenal glands and the testis. As the gestation of the mouse progressed, the RC cells had a tendency to localize in areas of the vascular pole of the metanephric glomerulus. In pig, when CRL was 0.8-2.0 cm, RC cells first appeared at the ventral walls of the dorsal aorta, the omphalo-mesenteric (i.e., the cranial mesenteric), the mesonephric, the mesonephric afferent glomerular arteries/arterioles and the inside of the mesonephric glomerulus. As the length of the pig embryo increased, no renin-immunoreactivity could be demonstrated at the degenerated mesonephros, while in the metanephros marked immunoreactivities were found only at the terminal regions of intralobular arteries, i.e., afferent arterioles or the vascular pole of the glomerulus.     

Adrenal steroid metabolism in birds: anatomy, physiology, and clinical considerations.

The hypothalamo-pituitary-adrenal system in birds is anatomically and functionally different from that in mammals. The adrenal gland structure and corticosteroid hormone physiology of birds will be reviewed. The anatomy and physiology sections of this article will be important for better understanding the pathogenesis, diagnosis, and possible treatment of primary or secondary adrenal gland disease. Causes of hyper- and hypoadrenocorticism in birds also will be reviewed. The article will conclude with current indications and complications to the clinical use of glucocorticoids in birds.     

MAGNETIC RESONANCE IMAGING OF THE PRESUMED NORMAL CANINE ADRENAL GLANDS

Forty-three dogs without evidence of endocrine disease that underwent spinal or abdominal magnetic resonance imaging (MRI) for clinical reasons were studied. Because the procedures were not optimized for inclusion of the adrenal glands, they were not always visible in all planes. Eighty-five of the 86 adrenal glands were seen and only the left gland in a 6-month-old Irish wolfhound could not be found. The right adrenal gland lay cranial to the left in all of the animals in which both glands were seen. The best landmarks for localization of the glands were vascular; both adrenal glands were always cranial to the ipsilateral renal vessels and in the region of the celiac and cranial mesenteric arteries. Various measurements were made on all the available scan planes. In some dogs the whole adrenal gland was difficult to visualize clearly, and this hindered the measuring process, especially when the right adrenal gland was in close contact with the caudal vena cava. The adrenal glands were mainly linear in shape but also had a variable degree of modification of their poles, especially the cranial pole of the right adrenal gland, which tended to be consistently wider and to present different shapes (rounded, arrowhead, inverted P, hook-shaped, triangular, or dome-shaped). Two main patterns of signal intensity were seen on fast spin echo (FSE) sequences (T2-weighted, T1-weighted, and T1-weighted after administration of a paramagnetic contrast medium): homogeneous and hypointense to surroundings or a corticomedullary type pattern with a hyperintense central area surrounded by a hypointense rim of tissue. The outline of the left adrenal gland was always very clear. The clarity of outline of the right adrenal gland was more variable, especially if it was in contact with the liver or the caudal vena cava. It was felt that the amount of retroperitoneal fat was not as important as stated in the human literature for visualization of the adrenal glands and that with an appropriate selection of scan planes and pulse sequences good assessment of the adrenal glands can be performed with MRI in canine patients.     

ULTRASONOGRAPHIC EVALUATION OF ADRENAL GLAND SIZE COMPARED TO BODY WEIGHT IN NORMAL DOGS

The accepted cut‐off value for adrenal gland maximum diameter of 0.74 cm to distinguish adrenal gland enlargement in dogs regardless of body weight may not be appropriate for small to medium breed dogs. The purpose of the current retrospective study was to examine adrenal gland dimensions as a function of body weight in healthy dogs in three weight categories (< 10 kg, 10–30 kg, and > 30 kg) representing small, medium, and large breeds, respectively, to establish greater confidence in determining if adrenal gland size is abnormal. The measurements of length (sagittal plane), cranial and caudal pole thickness (sagittal and transverse planes), and caudal pole width (transverse plane) of both adrenal glands were obtained ultrasonographically in clinically healthy dogs (n = 45) with 15 dogs in each weight group. Findings support our hypothesis that adrenal gland size correlates with body weight in normal dogs, and more precise reference intervals should be created for adrenal gland size by categorizing dogs as small, medium, or large breed. The caudal pole thickness of either adrenal gland in a sagittal plane was the best dimension for evaluating adrenal gland size based on low variability, ease, and reliability in measurement.     

Ultrasonographic adrenal gland measurements in clinically normal small breed dogs and comparison with pituitary-dependent hyperadrenocorticism

Ultrasonography is a sensitive and specific screening method for assessing the adrenal glands. The upper limit of the normal adrenal gland width is used as 7.5 mm. It is not known if adrenal gland width remains consistent with body weight. A reliable criterion of adrenal gland width in small breed dogs should be established. Small breed dogs with body weights of less than 10 kg were divided into two groups: 189 normal dogs and 22 dogs with pituitary-dependent hyperadrenocorticism (PDH). A retrospective study was conducted on dogs seen between January 1, 2006, and February 10, 2008. One hundred eighty-nine dogs of 14 different small breeds were enrolled in the normal adrenal gland group; the median gland width was 4.20 mm. Twenty-two dogs were in the PDH group; the median gland width was 6.30 mm. The cut-off value between normal adrenal glands and PDH was 6.0 mm. This figure gave a sensitivity and specificity of 75 and 94%, respectively, for detecting PDH. The adrenal gland appeared as a peanut shape with homogeneous hypoechoic parenchyma in normal dogs and in most dogs with PDH as well. This study was performed in a large population of small breed dogs and suggests that the normal adrenal gland size in small breed dogs is smaller than previously reported. We believe that a cut-off of 6.0 mm may be used as the criterion for differentiating a normal adrenal gland from adrenal hyperplasia.     

Hormone interactions confer specific proliferative and histomorphogenic responses in the porcine mammary gland

Mammary gland growth and morphogenesis are regulated by interactions between hormones as much as by their individual actions. The effect of these interactions on the mammary gland phenotype in species other than rodents is relatively undefined. We investigated the individual and combined effects of estrogen (E), progestin (P), and prolactin (PRL) on mammary gland development in gilts. Pigs were shown to have a ductal-lobular parenchyma that underwent hormone-stimulated progression of terminal ductal lobular unit (TDLU) morphogenesis similar to that in the human breast. Ovariectomy plus hypoprolactinemia abolished mammary gland growth. Estrogen alone stimulated mammary epithelial cell proliferation, terminal bud formation, and the progression of TDLU1 structures to a TDLU2 morphotype. Maximal epithelial cell proliferation, DNA content, parenchymal area, and morphological development of the porcine mammary gland were realized following treatment with E + PRL or E + P + PRL. In contrast, P alone did not promote epithelial cell proliferation, TDLU type progression, mammary gland growth, or morphogenesis. These data indicate that interactions between E and PRL are the main determinants of growth and morphogenesis in the porcine mammary gland.     

Acetylcholinesterase activity, and neurofilament protein, and catecholamine synthesizing enzymes immunoreactivities in the mouse adrenal gland during postnatal development.

The present study showed the acetylcholinesterase (AChE) activity, and neurofilament protein (NFP), catecholamine-synthesizing enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) immunoreactivities in the mouse adrenal gland during postnatal development. From birth to postnatal-1-day, AChE activity was weakly and diffusely found in some medullary cells and in very few nerve fibers whereas strong NFP immunoreactivity was seen in a few ganglion cells and in remarkably numerous nerve fibers in the medulla. Almost all meduallary cells were reactive for both DBH and PNMT during this period. From postnatal-2- or -3-day to postnatal-1-week, strong AChE activity was observed in a few large ganglion cells, but the reaction was weak in clusters of chromaffin cells, and the number of strong AChE-active nerve fibers in the medulla was rapidly increased. From postnatal-2-day onwards, the number of NFP-immunoreactive nerve fibers in the medulla were remarkably numerous. Numerous chromaffin cells were reactive for both DBH and PNMT whereas some chromaffin cells were reactive for only DBH from postnatal-2-day onwards. These results suggest that drastic changes such as an increase of acetylcholine in the nerve fibers, differentiation of noradrenaline and adrenaline cells of the medulla may occur during this period. From postnatal-2-week to postnatal-3-week, weak AChE activity was seen in the clusters of several chromaffin cells and a few ganglion cells, and the number of AChE-active nerve fibers in the medulla was gradually increased. From postnatal-4-week to postnatal-8-week (adult), the distribution and frequency of AChE activity in the adrenal gland were similar to those at postnatal-3-week. In the adult, AChE activity was weakly seen in the clusters of several chromaffin cells showing noradrenaline fluorescence in the adrenal medulla. The noradrenaline cells were contacted by denser AChE-reactive nerve fibers than adrenaline cells. These results suggest that the development of cholinergic nervous system in the mouse adrenal medulla may be completed by postnatal-3-week.     

Steroid hormone receptors ERα and PR characterised by immunohistochemistry in the mare adrenal gland

Background

Sex steroid hormone receptors have been identified in the adrenal gland of rat, sheep and rhesus monkey, indicating a direct effect of sex steroids on adrenal gland function.

Methods

In the present study, immunohistochemistry using two different mouse monoclonal antibodies was employed to determine the presence of oestrogen receptor alpha (ERalpha) and progesterone receptor (PR) in the mare adrenal gland. Adrenal glands from intact (n = 5) and ovariectomised (OVX) (n = 5) mares, as well as uterine tissue (n = 9), were collected after euthanasia. Three of the OVX mares were treated with a single intramuscular injection of oestradiol benzoate (2.5 mg) 18 – 22 hours prior to euthanasia and tissue collection (OVX+Oe). Uterine tissue was used as a positive control and showed positive staining for both ERalpha and PR.

Results

ERalpha staining was detected in the adrenal zona glomerulosa, fasciculata and reticularis of all mare groups. Ovariectomy increased cortical ERalpha staining intensity. In OVX mares and one intact mare, positive ERalpha staining was also detected in adrenal medullary cells. PR staining of weak intensity was present in a low proportion of cells in the zona fasciculata and reticularis of all mare groups. Weak PR staining was also found in a high proportion of adrenal medullary cells. In contrast to staining in the adrenal cortex, which was always located within the cell nuclei, medullary staining for both ERalpha and PR was observed only in the cell cytoplasm.

Conclusion

The present results show the presence of ERalpha in the adrenal cortex, indicating oestradiol may have a direct effect on mare adrenal function. However, further studies are needed to confirm the presence of PR as staining in the present study was only weak and/or minor. Also, any possible effect of oestradiol treatment on the levels of steroid receptors cannot be determined by the present study, as treatment time was of a too short duration.     

Ultrasonographic evaluation of the adrenal glands in healthy dogs: repeatability, reproducibility, observer-dependent variability, and the effect of bodyweight, age and sex

Adrenal length and width were determined from two-dimensional ultrasound longitudinal images. In study 1, 540 measurements of adrenal glands were attempted from five healthy beagle dogs by three different observers with different levels of expertise in ultrasonography, to determine the variability of adrenal gland measurements. Of these, 484 measurements were included in the statistical analysis, since 16 measurements of the left adrenal gland and 40 for the right could not be visualised by the observer. In study 2, a single measurement of both adrenal glands was taken from each of 146 dogs by the most trained observer from study 1, and the effects of different health status (healthy dogs v dogs with non-adrenal diseases), bodyweight, age and sex were assessed. A total of 267 measurements were included in the statistical analysis. The lowest intra- and inter-day coefficient of variation values were observed for the left adrenal gland and by the most trained observer. The health status had no statistically significant effect on adrenal gland length or width, whereas age had a significant effect only for the left adrenal gland (the greater the age, the greater the width or length) and sex had a significant effect only for the right adrenal gland (the width was larger in males and the length larger in females). The bodyweight had a significant effect for the length of both adrenal glands (the greater the bodyweight, the greater the length), but not the width. The differences between sd and coefficient of variation values for the width of the left adrenal gland were not statistically significant between the three observers, whereas they were statistically significant for the right adrenal gland.     

Regulation of pituitary somatotroph differentiation by hormones of peripheral endocrine glands

Anterior pituitary somatotroph differentiation occurs during chick embryonic and rat fetal development. A number of findings support the hypothesis that differentiation of these growth hormone (GH) producing cells in the chick and the rat is regulated by adrenal glucocorticoids and thyroid hormones. Somatotroph differentiation can be induced in cultures of chick embryonic and rat fetal pituitary cells with adrenal glucocorticoids and this effect can be modulated by concomitant treatment with thyroid hormones. Plasma levels of thyroid hormones, corticosterone and adrenocorticotropic hormone increase during development, consistent with the ontogeny of somatotrophs. Treatment of chick embryos or rat fetuses with glucocorticoids in vivo induces premature somatotroph differentiation, indicating that the adrenal gland, and ultimately anterior pituitary corticotrophs, may function to regulate pituitary GH cell differentiation during development. Administration of thyroid hormones in vivo also increases somatotrophs prematurely, and administration of the thyroid hormone synthesis inhibitor methimazole inhibits somatotroph differentiation in vivo, suggesting that endogenous thyroid hormone synthesis contributes to normal somatotroph differentiation. Our working model for the regulation of somatotroph differentiation during normal development includes modulation by elements of the hypothalamo-pituitary-adrenal and hypothalamo-pituitary-thyroid axes. Additional research is reviewed defining the mechanism of action for these peripheral hormones in induction of pituitary GH gene expression during development.     

Hyponatremia and hyperkalemia associated with peritoneal effusion in four cats

Four cats with considerable peritoneal effusion and corresponding hyponatremia and hyperkalemia were evaluated. The Na:K ratio in all cats was < 25, which is suggestive of adrenal insufficiency. An ACTH stimulation test was performed on 3 cats for evaluation of adrenal gland function. Serum cortisol and aldosterone concentrations did not support a diagnosis of adrenal gland insufficiency. In 1 cat, histologic evaluation of the adrenal glands at necropsy also failed to support a diagnosis of hypoadrenocorticism. On the basis of these findings, and because hyponatremia and hyperkalemia could not be readily explained by another cause, the electrolyte abnormalities were presumed to be secondary to peritoneal effusion.     

Computed tomographic adrenal gland quantification in canine adrenocorticotroph hormone-dependent hyperadrenocorticism

We conducted a retrospective study to determine whether multidetector computed tomography (CT) could be of value for adrenal gland assessment in dogs with pituitary-dependent hyperadrenocorticism. Adrenal gland attenuation and volume values of 49 dogs with hyperadrenocorticism were recorded and age, body weight, and gender were examined to determine if a relationship existed between these variables and adrenal gland morphology. There was not a statistically significant difference in mean X-ray attenuation of the left vs. right adrenal gland in normal dogs (35.3 +/- 6.1 HU), or in dogs with hyperadrenocorticism. The mean adrenal X-ray attenuation (+/- standard deviation [SD]) in dogs with microadenoma was 33.1 +/- 6.8 vs. 31.8 +/- 12.7 HU for dogs with macroadenoma, and these values were not statistically different. The mean volume of the left adrenal gland in normal dogs (0.59 +/- 0.17 cm3) was greater than that of the right adrenal gland (0.54 +/- 0.19 cm3) (P < 0.05). The mean CT volume (+/- SD) of the adrenal glands in dogs with microadenoma vs. macroadenoma were 1.60 +/- 1.25 vs. 2.88 +/- 1.60 cm3, respectively. There was no effect of age or gender on adrenal gland morphology or X-ray attenuation. The weight effect was the most important source of variation for the volume measurement in dogs with hyperadrenocorticism.     

Ultrasonographic characteristics of both adrenal glands in 15 dogs with functional adrenocortical tumors.

Ultrasonographic examination of both adrenal glands was performed in 15 dogs with functional adrenocortical tumors (FAT). Bilateral adrenal tumors were diagnosed in three of 15 dogs, and unilateral tumors were diagnosed in 12 of 15 dogs. Adrenal tumors were characterized by adrenal gland enlargement with loss of the normal shape and parenchymal structure. The contralateral adrenal gland could be imaged in all dogs with unilateral tumors. Based on size, shape, and parenchymal structure, the contralateral adrenal gland was similar to adrenal glands of normal dogs. The results of this study show that: 1) both adrenal glands should be imaged routinely in dogs with hyperadrenocorticism; 2) bilateral adrenocortical tumors seem to be more frequent than previously assumed; 3) one normal adrenal gland does not exclude the existence of a contralateral FAT; and 4) the functional atrophy of the contralateral adrenal gland in dogs with FAT may not be apparent ultrasonographically.