Dog Pituitary Gland Research Articles

Bone morphogenetic protein 4 and bone morphogenetic protein receptor expression in the pituitary gland of adult dogs in healthy condition and with ACTH-secreting pituitary adenoma

The purpose of this study was to investigate the expression of bone morphogenetic protein 4 (BMP4) and its receptors, bone morphogenetic protein receptor I (BMPRI) and BMPRII, in the pituitary gland of healthy adult dogs and in those with ACTH-secreting pituitary adenoma. Quantitative polymerase chain reaction analysis showed that the BMP4 messenger RNA expression level in the ACTH-secreting pituitary adenoma samples was significantly lower than that in the normal pituitary gland samples (P = 0.03). However, there were no statistically significant differences between samples with respect to the messenger RNA expression levels of the receptors BMPRIA, BMPRIB, and BMPRII. Double-immunofluorescence analysis of the normal canine pituitary showed that BMP4 was localized in the thyrotroph (51.3 ± 7.3%) and not the corticotroph cells. By contrast, BMPRII was widely expressed in the thyrotroph (19.9 ± 5.2%) and somatotroph cells (94.7 ± 3.6%) but not in the corticotroph cells (P < 0.001, thyrotroph cells vs somatotroph cells). Similarly, in ACTH-secreting pituitary adenoma, BMP4 and BMPRII were not expressed in the corticotroph cells. Moreover, the percentage of BMP4-positive cells was also significantly reduced in the thyrotroph cells of the surrounding normal pituitary tissue obtained from the resected ACTH-secreting pituitary adenoma (8.3 ± 7.9%) compared with that in normal canine pituitary (P < 0.001). BMP4 has been reported to be expressed in corticotroph cells in the human pituitary gland. Therefore, the results of this study reveal a difference in the cellular pattern of BMP4-positive staining in the pituitary gland between humans and dogs and further revealed the pattern of BMPRII-positive staining in the dog pituitary gland. These species-specific differences regarding BMP4 should be considered when using dogs as an animal model for Cushing's disease.

A dog growth hormone cDNA codes for a mature protein identical to pig growth hormone

Although methods for purification of dog ( Canis familiaris) growth hormone (cfGH) were described in the late Sixties, the cloning of its cDNA has not been achieved until now. In order to clone the cfGH cDNA, we capitalized on the high degree of nucleotide sequence conservation among mammalian G H genes to design a pair of consensus oligodeoxyri bonucleotide primers. With these, and starting with dog pituitary gland total RNA, we specifically amplified the cfGH cDNA using the reverse transcription-polymerase chain reaction. Its coding sequence (651 bp), as well as its 3' untranslated region (101 bp), resemble those of a typical mammalian GH cDNA. Interestingly, its encoded mature protei is identical to pig growth hormone (pGH).

Histochemical characterization and functional significance of the hyperintense signal on MR images of the posterior pituitary.

MR imaging of the pituitary fossa characteristically shows a well-circumscribed area of high signal intensity in the posterior lobe on T1-weighted images. We used a combination of high-field MR, electron microscopy, and histologic techniques in experimental animals to determine whether the hyperintensity of the posterior lobe might be functionally related to hormone neurosecretory processes, and to attempt to establish its chemical nature. Histologic sections of a dog's pituitary gland processed with lipid-specific markers showed intense staining in the posterior lobe but not in the anterior lobe, thus documenting the location of fat in the posterior pituitary. Administration of vasoactive drugs known to influence vasopressin secretion to anesthetized cats produced changes in the volume of high-intensity signal in the posterior pituitary. Subsequent electron microscopy showed a significant increase in posterior lobe glial cell lipid droplets and neurosecretory granules in dehydration-stimulated cats. The data suggest that the pituitary hyperintensity represents intracellular lipid signal in the glial cell pituicytes of the posterior lobe or neurosecretory granules containing vasopressin. The volume of the signal may, in turn, reflect the functional state of hormonal release from the neurohypophysis.

The utility of antisera to different synthetic adrenocorticotrophins (ACTH) and melanotrophins (MSH) for immunocytochemical staining of the dog pituitary gland.

Using the immunoperoxidase technique and specific antisera to synthetic ACTH beta (1-24), ACTH beta (17-39) and bMSHbeta1, selective immunocytochemical staining was localized in a distinctive cell type in the pars distalis and pars tuberalis of the dog pituitary gland. Except for a rare cell, the pars distalis and pars tuberalis did not stain with an anti-bMSH alpha serum. In the pars intermedia immunoreactive cells containing ACTH beta(1-24), ACTHbetap(17-39), bMSHbeta and/or bMSH alpha were observed. The specificity and validity of the antisera were demonstrated by elimination of their immunostaining capacity after prior absorption with their respective antigens, while absorption with other antigens failed to decrease staining intensity. The cytoplasm of the ACTH/MSH cells showed a positive reaction to periodic-acid-Schiff and assumed a pale aniline blue colour, whilst the granules were stained with carmoisine L and acid alizarine blue. These ACTH/MSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. It is concluded that ACTH and MSH beta were present and most probably produced by the corticomelanotrophs of the pars distalis and pars tuberalis. In addition to corticomelanotrophs analogous to those of the pars distalis and pars tuberalis, the pars intermedia showed many cells which contain MSH alpha alone or together with MSH beta and/or ACTH.

Effect of 17?-estradiol on cells stained for FSH? and/or LH? in the dog pituitary gland

The effect of long-term treatment (52 weeks) with high doses of 17β-estradiol (1.28 mg/kg/week intramuscularly) on gonadotrophs was studied in the pituitary gland of the beagle bitch. For immunochemical staining the immunoperoxidase technique and antisera to the specific beta (β) subunits of FSH and LH were employed. For control purposes antisera to the following hormones were also used: bovine TSHβ, canine GH, canine PRL and porcine ACTH1. In the pars distalis and pars tuberalis of control bitches, in addition to the cells which react solely with antisera to either LHβ or FSHβ, most cells were reactive to both antisera. The cells stained for FSHβ were less numerous than those shown to contain LHβ. TSHβ, PRL, GH and ACTH/MSH were localized in distinctly different cell types in the pars distalis of all control animals. In the treated bitches, almost complete regression of cells classically identified as gonadotrophs and stained for LHβ was observed. On the other hand, using the antiserum to FSHβ, selective immunochemical staining was localized in cells fitting the morphological characteristics of TSH cells. All these cells were also stained for TSHβ. However, a few cells were also shown to react solely with the antiserum to TSHβ. These cells, which seem to contain both TSHβ and FSHβ, were further clearly differentiated from PRL, GH and ACTH/MSH cells on the basis of their cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not necessarily apply to the glycoprotein hormones of the dog pituitary gland.

Localization of thyrotropin (TSH) in the dog pituitary gland.

Using the immunoperoxidase technique and antisera to the specific beta (beta) subunits of bovine and rat TSH, selective immunocytochemical staining was localized in a specific cell population in the pars distalis of the dog pituitary gland. These TSH cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. With the performic acid-alcian blue (pH 0.2) -PAS-orange G procedure these cells stained blue-purple, demonstrating FSH/LH cells (blue or turquoise), ACTH/MSH cells (red-purple) and PRL cells (orange-red). The TSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunocytochemical double staining. In the pars distalis of adult male dogs the TSH cells were mostly shown to be smaller in size and less numerous than in bitches in the anestrous phase of the sexual cycle. Moreover, cytological alterations in the immunoreactive thyrotrophs in the pituitary of male and female dogs generally paralleled the spontaneous changes in thyroid function associated with thyroid atrophy and/or pituitary insufficiency, and thyroid hyperplasia or goiter. In conclusion, because of their specificity and high potency, the antisera to the beta-subunits of bovine and rat TSH represent an effective tool for the selective immunocytochemical localization of TSH in the dog pituitary. This allows the study of the morphology and function of TSH cells under different physiological, pathological and experimental conditions.

Localization of gonadotrophic hormones in the dog pituitary gland. A study using immunoenzyme histochemistry and chemical staining.

Using the immunoperoxidase technique and antisera to the specific beta (beta) subunits of FSH and LH1, selective immunochemical staining was localized mostly in the same cell type in the pars distalis and pars tuberalis of the dog pituitary gland. However, some cells were consistently shown to react solely with antisera to either LH beta of FSH beta. The cells stained for FSH beta were at least 1.5 times less numerous than those shown to contain LH beta. In the pars distalis of adult male dogs the immunoreactive gonadotrophs varied greatly in their relative proportion and were mostly shown to be much less numerous than in bitches in the anestrus phase of the sexual cycle. These cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. The performic acid-alcian blue (pH 0.2)-PAS-orange G procedure stained the FSH/LH cells blue or turquoise, demonstrating TSH cells (blue-purple), ACTH/MSH cells (red-purple) and PRL cells (orange-red). The FSH/LH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not apply to gonadotrophic hormones, although some cells seem to be the source of either FSH or LH.

The utility of antisera to canine growth hormone and canine prolactin for immunocytochemical staining of the dog pituitary gland.

Using the immunoperoxidase technique and specific homologous antisera to highly purified canine prolactin (PRL) and canine growth hormone (GH), selective immunocytochemical staining was localized in two different cell types in the pars distalis of the dog pituitary gland. The specificity and validity of both antisera were demonstrated by elimination of their immunostaining capacify after prior absorption with their respective antigens, while absorption with other antigens failed to decrease staining intensity. This was further confirmed by studying the immunologic behavior of both antisera in the agar gel double diffusion immunoprecipitin test and by competitive radioactive binding analysis. The specific heterologous antisera to ovine PRL and human GH were shown to be just as useful as antisera to canine hormones for immunocytochemical demonstration of the individuality of PRL and GH cells in the dog pituitary. This confirms the validity and specific of our hitherto heterologous immunocytochemical procedure. However, because of their high potency the anti-canine GH and anti-canine PRL sera seem to be more efficient for immunocytochemical staining of the dog pituitary. The immunoreactive PRL and GH cells were also distinguished with reasonable certainity in two different populations of acidophils according to their tinctorial affinity. Both cell types differed further from each other in respect of their relative proportion, morphology and topography, which were variously pronounced for each sex.

Prolactin content of canine pituitary gland.

SUMMARY The prolactin concentration in the dog pituitary gland was determined by isoelectric focusing of adenohypophysial extract in polyacrylamide gels followed by densitometry of the isolated stained hormone band. Dogs of both sexes and various ages (excluding newborn pups and weanlings) were studied. The bitches comprised animals at different stages of the oestrous cycle and also included a small number of pregnant, lactating or ovariectomized animals. Low pituitary prolactin concentrations were found in males, sexually immature females and dioestrous females. Concentrations about 1·5 times as high occurred in oestrous, metoestrous (luteal) and ovariectomized females. Post-partum lactating bitches had the highest pituitary prolactin concentrations, these being double those occurring at dioestrus. With the exception of relatively high concentrations in ovariectomized bitches, these results are in good agreement with findings in the rat, mouse and rabbit. The persistence of high pituitary prolactin levels throughout metoestrus was believed to be associated with differences between the canine and murine reproductive cycle. Age did not influence pituitary prolactin levels in either males or females.

The follicular cell in the pars distalis of the dog pituitary gland: an electron microscope study.

The presence of follicular cells has been verified in the pars distalis of the dog pituitary gland through the use of electron microscopy. These cells are stellate; they extend cytoplasmic processes among parenchymal cells, and surround colloid-containing follicular cavities in some places. Around the cavities, apposed surfaces of adjacent follicular cells are connected with each other by prominent terminal bars and desmosomes. These cells project microvilli, as well as cilia in occasional cells, into the follicular cavity. A small number of granules, 150-200 m∧u in diameter, are sometimes contained in the cytoplasm subjacent to the follicular surface. (Endocrinology 77: 1053, 1965)

The subcellular distribution of histamine in brain tissue

The subcellular distribution of histamine has been analysed in guinea pig whole brain, dog hypothalamus and dog pituitary gland. In whole brain, histamine is found in both the nuclear and crude mitochondrial fractions. In brain tissue lacking mast cells (hypothalamus) “bound” histamine precipitates with the 17,000 g crude mitochondrial fraction, whereas 94 per cent of the “bound” histamine of the pituitary precipitates with the mast cells in the low speed nuclear fraction. Electron microscopic examination of subfractionated mitochondrial preparations from hypothalamus do not reveal structures comparable to the histamine containing granules of mast cells and a different type of histamine storage granule is suggested.