Effect of cyproterone acetate on cells of the pars distalis of the adenohypophysis in the Beagle Bitch

Summary The effects of oral administration of 100 mg per kg per day cyproterone acetate (CPA) for four weeks on cells of the pars distalis, as revealed by the immunoperoxidase technique and chemical staining, were studied in the ovariectomized beagle bitch. For immunochemical staining antisera to the following hormones were used: canine GH, canine PRL, porcine ACTH, bovine TSH, bovine LH and human FSH¹. The most striking effects of the treatment were an overall increase in the relative proportion of GH cells and a marked morphological indication of high secretory activity in these cells. In contrast, PRL cells were not affected significantly. In all ovariectomized control bitches a marked atrophy of the cells stained for FSH (FSH cells) and hypertrophy of the cells shown to contain LH(LH cells) were observed. FSH cells became enlarged, while LH cells appeared reduced in size by administration of CPA. In some treated bitches ACTH/MSH cells showed atrophy and regressive changes, whereas TSH cells seemed to become enlarged and were more densely arranged. These structural responses indicate that, in addition to its partial antigonadotropic properties, CPA as a synthetic progesterone derivative may stimulate GH secretion and possibly suppress CRH-ACTH activity in the ovariectomized beagle bitch.Abbreviations of Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin - CRH Corticotropin Releasing Hormone The authors are grateful to Dr. Christel Schoebel and Mrs. P. Kurth for carrying out the experimental work on animals, to Dr. H. Wiemann for the statistical analysis, to Mrs. B. Schilk, G. Soulioti and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Guenzel for his advice and encouragementRecipient of a Research Scholarship from the Arabic Republic of Egypt     

Effect of cyproterone acetate,d-norgestrel and progesterone on the canine mammary gland

Summary The effects of short-term (8 weeks) treatment with different doses of cyproterone acetate (CPA), d-norgestrel (d-N) and progesterone (PRO) on the mammary gland were studied in cycle-synchronized beagle bitches (first anoestrus). Mammary glands from non-treated primiparous beagle bitches in the 6th and 9th weeks of pregnancy were also included. The results were evaluated using the whole-mount technique, histologic, histochemical and biochemical methods. CPA, d-N and PRO were shown to cause dose-dependent mammary growth accompanied by an increase in the mammary weight, DNA content and activity of several histochemically demonstrable dehydrogenases. These changes resembled in some aspects mammary development observed in the last third of pregnancy. A single human oral contraceptive dose (HCD) of CPA as well as a dose as low as 1.0mg/kg/day subcutaneously of PRO was capable of stimulating complete mammary development. A comparable effect was first observed as a result of treatment with as much as 100 times the HCD of d-N. However, d-N and CPA were shown to be more effective than PRO in stimulating ductal proliferative activity. These structural and biochemical responses indicate that quantitative and/or qualitative differences may exist between PRO, the synthetic progesterone derivative CPA and the synthetic nortestosterone progestagen d-N with regard to their growth-promoting effect on the canine mammary gland. This may be explained by possible differences in their potency and range of biological activity, pharmacodynamics and effects on pituitary hormone secretion.The authors are grateful to Dr. Christel Schöbel and Mrs. P. Kurth for carrying out the experimental work on animals, to Dr. Y. Nishino and Mr. M. Leidecker for biochemical determination of DNA, to Dr. J. Kaufmann for statistical analysis, to Miss E. Fallenbacher, Mrs. B. Schilk, Mrs. G. Soulioti and Miss. U. Tüshaus for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

Immunocytochemical studies on the pituitary pars distalis of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary Immunocytochemical studies were performed to describe the characteristics of cell types and their distribution in the pars distalis of Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, collected at various stages of the reproductive cycle. Six distinct cell types have been identified in the pars distalis by the unlabeled immunoperoxidase technique and by the ABC method. Growth hormone (GH) and prolactin (PRL) cells were immunostained with antisera against chicken GH and ovine PRL. The GH-immunoreactive cells were round or oval orangeophilic cells distributed throughout the pars distalis with prominent aggregation in the posterolateral region. The PRL cells were pleomorphic carminophilic cells that occurred in small groups within the central and dorsocaudal regions of the pars distalis. They were sparsely distributed in the central region of the pars distalis in the hibernating bats, but increased significantly in the pregnant and lactating bats. The adrenocorticotropic (ACTH) cells were large round or polygonal amphophilic cells in the rostroventral and ventrolateral regions of the pars distalis. The thyrotropic (TSH) cells were small rounded or polygonal and distributed mainly in the ventrolateral region of the pars distalis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were identified immunocytochemically with antisera against the specific beta subunits of ovine LH and rat FSH. There were two populations of LH and FSH cells, one aggregated in the zona tuberalis and the other scattered singly throughout the rest of the pars distalis. The aggregated cells were immunoreactive with both antisera directed to LH and FSH, while scattered cells were reactive solely with antiserum to either LH or FSH and exhibited seasonal variations. In females, the proportional volume of the pars distalis occupied by LH cells was significantly reduced during pregnancy and lactation. No evidence of involution was observed in pars distalis cells except for PRL cells in males or females during hibernation.     

A comparative in vitro study on LHRH responsiveness of LH cells of the pars tuberalis and pars distalis

Summary The aim of the present study was to test whether the luteinizing-hormone (LH) cells in the pars tuberalis (PT) of the rat and mouse respond to LH-releasing hormone (LHRH) as do those of the pars distalis. A part of the basal hypothalamus containing the pituitary stalk, median eminence and the pars tuberalis (H-PT), was dissected out and incubated in vitro.The LH-secreting capacity of the PT was investigated after removal of the pituitary body (i.e., partes distalis, intermedia and nervosa). First, some rat and mouse H-PT tissues were treated with synthetic LHRH (100ng/ml), while others were incubated without LHRH. After 24 h of incubation, variable amounts of LH release were detected in the medium. This LH discharge, however, was not LHRH-dependent but proportional to the number of PT LH cells that were immunohistochemically detected in each incubated tissue. Since there was marked individual variation in the number of LH cells in the PT, the LH levels in the incubation medium were next compared before and after LHRH treatment using the same H-PT of the rat. An effect of LHRH could not clearly be shown in this experiment.Finally, the cytological response of the PT to LHRH was investigated by incubating both the H-PT and pituitary body connected to the intact pituitary stalk. Immunohistochemical examination of LHRH-treated tissues after 24 h revealed that, in females of both rats and mice, hormone depletion occurred in LH cells of the pars distalis but not in those of the PT. These results indicate that although LH cells in the PT can release LH in vitro, their mode of hormone synthesis and/or discharge differs from that of LH cells in the pars distalis. Since there was a marked individual variation and small LH-secreting capacity by the PT tissue, it seems unlikely, at least in rats and mice, that LH of PT origin plays an important role in the normal physiological state.     

Ultrastructure of STH cells in the pars distalis of the hypophysis of mice bearing transplanted hepatomas

Summary An electron microscopic analysis was performed on the pars distalis of the hypophysis of mice bearing transplanted hepatomas. Normal mice served as controls. In animals bearing a fast growing as well as in those bearing a slow growing hepatoma, the STH cells presented changes indicating increased release and synthesis of its secretory product. These changes can be summarized as follows: 1) Increased number of granuledepleted STH cells which were very irregular in size and shape. 2) Very irregular and sometimes bizarre nuclei. 3) Plasma membranes irregular and convoluted. 4) Increased quantity of endoplasmic reticulum which appeared sometimes dilated and contained material of varying electron density. 5) Abundant, irregular and frequently ramified mitochondria. 6) Hypertrophic Golgi system with increased number of microvesicles. 7) Increased number of large lysosomes of different type. 8) Increased number of fat droplets.Work carried out with the financial assistance of grants of the Consejo Nacional de Investigaciones Científicas y Técnicas de la Republica Argentina and the Comisión de Investigación Científica de la Universidad Nacional de La Plata. Thanks are due to the members of the technical Staff of the Institute for their technical assistance.     

Ultrastructure of STH cells of the pars distalis of castrated male mice after hepatectomy

Summary An electron microscopic study aimed at differentiation between castration gonadotrophs and posthepatectomy STH cells was performed on the pars distalis of the pituitary of castrated male mice, after partial hepatectomy. The ultrastructural features observed permit the distinction of both cell types. In the present experiments some remarkable ultrastructural changes, other than those described in a previous report, have been found in STH cells of hepatectomized mice with or without previous castration. Most of them contained masses of heterogeneous electron density, suggesting fusion of granules. These masses and some secretion granules were observed close to the plasma membrane, apparently in the process of discharging material into the pericapillary space. Granular extrusion was more frequent than normally. An increased number of lysosomes, probably related to the digestion of overproduced secretion material, was evident. The appearance of concentric lamellar formations might be related to an increase in cell movements. STH cells with severe cytoplasmic damage were also found, indicating an increased rate of cell loss.Work carried out with the financial assistance by a grant from the Comisión de Investigación Científica de la Universidad Nacional de La Plata. Thanks are due to the members of the Technical Staff of the Institute for their technical assistance.     

Effect of cyproterone acetate on prolactin secretion in the female rhesus monkey

Summary Adult female rhesus monkeys were given cyproterone acetate orally in doses of 0.04, 0.4, 4 and 40mg per kg per day for 12 weeks. Its effects were assessed on serum prolactin (PRL) concentration, the morphology of the PRL cells, and the development of the mammary glands. Serum PRL was relatively unchanged in the control animals from the fourth through the twelfth weeks of the study. In contrast, PRL was significantly elevated in each group of drug-treated animals during the same time periods. There was no development of the mammary glands nor was there any evidence of milk secretion in the control animals; however, in the monkeys given cyproterone acetate the mammary glands had extensive lobuloalveolar growth and milk-like secretion that could be expressed as early as the fourth or fifth week of the study. By immunocytochemistry and differential light microscopic staining techniques, the PRL cells in the pituitary glands of the experimental animals were found to be more numerous and much larger than those present in the controls. They displayed a well developed Golgi complex and had an abundance of cytoplasmic RNA. These data suggest that PRL secretion is markedly enhanced by cyproterone acetate.Supported in part by USPHS Grant AM12583     

Structure of the nongranulated cells in the hypophyseal rostral pars distalis of cyclostomes and actinopterygians

Summary The structure of the nongranulated cells in the sea lamprey adenohypophysis and similar cells of the rostral pars distalis in a number of actinopterygian fishes was examined with the aim of determining the role(s) of these cells in pituitary function.A number of possible roles are proposed for the nongranulated cells. In salmonids and Amia calva the cells may be involved in the active movement of material into or from the follicle lumina. The structure of the nongranulated cells in in vitro cultured and in in vivo transplanted trout rostral pars distalis also suggests a phagocytotic role for these cells. In teleosts with a non-follicular rostral pars distalis the nongranulated cells appear to play roles in the release of granules from the prolactin cells and in the subsequent dispersal of the hormone (and/or carrier substance) into the peripheral circulation.We wish to thank Dr. B.I. Baker who performed the trout pituitary in vitro cultures and Professor M. Hyder who was closely involved with the collection of the Tilapia specimens. We also wish to thank Mr. R. Lindsay and Mrs. L. Lin for their technical assistance and Drs. F.W.H. Beamish and R. Sonstegard for their help in supplying some of the animals used in this study. —The work was supported in part by a grant-in-aid of research from the National Research Council of Canada to J.F.L. and in part by a negotiated NRC grant. The paper is number 138 in the physiology of migration series.     

Cytological changes in the pars distalis of pituitary of the green frog,Rana esculenta L., during the reproductive cycle

Summary The cytological changes in the pars distalis of pituitary of male and female green frog, Rana esculenta during the reproductive cycle have been studied by examining the form, cytoplasmic granulation and numerical proportions of the different cell types. Some sex differences were recorded, particularly in relative total number of cells in a median sagittal section of the pars distalis. No extensive cytological changes occur during the hibernation period, but there are striking alterations at the time of spring awakening, during spawning months and during replenishment of the spent gonads i.e. during late summer and autumn months. Important changes are to be observed in the acidophils type one (A1) and basophils type two (B2) and three (B3). Changes in the A1 cells during breeding season have been speculated as being in support of the spawning activity of the animal. The B2 cells exhibit most profound changes which correlate precisely with the morphological changes in gonads, and these cells are considered as the FSH-gonadotrops. The functional significance of the B3 cells is discussed and it is suggested that they might be the source of an ICSH- or LH-like hormone.The work has been supported by grant from the National Research Council of Italy.The award of postdoctoral fellowship by the National Research Council of Italy is gratefully acknowledged.     

An electron microscopic study of stellate cells and cavities in the pars distalis of frog pituitary

Stellate cells in the pars distalis of adult Rana ridibunda were observed electron microscopically under normal and experimental conditions (TRH injection). The stellate cells have lengthy processes extending into the intercellular spaces between the secretory cells and scanty cytoplasm surrounding the nucleus. Occasional desmosomes link stellate cells to adjacent secretory cells. In the pars distalis of animals injected with thyrotropic-releasing hormone (TRH), the stellate cells form large cavities (2-6 mum) filled with heterogeneous material. Their cytoplasm contains well-developed Golgi complexes and some lysosomes; these are the principal morphological alterations as compared to those observed in control animals. It is suggested that stellate cells could play an active role in addition to providing a structural framework for the pars distalis.     

Immunocytochemical effects of thyroxine stimulation on the adenohypophysis of dwarf (dw) mutant mice

The effects of dietary thyroxine on the immunoreactivity of cells in the pars distalis of the adenohypophysis in dwarf (dw/dw) mice were determined by ultrastructural immunocytochemistry. In nontreated dwarfs only adrenocorticotropic hormone (ACTH) cells and luteinizing hormone (LH) cells showed positive reactions to their respective antibodies, whereas no cells showed immunoreactivity to antibodies to growth hormone (GH), thyroid-stimulating hormone (TSH), or prolactin (Prl). In dwarfs supplemented postnatally with dietary thyroxine for 9 wks, the treatment failed to produced immunoreactive GH, TSH or Prl cells. However, LH cells became more prominent and fully developed, with denser concentrations of immunoreactive particles overlying the secretory granules than occurred in nontreated dwarfs. In thyroxine-treated dwarfs, ACTH cells were similar in ultrastructural features and immunoreactivity to those in nontreated dwarfs.     

Estrogen-alpha and progesterone receptor expression in cystic endometrial hyperplasia and pyometra in the bitch

Estrogen-alpha receptor (ER) and progesterone receptor (PR) were examined immunohistochemically in uteri of normal bitches, in uteri of bitches with cystic endometrial hyperplasia-mucometra (CEH-M) and in uteri of bitches with endometritis-pyometra (E-P), under exogenous progesterone treatment.In the CEH-M group, the ER- and PR-scores of all uterine cell types were higher than the ER- and PR-scores of normal uteri, although these differences were not always statistically significant. The ER-scores of E-P group were significantly lower than the ER-scores of the normal uteri and CEH-M group. The PR-scores of the E-P group tended to be higher than the PR-scores of the normal uteri, except for the surface epithelium, although these differences were not statistically significant. Exogenous progesterone treated bitches with CEH-M or E-P showed reduced ER- and PR-scores in the different uterine cell types, compared with the corresponding nontreated CEH-M or E-P group.The differences in ER and PR expression between CEH-M and E-P suggest different factors in the pathogenesis of both entities. Although, these changes in ER and PR expression do not seem to be directly involved in the pathogenesis of CEH-M and E-P. It is suggested that for CEH-M and progestin induced CEH-M a hormone dependent pathway is responsible. For P, the trigger may be bacterial infection.     

Localization of gonadotrophic hormones in the dog pituitary gland

Summary Using the immunoperoxidase technique and antisera to the specific 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 or FSH. The cells stained for FSH were at least 1.5 times less numerous than those shown to contain LH. 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.Abbreviations for Pituitary Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin The authors are grateful to Dr. H. Wiemann for the statistical evaluation and to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistanceRecipient of a Research Scholarship from the Arabic Republic of Egypt     

Immunohistochemical localization of FSH and LH in the pars distalis of vervet (Cercopithecus aethiops) and babbon (Papio hamadryas) pituitaries

Summary Antisera against oLH¹, oLH and hFSH were used to localize gonadotropic cells in the pars distalis of Cercopithecus aethiops and Papio hamadryas. Three separate cell types were observed for FSH and LH: 85% of immunohistochemically identified gonadotropic cells reacted to all the various antisera; 10% reacted with the anti-LH antibody only; and 5% with the anti-hFSH antibody only. Comparisons between adjacent serial sections treated with various antisera, other than anti-gonadotropic hormones, demonstrated that the gonadotropic cells of these monkeys did not respond to these antisera.

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Résumé Des anticorps anti-LH ovine, anti-LH ovine et anti-FSH humaine ont été utilisés pour localiser les cellules gonadotropes dans la pars distalis de l'hypophyse des Singes Cercopithecus aethiops et Papio hamadryas. Trois catégories cellulaires distinctes, réagissant avec des anticorps anti-hormones gonadotropes, ont été observées. 85% des cellules immunoréactives identifiées en tant que cellules gonadotropes réagissent simultanément avec les différents anticorps mentionnés; 10% des cellules gonadotropes réagissent seulement avec l'anticorps anti-oLH et 5% de ces cellules seulement avec l'anticorps anti-hFSH. La comparaison avec des coupes adjacentes traitées par divers anticorps autres que les anticorps anti-gonadotropines prouve que les cellules gonadotropes de ces Singes ne réagissent jamais simultanément avec l'un ou l'autre de ces anticorps.

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Abbreviations used in this Article oLH ovine luteinizing hormone - hFSH human follicle stimulating hormone - ACTH corticotropin - GH growth hormone - LPH lipotropin - TSH thyrotropin     

Proliferation of lamellar whorls in arcuate neurons of the hypothalamus of male rats treated with estradiol benzoate or cyproterone acetate

Summary The arcuate nucleus of the hypothalamus (AH) of male rats which had been treated either with estradiol benzoate (E²B) or cyproterone acetate (CPA) was examined ultrastructurally for the presence of whorls of endoplasmic reticulum. The incidence of whorl containing neurons (WCN) was 2–4 times higher in the AH of animals treated for 2–3 weeks with E²B or for 2 weeks with CPA than in the AH of oil treated controls. CPA is a powerful anti-androgen while E²B acts both peripherally and centrally to limit testosterone production. These findings, together with previous evidence that whorls proliferate in AH of male rats deprived of androgen by morphine treatment or castration, suggest that steroid feedback (androgen alone or both androgen and estrogen) plays an important role in AH whorl proliferation. The possibility that WCN may be LH-RH containing neurons is suggested by the close correspondence between the number and location of WCN within AH as determined in this study and the distribution of LH-RH containing cells reported by others.The authors are indebted to Schering AG for supplying cyproterone acetate for this study. This work was supported by grants DA-00259, NS-09156 and MH-14677 from U.S.P.H.S.Research Scientist Development Award MH-38894Research Scientist Development Award MH-70180     

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain     

Immunofluorescent identification of somatotropic and prolactin cells in the anterior lobe of the hypophysis (pars distalis) of the monkey,Macacus irus

Summary Using antibodies against human growth hormone and ovine prolactine, it is possible to identify somatotropic and prolactin cells in the anterior lobe of the hypophysis (pars distalis) of the monkey, Macacus irus. These cells are present in both adult and infant male and female monkeys. The density of the somatotropic cells is greater at the periphery of the anterior lobe. Prolactin cells are of two types: one type is dispersed throughout the pars distalis and consists of small and large cells, the other type forms clusters of large cells.We wish to express our thanks to Mrs. M. Raccurt (CNRS) for her highly skilled technical assistance     

Immunocytochemical identification of growth hormote (GH) cells in the pituitary of three anuran species using an antiserum against purified bullforg GH

An antiserum was prepared against the recently purified bullfrog (bf) growth hormone (GH); it was applied to sections of brain and pituitary of three urodele (Ambystoma, Pleurodeles and Cynops) and three anuran (Xenopus, Bufo vulgaris and B. japonicus) species. No immunostaining was obtained in the urodele pituitary, being consistent with the results of immunoblot analysis of the pituitary homogenate. In the three anuran species, strong immunoreactivity was observed in GH cells that were concentrated in the posterodorsal region of the pars distalis. No GH-like immunoreactivity was detectable in the brain of any of the species. A comparison using adjacent sections stained with anti-bf prolactin (PRL) confirmed the anteroventral localization of PRL cells. Colocalization of GH and PRL was not apparent. These data suggest that the molecular structure of amphibian GHs is considerably different between anurans and urodeles. The antiserum used in the present work shows a high species specificity, recognizing only anuran GHs. In contrast anti-bfPRLlabeled PRL cells in all the amphibian species studied in the present work, suggesting that PRLs possess common amino acid sequences recognized by the anti-bfPRL.     

Chromophobic cells and their role in extracellular transport of hormones in the bovine adenohypophysis (pars distalis)

Chromophobic cells form sheaths around secretory cells. They are in close contact with them, but occasionally leave open intercellular spaces bounded by desmosomes. It is suggested that 1. there is a reduced exposure of secretory cells to direct negative feedback exerted by hormone discharge of neighbouring cells--especially those of the same cell type, 2. chromophobic cells contribute to the formation of intralobular pathways for extracellular hormone transport.