Verónica Filippa

Seasonal variations of gonadotropins in the pars distalis male Viscacha pituitary. Effect of chronic melatonin treatment

The gonadotropes, LH and FSH cells, were immunohistochemically identified in the pituitary pars distalis of the adult male viscacha (Lagostomus maximus maximus) using specific antibodies against hLHbeta and hFSHbeta with the streptavidin-biotin-peroxidase complex. The distribution, size and percentage immunopositive area of these cells were analyzed by image analysis in viscachas captured during the annual reproductive cycle and after the chronic administration of melatonin. The LHbeta and FSHbeta cells showed seasonal changes in the distribution, size and percentage immunopositive area. The LHbeta cells were found widely distributed throughout the pars distalis during the reproductive period, and they were found in the ventro-medial region in the pars distalis during the gonadal regression and gonadal recovery periods. The LHbeta cells reached the largest size and immunopositive area during the reproductive period and the smallest size and immunopositive area during the gonadal regression period. The FSHbeta cells were found in the ventro-medial region during reproductive and gonadal regression periods. The FSHbeta cells were found widely distributed throughout the pars distalis during the gonadal recovery period when they showed the maximum percentage immunopositive area. A decrease in the size of LHbeta and FSHbeta cells was observed after the chronic administration of melatonin. Moreover, it produces a decrease in the immunopositive area occupied by the LHbeta cells but not in the immunopositive area occupied by the FSHbeta cells. Our results show great activity of LHbeta and FSHbeta cells in different moments of the annual reproductive cycle demonstrating that these cells do not secrete in parallel. Moreover, melatonin acts differentially on the activity of the gonadotrope cells.

Folliculostellate cells in pituitary pars distalis of male viscacha: immunohistochemical, morphometric and ultrastructural study

Folliculostellate cells (FSC) have been reported in pituitary of several mammalian species. FSC morphology and secreted substances have been instrumental to the understanding of their function. The purpose of this work was to perform an immunohistochemical, morphometric and ultrastructural study of the pituitary pars distalis FSC in adult male viscacha and to analyze their relation with hormone secreting cells. Immunohistochemistry and image analysis were carried out in different sectors of the gland, from the middle (sector 1) to the glandular periphery (sector 5). Transmission electron microscopy with lanthanum as electrodense tracer was used. FSC formed follicles with PAS-positive colloid inside. They expressed S-100 protein mainly in both nucleus and cytoplasm. FSC were stellate-like in shape and exhibited short cytoplasmic processes that contacted with blood vessels and endocrine cells. In addition, some follicular colloids were immunostained with anti-S-100 protein. A few FSC were immunostained with anti-glial fibrillary acidic protein (GFAP) and anti-vimentin. The morphometric parameters analyzed (percentages of S-100-positive total, cellular and colloidal areas) increased from sector 1 to sector 3 and then decreased to sector 5. Hormone secreting cells, mainly lactotrophs, gonadotrophs and corticotrophs were associated with FSC and follicles. The ultrastructural study demonstrated that FSC developed junctional complexes and desmosomes between their lateral membranes. Lanthanum freely penetrated the spaces between granulated cells and FSC, but did not penetrate into the follicular lumen. In conclusion: 1) the differential expression of S-100 protein, GFAP and vimentin may indicate different physiological stages of FSC; 2) the expression of these proteins suggests a neuroectodermic origin of these cells; 3) FSC spatial distribution, association with endocrine cells, and the generation of an intercellular communication network suggest that FSC are involved in the pituitary pars distalis paracrine regulation of the viscacha.

Androgen receptors in coeliac ganglion in late pregnant rat.

The ovarian function is controlled by endocrine factors and neural influence. In late pregnant rat, androstenedione, from the coeliac ganglion, has a luteotrophic effect in the ex vivo coeliac ganglion-superior ovarian nerve-ovary system. In this work we investigate the presence of androgen receptors in the coeliac ganglion of late pregnant rats by immunohistochemistry. We also explore, from a physiological point of view, the potential participation of these receptors in the androstenedione ganglionic action on progesterone release and metabolism, as well as on nitrites release in the ovary compartment. The coeliac ganglion was isolated after being fixed in situ and immunohistochemistry was performed. In the system, three experimental groups were used with the addition of (a) androstenedione, (b) flutamide, and (c) androstenedione plus flutamide in the ganglion compartment. Progesterone and nitrite concentrations were determined in the ovary compartment at different incubation times. Corpora lutea samples isolated at the end of incubation were used to determine the expressions and activities of the progesterone synthesis (3beta-hydroxysteroid-dehydrogenase, 3beta-HSD) and degradation (20alpha-hydroxysteroid-dehydrogenase, 20alpha-HSD) enzymes. Immunohistochemistry revealed cytoplasmatic androgen receptor immunoreactivity in neural somas in the coeliac ganglion. In the coeliac ganglion-superior ovarian nerve-ovary system, androstenedione addition increased 3beta-HSD and decreased 20alpha-HSD, showed a tendency to decrease 20alpha-HSD expression, and increased nitrites release in relation to control. Androstenedione plus flutamide decreased progesterone and nitrites release in relation to the androstenedione group. This work demonstrates the presence of androgen receptors in neurons of celiac ganglion and provides evidence for the luteotrophic action of androstenedione via a neural pathway that may be mediated by these receptors.

Morphological and morphometric changes of pituitary lactotrophs of viscacha (Lagostomus maximus maximus) in relation to reproductive cycle, age, and sex.

Lactotrophs in pituitary pars distalis (PD) of viscacha were studied by immunohistochemistry and morphometric analysis in the following groups: 1) adult males throughout the reproductive cycle (reproductive, gonadal regression, and recovery periods), 2) melatonin‐treated adults, 3) castrated adults, 4) prepubertal, 5) non‐pregnant females, and 6) pregnant females (early, mid, and late pregnancy). Immunopositive percentage area (%IA), cell percentage in PD (% PDC), number of cells per reference area (no.cell/RA), major cellular and nuclear diameters were analyzed. Lactotrophs were mainly localized in the ventro–medial region and the caudal extreme of PD. In the male viscachas, they were isolated in small and big groups, close to blood vessels and near follicles. These cells were pleomorphic and with a heterogeneous cytoplasmic immunolabeling pattern. In the adult males of the gonadal regression period the morphometric parameters were the lowest. Most parameters of lactotrophs in the prepubertal were significantly lower than in the adult males in the reproductive period. In the melatonin‐treated animals and in castrated animals there was a decrease in %IA, %PDC, and no.cell/RA. In the females, the morphometric parameters increased at the end of pregnancy. Non‐pregnant females exhibited a higher immunopositive area and number, but a smaller size of cells than males. Our results showed that in the adult male viscacha, lactotrophs vary seasonally, probably due to the photoperiod effect through melatonin. Besides the changes observed after castration, in prepubertal animals, in adults of different sex, and during pregnancy suggest that the gonadal steroid hormones might modify the lactotrophs activity. Anat Rec, 293:150–161, 2010.

Immunohistochemical and Morphometric Study of Pituitary Pars Distalis Thyrotrophs of Male Viscacha (Lagostomus maximus maximus): Seasonal Variations and Effect of Melatonin and Castration

The physiology and behavior of viscacha vary along the year according to the modifications of environmental signals such as the photoperiod length, temperature, rainfall pattern, food composition, and social interactions. The pituitary pars distalis thyrotrophs (TSH cells) of male viscacha were immunohistochemically identified, and the morphometric parameters: percentage of immunopositive area (% IA), cell percentage in pars distalis (% PDC), number of cells per reference area (no. cells/RA), and major cellular and nuclear diameters were analyzed. Three different groups of adult male viscachas were used: (1) captured in their natural habitat during the year, (2) melatonin‐administered, and (3) castrated. The thyrotrophs were localized in the ventromedial sector, mainly in the pars distalis cephalic extreme. They were oval or pyramidal in shape, and their immunostaining intensity was heterogeneous. The % IA, % PDC, and no. cells/RA exhibited a significant decrease in June–July (winter, gonadal regression period) in relation to February–March (summer–early autumn, reproductive period), and they were recovered in August–September (later winter–early spring, gonadal recovery period). No morphometric variations of TSH cells were observed in melatonin‐treated animals, whereas a decrease of the % IA, % PDC, and no. cells/RA was observed in castrated animals in relation to the intact animals. Our results show TSH cell morphometric variations during the year in agreement with the animals different physiological conditions during the reproductive cycle, and probably in response to the environmental signals changes. Melatonin does not have a direct effect on the TSH cells. However, castration modifies some thyrotroph morphometric parameters, reinforcing the hypothesis that androgens affect the cells activity. Anat Rec, 291:400–409, 2008.

Cellular associations of pituitary gonadotrophs in a rodent (Lagostomus maximus maximus) with photoperiod-dependent reproduction.

The morphological characteristics and percentage of the cellular associations between gonadotrophs (LH- and FSH-secreting cells) and other cellular types were studied in pituitary pars distalis of adult male viscachas (Lagostomus maximus maximus) by double immunohistochemistry using specific antibodies to LH, FSH, PRL, GH, ACTH, TSH and S-100 protein (by folliculostellate cells; FSC), during long and short photoperiods. Bihormonal gonadotrophs were observed in ventro-medial and dorsal regions, interspersed between monohormonal gonadotrophs, and their number increased in short photoperiod. LH- and FSH-gonadotrophs were found around lactotrophs, enclosed by somatotrophs in the dorsal region, and associated with irregular corticotrophs. Gonadotrophs and thyrotrophs were associated along blood vessels and follicular structures. The cytoplasmic prolongations of FSC were in contact with both gonadotrophs. The percentage of LH-FSH, LH-ACTH, LH-FSC, FSH-LH, FSH-PRL, FSH-GH, FSH-ACTH, FSH-TSH and FSH-FSC associations decreased, whereas LH-PRL increased in short as compared to long photoperiod. The most abundant associations were LH-GH and LH-TSH during long photoperiod, but LH-GH and LH-PRL during short photoperiod. FSH-GH and FSH-PRL were the most numerous associations, and LH-FSC and FSH-FSC were the less abundant ones in both photoperiods. These results provide the morphological evidence for specific cellular associations between gonadotrophs and other cellular types of viscacha pituitary.

The pituitary of non-pregnant and pregnant viscachas (Lagostomus maximus maximus): a comparative study by immunohistochemistry and morphometric analysis

The neuroendocrine hypothalamic-pituitary axis undergoes morphological and biochemical changes throughout gestation. In viscacha, pregnancy lasts approximately 154 days, and three stages can be described: early, mid- and late pregnancy. The aim of this work was to study the pituitary LH-gonadotrophs, FSH-gonadotrophs, somatotrophs, corticotrophs and thyrotrophs of non-pregnant and pregnant adult viscachas by immunohistochemistry and morphometric analysis. Immunopositive percentage area (%IA), cell percentage in the pars distalis (%PDC), number of cells per reference area (n°cell/RA), and major cellular (MCD) and nuclear (ND) diameters were analyzed. The different cell populations showed a well-defined morphology, immunolabeling patterns and regionalization in the pars distalis (PD). In the early pregnancy of animals the morphometric analysis demonstrated that %IA, %PDC and n°cell/RA increased in the FSH-gonadotrophs and decreased in the somatotrophs in relation to non-pregnant animals. In mid-pregnancy, there was an increase in %IA, %PDC, and n°cell/RA of LH-gonadotrophs, FSH-gonadotrophs, somatotrophs, and thyrotrophs. The MCD of LH-gonadotrophs and FSH-gonadotrophs increased. In late pregnancy, the %IA, %PDC and n°cell/RA of LH-gonadotrophs, FSH-gonadotrophs, somatotrophs and corticotrophs decreased whereas the values of the thyrotrophs remained constant. The MCD of LH-gonadotrophs, FSH-gonadotrophs and corticotrophs decreased. No significant changes were observed in the ND of the studied cell types. In conclusion, this work provides evidence for histological and morphometric changes in the different cell types of the pituitary PD in viscachas during pregnancy, probably according to the requirements of this physiological stage.

Androgen Receptors Expression in Pituitary of Male Viscacha in relation to Growth and Reproductive Cycle.

The aim of this work was to study the androgen receptors (AR) expression in pituitary pars distalis (PD) of male viscachas in relation to growth and reproductive cycle. AR were detected by immunocytochemistry and quantified by image analysis. Pituitary glands from fetus, immature, prepubertal, and adult viscachas during their reproductive cycle were used. In the fetal PD, the immunoreactivity (ir) was mainly cytoplasmic. In immature and prepubertal animals, AR-ir was cytoplasmic (ARc-ir) and nuclear (ARn-ir) in medial region. In adult animals, ARn-ir cells were numerous at caudal end. AR regionalization varied between the PD zones in relation to growth. In immature animals, the ARn-ir increased whereas the cytoplasmic expression decreased in relation to the fetal glands. The percentage of ARc-ir cells increased in prepubertal animals whereas the nuclear AR expression was predominant in adult viscachas. The AR expression changed in adults, showing minimum percentage in the gonadal regression period. The variation of nuclear AR expression was directly related with testosterone concentration. These results demonstrated variations in the immunostaining pattern, regionalization, and number of AR-ir cells throughout development, growth, and reproductive cycle, suggesting the involvement of AR in the regulation of the pituitary activity of male viscacha.

Effects of castration on androgen receptors and gonadotropins in the pituitary of adult male viscachas

The aims of the present study were to determine whether castration results in quantitative immunohistochemical changes in androgen receptors (AR), LH-immunoreactive (IR) cells and FSH-IR cells, and to analyse the colocalisation of AR and gonadotropins in the pituitary pars distalis (PD) of viscachas. Pituitaries were processed for light and electron microscopy. AR-IR, LH-IR and FSH-IR cells were detected by immunohistochemistry. In morphometric studies, the percentage of AR-IR, LH-IR, FSH-IR, LH-IR/AR-IR and FSH-IR/AR-IR cells was determined. In intact viscachas, AR were distributed throughout the PD; they were numerous at the caudal end, with intense immunostaining. LH-IR cells and FSH-IR cells were found mainly in the ventral region and at the rostral end of the PD. Approximately 45%-66% of LH-IR cells and 49%-57% of FSH-IR cells expressed AR in the different zones of the PD. In castrated viscachas, there was a significant decrease in the percentage of AR-IR, LH-IR, FSH-IR, and FSH-IR/AR-IR cells. Some pituitary cells from castrated viscachas also exhibited ultrastructural changes. These results provide morphological evidence that gonadal androgens are directly related to the immunolabelling of AR, LH and FSH. Moreover, the colocalisation of AR and FSH is most affected by castration, suggesting the existence of a subpopulation of gonadotrophs with different regulatory mechanisms for hormonal synthesis, storage and secretion.

Effect of fasting in the digestive system: histological study of the small intestine in house sparrows.

In birds and mammals the metabolic response to fasting has been studied and can be characterized by three consecutive phases reflecting metabolic and physiological adjustments. An effective way to minimize energy expenditure during food scarcity is to decrease the mass of the organs. As the digestive system is metabolically expensive to maintain, the small intestine and the liver are the most affected organs. We evaluated the effects of phase III starvation on the mass of the different organs and histological parameters on house sparrows, a small non-migrant bird. In a short period of time (34 h) we observed a larger reduction in the digestive organ mass when compared to the mass of the body and non-alimentary tissues. Furthermore, the intestinal mass was proportionally more reduced than its length and nominal surface area. A reduction on the intestinal mucosal layer also resulted in a shortening of villus (length and thickness) and crypt depth. Moreover, the morphology of the enterocytes changed from cylindrical to cubical, suggesting that the surface exposed to the lumen was conserved. This may indicate an adaptive response to the moment of refeeding. The nominal surface area/body mass remained constant in both groups and several histological parameters were reduced, suggesting that starving induces the atrophy of the small intestine. However, the goblet cells were conserved after fasting indicating a protective tendency.