Partial characterization of immunoreactive substance P in the rat pituitary gland

Two distinct carboxy-terminus-directed anti-substance P (SP) sera (R-1C and R-6G) were used to characterize immunoreactive SP (I-SP) in acetic acid extracts of anterior pituitary (AP) and posterior pituitary (PP) glands of adult male rats. The tissue concentrations of I-SP measured by R-1C and R-6G were comparable. The contents of I-SP were 600–1150 pg/AP and 25–52 pg/PP. I-luteinizing hormone releasing hormone and I-somatostatin (I-SOM) were undetectable in AP extracts, but PP extracts contained the equivalents of 325–785 pg I-SOM/gland. Serial dilutions of AP and PP extracts produced displacement curves with both SP antisera that were parallel to the respective synthetic SP standard and hypothalamic extract displacement curves. Gel filtrations of AP and PP extracts on a Sephadex G-25 column produced I-SP peaks eluting in the same fractions as synthetic SP and hypothalamic I-SP. However, the AP I-SP profile also revealed a side peak migrating between the void volume and the major I-SP peak. Neither immunoreactive species in the AP extract were eliminated when eluted with 6.0 M guanidine HCl, a strong denaturing agent. $\text{In vitro}$ incubation of paired anterior hemipituitaries for 30 min in the presence of a 56 mM K⁺ concentration resulted in a significant (p<.0001), 25-fold increase in the release of I-SP into the incubation medium above the mean control value. Radiofrequency lesions placed in the median eminence-arcuate region of male rats caused a significant (p<.001) reduction of I-SP in both the AP and PP. These reductions were inversely related to the plasma prolactin values. The elevation in plasma prolactin was taken as an index of completeness of lesions. We conclude that: 1) the rat pituitary contains I-SP as assessed by its immunologic and chromatographic behavior, 2) K⁺ depolarization is a potent stimulator of the release of AP I-SP $\text{in vitro}$, 3) the ME-arcuate region is important for the maintenance of pituitary I-SP levels in the rat.     

Expression of androgen receptor and estrogen receptor-alpha in the developing pituitary gland of male sheep lamb

To explore the expression of androgen receptor (AR) and estrogen receptor alpha (ERα) in the developing pituitary of male lamb, we detected AR and ERα expression in the anterior pituitary of lambs aged 2-7 months old by immunohistochemistry. The results showed that both AR immunoreactivity (AR-ir) and ERα immunoreactivity (ERα-ir) were localized in the nuclei of anterior pituitary cell. The percentage of the anterior pituitary cells expressing ERα fluctuated from 8.79±0.02% to 11.80±0.04% during the examined stages, but fell significantly to the lowest level at 6 months. While the proportion of AR-ir showed significant changes, it was in 11.52±1.26% at 2 months, it firstly increased to 19.86±1.03% at 3 months, and then significantly decreased to 8.18±1.17% at 6 months (P<0.05). The expression of both AR-ir and ERα-ir were the lowest level at 6 months old. By staining for PCNA, we observed that the changes in expression of AR and ERα at different lamb ages did not result from cell proliferation of anterior pituitary cells. These results indicate that both AR and ERα are important in regulation of secretary function of anterior pituitary in sheep lamb, although the related mechanism needs to be elucidated further.     

Immunohistochemical distribution of simple-epithelial-type keratins and other intermediate filament proteins in the developing human pituitary gland

Summary An immunohistochemical study of the production of the intermediate filaments [vimentin, cytokeratin, and glial filament acidic protein (GFAP)] during development of the pituitary gland was made by use of fetal and adult human pituitary tissue. Among these intermediate filament proteins in the anterior and intermediate lobes of the pituitary, cytokeratin is the first to appear, followed by GFAP and vimentin. However, only cytokeratin is seen during the period of morphogenesis of the pituitary gland, with the type-II subfamily cytokeratin 8 being the earliest to appear. Among the simple-epithelial-type cytokeratins, cytokeratins 8 and 19 were observed within the pituitary primordium during morphogenesis. Cells immunoreactive for cytokeratins 8 and 19 showed a heterogeneous three-dimensional distribution pattern in Rathke's pouch. Both cytokeratins 8 and 19 tended to be strongly positive at sites in the pituitary primordium where cells had become more loosely arranged (i.e., areas far from the diencephalon) but were only weakly positive in areas in which the epithelial cells were densely packed (i.e., areas closely associated with the diencephalon). It is concluded that, during the period of morphogenesis, Rathke's pouch has the intermediate filaments characteristic of simple epithelium and shows different immunoreactivity for simple-epithelial-type cytokeratins from place to place according to the extent of cellular differentiation.     

Cre-mediated recombination in the pituitary gland

Organ-specific expression of a cre recombinase transgene allows for the analysis of gene function in a particular tissue or cell type. Using a 4.6 kb promoter from the mouse glycoprotein hormone alpha-subunit (alphaGSU or Cga) gene, we have generated and characterized a line of transgenic mice that express cre recombinase in the anterior and intermediate lobes of the pituitary gland. Utilizing a cre-responsive reporter transgene, alphaGSU-cre transgene expression was detected in the pituitary primordium and in all five cell types of the adult anterior pituitary. alphaGSU-cre transgene activity was also detected in the cardiac and skeletal muscle. Little or no activity was evident in the gonads, adrenal glands, brain, ventromedial hypothalamus, or kidneys. The alphaGSU-cre transgenic mice characterized here will be a valuable tool for examining gene function in the pituitary gland.     

Expression of androgen receptor and its co-localization with estrogen receptor-alpha in the developing pituitary gland of sheep fetus

No information is known concerning the expression of androgen receptor (AR) and its co-localization with estrogen receptor alpha (ERα) in the developing pituitary of sheep fetus. In the present study, we detected AR expression and its co-localization with ERα in the anterior pituitary of sheep fetus from day 60 of gestation to the postnatal by dual immunochemistry. The results showed that both AR immunoreactivity (AR-ir) and ERα immunoreactivity (ERα-ir) were predominantly localized in the nuclei of LH positive gonadotropes. The cell counting results showed that the percentage of the anterior pituitary cells expressing AR fluctuated from 13.51 ± 0.92 to 17.05 ± 1.83% during the examined stages, but there were no significant differences between sexes and among ages examined (P > 0.05). However, the proportion of AR-ir cells containing LH markedly increased from day 60 of gestation to the neonatal (P < 0.05). The percentage of AR-ir cells expressing ERα-ir significantly increased from day 60 of gestation to the neonatal, respectively (P < 0.05), but no significant differences were seen between genders at each stage examined. These results indicate that both AR and ERα are mainly expressed in the gonadotropes of anterior pituitary gland of sheep fetuses, whereas the functions and interaction of AR and ERα expressions in the developing pituitary gland are required to be elucidated further. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hypothalamus and cytodifferentiation of the foetal pituitary gland

Summary To investigate whether the hypothalamus is involved in the cytodifferentiation of the anterior pituitary gland, rat foetuses were encephalectomized in utero on day 16 of pregnancy.Pituitary sections from encephalectomized and normal littermate foetuses were studied on day 21 with the immunofluorescence technique using antibodies and -MSH, anti -MSH, anti -(17–39) ACTH and anti -(1–24) ACTH. On day 16, only the anti -MSH revealed a few cells in the pars distalis but not in the pars intermedia. On the other hand, on day 21, the pituitary cells reacting with antibodies anti -MSH, anti -MSH and anti -(17–39) ACTH were as numerous in the encephalectomized foetuses as in the normal littermate foetuses. The cells revealed with the antibody anti -(1–24) ACTH were less numerous and less fluorescent in the pars distalis and intermedia of the hypophysis of the encephalectomized foetuses.On day 21, the adrenals of the encephalectomized foetuses were atrophied in comparison with those of the normal littermate foetuses but they were larger than on day 16.These data suggest that the cytodifferentiation of the corticotroph and melanotroph cells of the hypophysis occurs without the influence of the hypothalamus which is necessary for the normal release of ACTH.