Role of pituitary POMC-peptides and insulin-like growth factor II in the developmental biology of the adrenal gland

During fetal life, it is critical that there is coordinate regulation of the growth, zonation and differentiation of the fetal adrenal cortex to ensure that cells in key tissues and organs are exposed in a programmed temporal sequence to the actions of glucocorticoids. Glucocorticoids are essential for maturation of key target organs before birth, including the lung, brain, liver, gut, kidney and adrenal, and the prepartum increase in glucocorticoid synthesis and secretion by the fetal adrenal gland is critical for the successful transition to postnatal life. It is also evident that premature or abnormal exposure of embryonic or fetal tissues to glucocorticoids during critical windows of development can irreversibly alter the programmed development of organ systems. Premature or abnormal exposure of the fetus to excess glucocorticoids may occur either as a consequence of endogenous stimulation of the fetal hypothalamo-pituitary-adrenal axis (HPAA) or as a consequence of exposure to exogenous glucocorticoids in a therapeutic context. Administration of synthetic glucocorticoids to women at risk of preterm labour, for example, is a routine clinical practice designed to improve respiratory function and neonatal outcome. It is clearly important to understand what endogenous factors regulate the growth and functional maturation of the adrenal cortex during development and the consequent likelihood of exposure of developing tissues to excess corticosteroids. To date, investigations have centred on the role of ACTH 1-39 in the stimulation of adrenal growth and steroidogenesis in long gestation species, such as the primate and sheep, where maturation and differentiation of organ systems occurs predominantly before birth. In this review, we will focus on the evidence that in addition to ACTH 1-39, other pro-opio-melanocortin (POMC) derived peptides, which are synthesized, processed and secreted by the fetal pituitary, play a role in the coordinate regulation of the specific phases of growth and functional development of the fetal adrenal gland in vivo. We will discuss our recent findings on the direct in vivo actions of N-POMC 1-77 and separately, insulin like growth factor II (IGF-II), as adrenal growth factors. These studies provide an understanding of the separate regulatory mechanisms which control activation of adrenal growth and stimulation of adrenal steroidogenesis in the late gestation fetus.     

Lipocortin-1 immunoreactivity in the human pituitary gland

We evaluated the distribution of lipocortin-1 immunoreactivity in 118 immature or mature human hypophyses using the peroxidase-antiperoxidase (PAP) technique with a polyclonal rabbit antiserum against lipocortin-1. Serial sections were evaluated for five pituitary hormones and S-100 protein immunoreactivity to compare their distributions with that of lipocortin-1. Scattered or moderate numbers of cells exhibited lipocortin-1 immunoreactivity in the pars distalis of 89 subjects ranging in age from 27 weeks' gestation to 83 years. Seven immature and seven aged specimens exhibited no immunostaining, while 15 specimens from older individuals exhibited only rare immunostaining. Immunostaining did not appear to co-localize selectively with any specific pituitary hormone, although the distribution of immunoreactivity did overlap that of some corticotrophs and was seen in elongated processes of S-100-containing folliculostellate cells. Lipocortin-1 was also found in epithelial cells lining colloid cysts of the residual pars intermedia in 115 of 118 pituitaries ranging in age from 23 weeks' gestation to 83 years. In many intermediate lobe cysts, lipocortin-1 exhibited a pattern of immunoreactivity that partially overlapped the distribution of S-100 protein immunostaining, although the pattern was not identical. Pre-absorption of anti-lipocortin-1 antiserum with lipocortin-1-coupled Sepharose-4B immunoreactivity resulted in loss of immunoreactivity in both lobes. No lipocortin-1 immunoreactivity was seen in the neurohypophysis.     

Stress-induced testicular hyposensitivity to gonadotropin in rats. Role of the pituitary gland

The time course of stress-induced testicular hyposensitivity to gonadotropins was studied in hypophysectomized or naloxone-treated rats exposed to various periods of immobilization. Blood was collected from a chronically indwelling intra-atrial catheter every hour for luteinizing hormone (LH) and testosterone (T) measurement. Eight hours of immobilization completely suppressed T secretion without significant effect on LH. Human chorionic gonadotropin (hCG, 5 IU/rat, i.m.) induced a marked increase in plasma T levels in normal control groups 3 h post-injection while in immobilized rats the response was completely abolished, even after only 30 min of stress. In hypophysectomized rats, as expected, plasma T levels were undetectable, but, contrary to results obtained in normal animals, hCG induced a similar increase of plasma T levels both in control and stressed rats. Immobilization stress failed to inhibit plasma T values in hypophysectomized rats pretreated for 4 days with human menopausal gonadotropin (hMG) + hCG, while it did so in similarly treated normal animals. Naloxone induced a rise of plasma LH and T levels in control rats, but did not antagonize the stress-induced fall of plasma T concentration. In all groups, steroid testicular content mimicked variations of plasma T values. In particular, in stressed animals the lack of accumulation of testicular 17-hydroxyprogesterone probably reflected a normal activity of 17-20 lyase. These results indicate that stress induces very rapidly a state of Leydig cell hyposensitivity to gonadotropins and a blockade of T biosynthesis. The causal relationship between the two effects is presently not clear but these events seem to be due to stress-induced release of an inhibitory factor of pituitary origin other that endorphin.     

Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of [125I]EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of [125I]EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of [125I]EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland.     

A "20K" form of growth hormone in the murine pituitary gland

Extracts of mouse and rat adenohypophyses have been analyzed for a low-molecular-weight variant of growth hormone (GH) known to occur in humans, the so-called "20K"-GH (mol wt = 20,000 vs 22,000 for traditional human GH). Pituitary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostained with an antiserum raised against murine GH. Reactive and unreactive bands in close vicinity of the major GH band were fingerprinted by a peptide-mapping technique that reveals only the tyrosine-containing peptides, but can be applied to fingerprint single protein bands within gels. We found a protein band 2000 mol wt smaller than the major murine GH in both mouse and rat pituitary glands whose fingerprint resembled that of major GH. The peptide-mapping results are consistent with the interpretation that the internal deletion of amino acid residues most likely is in the same region of the molecule as in the human 20K-GH. Unlike the human 20K-GH, however, the murine counterpart showed no cross-reactivity with an antiserum raised against 22K-GH in the test system used here.     

Glycosylated growth hormone: detection in murine pituitary gland and evidence of physiological fluctuations

We examined murine pituitary glands for glycosylated growth hormone using a lectin-binding immunoassay. Every gland tested exhibited significant concanavalin A-binding growth hormone immunoreactivity. The activity was lower in female rats than in males, and lower in lactating than in virgin females. The activity increased following ovariectomy, although total immunoreactive growth hormone was not altered. Administration of estradiol benzoate decreased the concentrations of both types of growth hormone. Progesterone administration, in contrast, did not change concanavalin A-binding growth hormone, but it lowered total growth hormone. Western blotting revealed a growth hormone immunoreactive band 4-5K heavier than the growth hormone monomer in both rat and mouse pituitary. These data suggest the existence in the murine pituitary of a glycosylated form of growth hormone that undergoes physiological fluctuations.     

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.     

A lectin-binding immunoassay indicates a possible glycosylated growth hormone in the human pituitary gland

By means of a lectin binding immunoassay we detected a glycosylated human growth hormone-immunoreactive substance in the human pituitary gland. The assay involves reacting the test substance with concanavalin A immobilized on a solid support followed by treatment with human growth hormone antibodies. Quantitation is achieved by reaction of the concanavalin A-glycoprotein-antibody complex with an 125I-labeled second antibody. Specificity of the assay for a glycosylated human growth hormone was indicated by inhibition of binding by N-acetyl-D-glucosamine. Furthermore, use of antiserum that had been absorbed with human growth hormone reduced the amount of radioactivity bound to the tubes. Western blotting of pituitary immunoprecipitates after gel electrophoresis in sodium dodecyl sulfate revealed human growth hormone-immunoreactive components that also reacted with concanavalin A.