ABSTRACT

There is evidence that prolactin (PRL) influences gastrointestinal function. However, the sites at which prolactin exerts these effects are not known. A monoclonal antibody was therefore generated against the rabbit mammary gland prolactin receptor (MAb 218) and used to study the distribution of the prolactin receptor in the rabbit gastrointestinal tract (GIT) by immunohistochemistry. MAb 218 is an IgG 1 κprecipitating antibody which precipitates major affinity cross-linked mammary gland prolactin receptor subunits of molecular masses 45 and 80 kDa. It has an affinity of 0·8 × 10⁹ mol/l for the prolactin receptor and does not react with GH or insulin receptors in precipitation assays. MAb 218 immunoreactivity was observed in classical prolactin target cells such as mammary gland epithelium, and this immunoreactivity was abolished by preincubation of MAb 218 with purified prolactin receptor but not by preincubation with purified GH receptor.

In the GIT, the most intense immunoreactivity was associated with the oesophageal epithelium, chief (zymogenic) cells of the fundic mucosa, pancreatic islets of Langerhans and surface epithelial cells of the duodenum and jejunum. Other specific elements of the GIT were immunoreactive at lower levels or were immunonegative. No immunoreactivity was observed in these locations with a control monoclonal antibody (MAb 50·8) of identical isotype to 218.

To support the immunohistochemical findings, rabbit gastric mucosal membranes were used to show the presence of lactogen-specific binding. Scatchard analysis of ¹²⁵I-labelled human GH binding to the gastric mucosal membranes with rat prolactin as displacing ligand yielded an affinity constant (K _a) of 1·0 ± 0·2 × 10⁹ mol/l with a capacity of 3·5 ± 0·4 fmol/mg protein. Affinity cross-linking and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the gastric receptor revealed lactogenic hormone-binding subunits of molecular masses 43, 68 and 83 kDa. The 68 kDa subunit was not seen in rabbit mammary gland or ovarian tissue, and may be unique to gastric mucosa.

In conclusion, we have demonstrated the presence of a high affinity lactogenic receptor in specific epithelial cell subpopulations of the GIT. This localization of the prolactin receptor in the GIT will assist in further functional assignment of prolactin to gastrointestinal physiology.

Journal of Endocrinology (1993) 139, 371–382

Androgenic status affects rat preadipocyte adipose conversion from two deep intra-abdominal (epididymal and perirenal) fat depots differently. The aim of this study was to establish whether these site-specific alterations of adipogenesis are related to altered expressions of the transcriptional factors regulating proliferation and differentiation of preadipocytes, c-myc and CCAAT/enhancer binding proteins (C/EBPs: C/EBPalpha and beta). The increased proliferation of epididymal and perirenal preadipocytes from castrated rats was not linked to variations in c-myc mRNA and protein levels. The expression of the early marker of adipogenesis, lipoprotein lipase (LPL), was decreased by androgenic deprivation in epididymal cells but remained insensitive to the androgenic status in perirenal preadipocytes. In contrast, LPL expression increased in subcutaneous preadipocytes from castrated rats, an effect which was partly corrected by testosterone treatment. Expression of C/EBPbeta was unaffected by androgenic status whatever the anatomical origin of the preadipocytes. In contrast, the mRNA and protein levels of C/EBPalpha were greatly decreased by androgenic deprivation in epididymal cells, an alteration which could not be corrected by in vivo testosterone administration. Altogether these results demonstrated that in preadipocytes androgenic deprivation affects site-specifically the expression of LPL, an early marker of adipogenesis and of C/EBPalpha, a master regulator of adipogenesis. These observations contribute to an explanation of why castration induces defective adipose conversion in rat epididymal preadipocytes specifically.

ABSTRACT

Acromegaly is characterized by coarsening of facial features, acanthosis nigricans, hypertrichosis and oily skin. To determine the site through which GH exerts these effects, we have used immunohistochemistry to localize the GH receptor/binding protein (BP) in rat, rabbit and human skin. Three monoclonal antibodies (MAb 1, 43, 263) were immunoreactive in identical locations, whereas no immunoreactivity was evident when control monoclonal antibodies (MAb 50.8 and MAb 7 (rat)) were used. Skin from neonatal and adult animals was used to determine whether GH receptor/BP expression was developmentally regulated.

Immunoreactivity of the GH receptor/BP in the three species was consistently localized in the stratum basale and stratum spinosum. Intermittent staining was observed in the stratum granulosum. Scattered basal epidermal cells often displayed more intense immunoreactivity. This distribution was observed at all maturational stages examined. Intense GH receptor/BP immunoreactivity was observed in all histological layers of the lower one-third of hair follicles and in hair matrix cells of the dermal papillae. Immunoreactivity was also detected in the outer epithelial root sheath of the upper two-thirds of hair follicles, in sebaceous glands and in fibroblasts of the connective tissue sheath surrounding the follicle. GH receptor/BP immunoreactivity was also present in the secretory duct and myoepithelial cells of human eccrine sweat glands. Fibroblasts, Schwann cells of peripheral nerve fascicles, skeletal muscle cells and adipocytes of the dermis were also immunoreactive as were medial smooth muscle and endothelial cells of arteries.

These results provide evidence that GH acts locally on the epidermis and epidermal appendages concordant with our recent localization of GH receptor/BP to epithelial cell types of the gastrointestinal and reproductive systems.

Journal of Endocrinology (1990) 126, 467–472

ABSTRACT

Orchidectomy has been reported to decrease concentrations of thyrotrophin (TSH) in the circulation of male rats without affecting serum levels of thyroid hormones. To understand the mechanism underlying this observation, we have measured the effect of gonadal status on the in-vitro release of TSH-releasing hormone (TRH) by male rat hypothalamic fragments. Because hormone release rates can be affected by changes in the post-translational processing of the hormonal precursors, we have also studied the corresponding changes in the concentrations of TRH and TRH-Gly, a TRH precursor peptide in hypothalamus and pituitary, by radioimmunoassay.

We observed a significant decline in the in-vitro release of TRH from incubated hypothalami 1 week after castration, which was quantitatively reversed by testosterone replacement. Concentrations of TRH and TRH-Gly in the posterior pituitary, on the other hand, which derive from neurones of hypothalamic origin, increased significantly with castration and were returned to the normal range by testosterone replacement.

We conclude that the primary effect of testosterone is the stimulation of hypothalamic TRH release, resulting in the depletion of TRH and TRH precursors from TRH-containing neurones which project into the median eminence and posterior pituitary.

Journal of Endocrinology (1990) 125, 263–270

Abstract

Testicular macrophages are a relevant cell type for the regulation of Leydig cell steroidogenesis. The availability of liposome technology allows in vivo manipulation of macrophages in order to analyze their role in the regulation of the hypothalamic-pituitary-testicular axis. In this study, adult (70 days of age) and prepubertal (22 days of age) rats were injected intratesticularly with liposomes containing either dichloromethylene diphosphonate (C12MDP) to deplete testicular macrophages or muramyl tripeptide (MTP-PE) to activate them. Control rats were injected with the corresponding volumes of 0·9% NaCl. Animals were killed 10 days after treatment. Adult rats injected bilaterally or unilaterally with C12MDP liposomes showed increased serum LH and testosterone concentrations, as well as increased testosterone concentrations in the testicular interstitial fluid. In unilaterally injected rats, testosterone concentrations in the interstitial fluid were higher in the macrophage-containing testes than in the contralateral, macrophage-depleted testes. Adult rats treated bilaterally with MTP-PE liposomes showed increased numbers of testicular macrophages, whereas the number of Leydig cells was unchanged. Serum LH concentrations were decreased, but no changes were found in testosterone concentrations. Prepubertal rats treated bilaterally with C12MDP liposomes showed decreased numbers of Leydig cells. However, serum LH and testosterone concentrations were increased. Otherwise, prepubertal rats treated bilaterally with MTP-PE liposomes showed increased numbers of macrophages and Leydig cells, as well as increased serum testosterone concentrations. These data suggest that testicular macrophage-derived factors act at two different levels in the pituitary-testicular axis: first, at a central level by inhibiting LH secretion, and secondly, at a local level by stimulating Leydig cell steroidogenesis.

Journal of Endocrinology (1996) 150, 57–65

We observed disability of movement in 6-month-old transgenic mice expressing the fusion gene coding for the bovine GH (bGH) under the transcriptional control of phosphoenolpyruvate carboxykinase promoter (PEPCK-bGH). Histological study of the knee joint showed altered synovial and tibial articular cartilage tissues. In the cartilage the following observations were made: (i) generalized loss of the normal zonal structure and presence of clefts, and (ii) profound alterations in chondrocyte growth/differentiation processes consistent with hypertrophy. The synovial tissue showed a reduced number of adipocytes, and a significant thickening of synovial lining tissue and pannus. These findings indicate that transgenic mice suffer damage to diarthritic joints with osteoarthritic appearance. As changes in synovial membrane in osteoarthritis are almost indistinguishable from those seen in inflammatory arthritis, we determined the potential correlation with an immunological disorder. Serological determination of self-antibodies measured as a function of age and sex showed anti-nuclear, anti-single-stranded DNA, anti-double-stranded DNA and anti-70K antibodies, and an altered immunoglobulin typing. These results suggest that transgenic mice expressing bGH develop an arthritic process which is correlated with an immune disorder. The results also indicate that these mice are a suitable animal model to study the specific role of GH-driven processes in immune cells and arthritis.

We have demonstrated and localized human GH (hGH) gene expression in surgical specimens of normal human mammary gland and in proliferative disorders of the mammary gland of increasing severity using sensitive in situ RT-PCR methodology. hGH mRNA identical to pituitary hGH mRNA was first detected by RT-PCR of RNA derived from samples of normal human mammary gland. Cellular localization of hGH gene expression in the normal mammary gland exhibited restriction to luminal epithelial and myoepithelial cells of the ducts and to scattered stromal fibroblasts. We subsequently examined the expression of the hgh gene in three progressive proliferative disorders of the human mammary gland, i.e. A benign lesion (fibroadenoma), a pre-invasive stage (intraductal carcinoma) and an invasive ductal carcinoma. hGH mRNA was readily detected in the tumoral and non-tumoral epithelial components and also in cells of the reactive stroma including fibroblasts, myofibroblastic and myoepithelial cells, inflammatory infiltrate lymphocytes and endothelial cells in areas of neovascularization. In all three proliferative disorders examined, the intensity of the cellular labeling observed in both the epithelial and stromal compartments was always stronger compared with that in adjacent normal tissue. hGH protein was also present in significantly higher concentration in extracts derived from proliferative disorders of the mammary gland compared with extracts derived from normal mammary gland. We also examined hGH gene expression in axillary lymph nodes not containing and containing metastatic mammary carcinoma. hGH gene expression was evidenced in metastatic mammary carcinoma cells and in reactive stromal cells by both in situ hybridization and in situ RT-PCR. In contrast, in lymph nodes not containing metastatic mammary carcinoma, hGH mRNA was detected only by use of in situ RT-PCR. Thus, increased expression of the hGH gene in the epithelial component and the de novo stromal expression in proliferative disorders of the mammary gland are suggestive of a pivotal role for autocrine hGH in neoplastic progression of the mammary gland.

In mammals, a complex interaction between the immune and the reproductive systems has been described, in which testicular immune cells produce cytokines and growth factors which modulate gonad functions, while specific gonad cells influence the immune response in this organ. In this study we describe the presence of acidophilic granulocytes in the testis of the hermaphrodite teleost fish gilthead seabream (Sparus aurata L.) by using a specific monoclonal antibody. During the post-spawning stage of the testis, this cell type appears in the germinal compartment, accumulates interleukin (IL)-1beta and does not seem to be involved in the phagocytosis of degenerating cells. Moreover, in vitro, 11-ketotestosterone and 17beta-oestradiol, the principal fish sexual steroids, regulate the respiratory burst activity of acidophilic granulocytes obtained from the head-kidney (the bone marrow equivalent in fish) and the intracellular accumulation of IL-1beta by these cells. It is likely, therefore, that IL-1beta produced by testicular acidophilic granulocytes regulates important functions of the testis in fish.

This study was carried out to investigate the clinical and biochemical factors which might be of importance in predicting the outcome of patients with myxoedema coma. Eleven patients (ten female) aged 68.1+/-19.5 years attended our institution over a period of 18 years.Glasgow and APACHE II scores and serum free thyroxine and TSH were measured in all the patients on entry. Patients were selected at random to be treated with two different regimens of l-thyroxine.Four patients died with the mortality rate being 36.4%. The patients in coma at entry had significantly higher mortality rates than those with minor degrees of consciousness (75% vs 14.3% respectively, P=0.04). The surviving patients had significantly higher Glasgow scores than those who died (11.85+/-2.3 vs 5.25+/-2.2 respectively, P<0.001). Comparison of the mean values of APACHE II scores between the surviving group and those who died was significantly different (18.0+/-2.08 vs 31.5+/-2.08 respectively, P<0.0001).The degree of consciousness, the Glasgow score and the severity of the illness measured by APACHE II score on entry were the main factors that determined the post-treatment outcome of patients with myxoedema coma.

Hyperstimulation of ovarian function with human FSH (hFSH) attenuates the preovulatory surge of LH. These experiments aimed at investigating the mechanism of ovarian-mediated FSH suppression of the progesterone (P₄) receptor (PR)-dependent LH surge in the rat. Four-day cycling rats were injected with hFSH, oestradiol benzoate (EB) or vehicle during the dioestrous phase. On pro-oestrus, their pituitaries were studied for PR mRNA and protein expression. Additionally, pro-oestrous pituitaries were incubated in the presence of oestradiol-17β (E₂), and primed with P₄ and LH-releasing hormone (LHRH), with or without the antiprogestin RU486. After 1 h of incubation, pituitaries were either challenged or not challenged with LHRH. Measured basal and LHRH-stimulated LH secretions and LHRH self-priming were compared with those exhibited by incubated pituitaries on day 4 from ovariectomized (OVX) rats in metoestrus (day 2) injected with hFSH and/or EB on days 2 and 3. The results showed that: i) hFSH lowered the spontaneous LH surge without affecting basal LH and E₂ levels, gonadotroph PR-A/PR-B mRNA ratio or immunohistochemical protein expression; ii) incubated pro-oestrous pituitaries from hFSH-treated rats did not respond to P₄ or LHRH, and lacked E₂-augmenting and LHRH self-priming effects and iii) OVX reversed the inhibitory effects of hFSH on LH secretion. It is concluded that under the influence of hFSH, the ovaries produce a non-steroidal factor which suppresses all PR-dependent events of the LH surge elicited by E₂. The action of such a factor seemed to be due to a blockade of gonadotroph PR action rather than to an inhibition of PR expression.