Unimpaired postreceptor regulation of luteinizing hormone secretion by gonadotropin-releasing hormone and estrogen in aged rat anterior pituitary cells

Delayed, attenuated, or absence of the proestrous LH surge occurs in aging rats. To assess how aging affects the positive feedback action of 17 beta-estradiol (E2) on the pituitary, we determined the responsiveness of rat pituitary cells to GnRH and the secretagogues affecting intracellular signal transduction mechanisms in the presence or absence of E2. We also correlated the LH response to pituitary LH content. Anterior pituitaries excised from ovariectomized Sprague-Dawley rats, either young (3-4 months) or old (19-20 months), were enzymatically dispersed and then pretreated with or without E2 (0.6 nM) for 48 h, followed by incubation for 3 h with or without various secretagogues. The secretagogues included GnRH (1 and 10 nM), veratridine (increases Ca2+ influx; 5 and 10 microM), and phorbol 12-myristate 13-acetate (a protein kinase-C activator; 10 and 100 nM). LH in media and cells were measured by RIA and expressed on the basis of cellular DNA. GnRH, veratridine, and phorbol 12-myristate 13-acetate at all doses stimulated (P < 0.01) LH release in cells from both young and old rats. E2 stimulated (P < 0.05 to P < 0.01) all secretagogue-induced LH release in cells from both young and old rats, but only basal LH release (P < 0.05) in cells from young rats. The magnitude of both basal and secretagogue-induced LH release in either the presence or absence of E2 was smaller (P < 0.01) in cells from old than in those from young rats. The initial cellular LH was lower (P < 0.01) in cells from old than in those from young rats. The LH-releasing ability (expressed as a percentage of total cellular LH) of cells from old rats was identical (P > 0.05) to that of cells from young rats under all conditions studied. These results suggest that the reduced magnitude of LH release by cells from old rats may be attributed to reduced cellular LH, rather than to impaired estrogen feedback or impaired signal transduction mechanisms. It remains to be determined whether LH biosynthesis per cell and/or the number of gonadotropes decrease with age.     

Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland

CART peptide specific polyclonal antisera were raised in rabbits. The antisera were raised to CART peptide fragments that span most of the predicted CART protein. The specificity of each antisera was demonstrated by blockade of immunostaining by the immunizing peptide but not by the other CART peptide fragments. In the hypothalamus and pituitary of colchicine and noncolchicine treated rats, immunostaining was observed in cell bodies, fibers and varicosities. Clusters of cells were also stained in the adrenal medulla. It is noteworthy that cellular immunostaining was only found in areas previously shown to express CART mRNA. These findings indicate the presence of CART peptide(s) in the hypothalamus, pituitary, and adrenal gland. Furthermore, we also present evidence for the possible processing of the CART pro-peptide into smaller peptide fragments. These neuroanatomical findings suggest a role of CART peptides in hypothalamic, pituitary and adrenal function.     

Leptin receptors in estrogen receptor-containing neurons of the female rat hypothalamus

This study was undertaken to reveal whether integration of the peripheral signals, leptin and estradiol, that convey information on the metabolic state and gonadal function, respectively, might occur in the same hypothalamic neuronal perikarya. Light and electron microscopic immunolabeling for leptin receptors (LRs) and estrogen receptors (ERs) was carried out on hypothalamic sections of female rats. In the medial preoptic area, periventricular regions, including the parvicellular paraventricular nucleus, the arcuate nucleus and the ventromedial hypothalamic nucleus, all of the cells that expressed immunoreactivity for ERs were also immunopositive for LR. On the other hand, only a subpopulation of LR-containing cells was found to express ERs. The extensive colocalization of receptors for leptin and estrogen in neuronal perikarya of all parts of the hypothalamus suggests a closely coupled interaction between these peripheral signals in the regulation of a variety of behavioral and neuroendocrine mechanisms.     

Regulation of c-fos expression by EGF and GnRH in specific anterior pituitary cells from proestrous female rats

C-fos is an early expression oncogene that can be stimulated by a variety of regulators. It is expressed by subsets of all pituitary cells, with increased expression seen in proestrous rats. However, in freshly dispersed pituitary cells studied during different stages of the cycle, there is limited expression of fos by luteinizing hormone (LH) cells and little basal expression by cells with follicle-stimulating hormone (FSH) antigens. Proestrus is a time during which pituitary gonadotropes express peak levels of receptors for gonadotropin-releasing hormone (GnRH) and epidermal growth factor (EGF). We hypothesized that if GnRH or EGF stimulated fos activity in gonadotropes they would be most effective during the peak expression of their receptors. Anterior pituitaries were removed, cut into small pieces, and stimulated for 30 min. Total RNA was then collected and analyzed by Northern analysis. Both EGF and GnRH caused an increase in c-fos mRNA levels in the anterior pituitary gland compared with unstimulated pituitary glands assayed immediately after removal from the pituitary. However, the stimulatory effects were no greater than those seen with medium alone. This suggested that fos expression could be stimulated by local factors either in the pituitary or the medium itself. The second phase of the study focused on pituitary cells plated for 1 hr and then stimulated with EGF and GnRH for 15 min. Dual immunocytochemistry was done to learn which cell types expressed the fos proteins. After 15 min, EGF and GnRH both increased the percentages of fos-bearing cells above levels seen in medium alone. EGF stimulated fos proteins in subsets of FSH, adrenocorticotropin (ACTH), and growth hormone (GH) cells. GnRH increased fos proteins in subsets of ACTH and GH cells. These results suggest that EGF and GnRH may regulate fos expression, but not necessarily in gonadotropes. They also highlight the need for carefully timed experiments because endogenous factors in the pituitary itself may stimulate immediate early gene expression. (J Histochem Cytochem 46:935-943, 1998)     

In vitro effects of CINC/gro, a member of the interleukin-8 family, on hormone secretion by rat anterior pituitary cells

We investigated the effects of CINC/gro on hormone secretion using normal rat anterior pituitary cells. In normal anterior pituitary cells, 10-100 ng/ml of CINC/gro significantly increased the secretion of PRL within 3 h of incubation, and two-fold enhancement of PRL secretion was induced by 100 ng/ml of CINC/gro within 24-h incubation, while the response of GH and ACTH secretions to CINC/gro was weak. On the other hand, CINC/gro suppressed basal LH and FSH secretions in a concentration-dependent manner. The percent inhibition of basal secretion by CINC/gro (50 ng/ml) within 24-h incubation was 70% for LH and 43% for FSH. Twenty-four-hour incubation with 100 ng/ml of IAP completely blocked the CINC/gro-stimulated PRL and GH secretions and CINC/gro's suppression of both basal LH and FSH secretions. These data demonstrate a new biological activity for CINC/gro and provide evidence for immune system regulation of anterior pituitary hormone secretion.     

Calcitonin gene-related peptide- and substance P-like-immunoreactive innervation of the anterior pituitary in the rat

In our previous studies substantial amounts of substance P- and calcitonin gene-related peptide-like-immunoreactive nerve fibers have been identified in the anterior pituitary of the monkey and the dog. They were found to be in close proximity to the gland cells, even making synaptic contacts with some types of the gland cells. The present study investigated in detail the calcitonin gene-related peptide- and substance P-like immunoreactivities of the anterior pituitary in the rat. Though the immunoreactive fibers were not as abundant as in the anterior pituitary of the monkey and the dog, they still appeared in notable amounts. The calcitonin gene-related peptide- and substance P-like-immunoreactive nerve fibers occurred mostly as thin, tortuous, and densely varicose fibers, weaving among the gland cells. They are widely distributed, more in the central part of the gland. Double-immunostaining proved nearly complete co-localization of these two peptides in the nerve fibers. It is hypothesized that the anterior pituitary can be regulated by direct neural factors as well as humoral factors.     

Cellular distribution and gene regulation of estrogen receptors alpha and beta in the rat pituitary gland

The pituitary gland is a heterogeneous tissue comprised of several hormone secreting and supporting cells, most of which are targeted by estrogens. Estrogen-induced changes in the pituitary are presumably mediated via the classical estrogen receptor, ER alpha. However, a novel receptor, ER beta, and pituitary-specific truncated estrogen receptor products (TERPs) were recently identified. The objectives of this study were to examine the distribution of these receptors in the rat pituitary and compare their regulation by estradiol in Sprague-Dawley and the estrogen-sensitive Fischer 344 rats. Pituitary cryosections were subjected to immunocytochemistry for specific cell types, followed by in situ hybridization for ER alpha or ER beta. ER alpha was expressed by approximately 45% of the lactotrophs and melanotrophs, 35% of the corticotrophs and folliculo-stellate cells, and 25% of the gonadotrophs. The expression of ER beta showed a similar pattern but was generally lower than ER alpha. In two cell types, melanotrophs and gonadotrophs, ER beta expression was significantly lower than ER alpha. In the second experiment, pituitary sections were immunostained for ER alpha, followed by in situ hybridization for ER beta. Only a minute population (6-10%) of either anterior or intermediate lobe cells coexpressed ER alpha and ER beta. In the next experiment, Fischer 344 and Sprague-Dawley rats were injected with oil or estradiol for 24 h. Total RNA from dissected anterior and posterior (neurointermediate) pituitaries was subjected to RT-PCR for ER alpha, ER beta, or TERPs. Interestingly, ER alpha and ER beta were unchanged by estradiol in either lobe of the pituitary. In contrast, estradiol increased pituitary TERP messenger RNA levels 4- to 7-fold. A 20-kDa TERP protein was detected by Western blots in the pituitary but not the uterus. There were no differences in the estradiol-induced expression of any of the receptors between the two strains of rats. We conclude that: 1) ER beta is expressed in all anterior and intermediate lobe cell types examined, albeit at a lower level than ER alpha; 2) no more than 10% of pituitary cells coexpress ER alpha and ER beta; and 3) estradiol markedly increases TERP messenger RNA levels but does not alter the expression of ER alpha or ER beta. We propose that estrogen receptor heterogeneity contributes to the diversity of pituitary cell responsiveness to estrogens.     

An ecto-ATPase and an ecto-ATP diphosphohydrolase are expressed in rat brain

Extracellular nucleotides acting as signaling molecules are inactivated by hydrolysis catalyzed by ecto-nucleotidases. ATP is sequentially degraded via ADP and AMP to adenosine. Enzymes that can be involved in the extracellular hydrolysis chain are ecto-ATP diphosphohydrolase (ecto-apyrase), ecto-ATPase, ecto-ADPase and 5'-nucleotidase. Mammalian ecto-ATP diphosphohydrolase is a member of a family of apyrases sharing four "apyrase conserved regions" that presumably participate in the formation of the catalytic site. We report the presence of ecto-ATP diphosphohydrolase in rat brain and the primary structure of a new mammalian member of the apyrase family. Expression in CHO cells shows that it represents an ecto-ATPase. As revealed by Northern analysis of rat tissues, the ecto-ATPase is co-expressed with ecto-ATP diphosphohydrolase in heart, kidney, spleen, thymus, lung, skeletal muscle and brain. Signals for both ecto-nucleotidases are very weak in liver. mRNAs for both proteins are present in PC12 cells, suggesting that the two nucleotidases may be co-expressed in the same neural cell. Using computer-aided sequence analysis, primary structure and membrane topography are compared with those of other members of the apyrase family.     

Identification of genes expressed in the rat prostate that are modulated differently by castration and Finasteride treatment

In mammals, testosterone and 5alpha-dihydrotestosterone (DHT) are the principal male hormones (androgens). Testosterone is the most abundant circulating androgen, and is converted in specific tissues to DHT by the 5alpha-reductase enzymes. Although each of these androgens binds to the same receptor protein (androgen receptor, AR), each exerts biologically distinct effects. Theories to explain the specific effects of testosterone and DHT have centered on kinetic differences of binding of androgens to the receptor or differences in the metabolic fates of the two hormones. In the current experiments, differential display PCR (ddPCR) was used to identify genes regulated differently by testosterone and DHT. Adult male rats were treated as follows: castrated, treated with Finasteride (an inhibitor of 5alpha-reductase) or left intact for ten days. RNA was prepared from the dissected prostates of these animals and used for ddPCR. Genes exhibiting four distinct patterns of regulation were observed among the mRNAs. Class 1 genes showed equivalent expression in intact and Finasteride-treated animals, but were absent in castrated animals (mRNAs D1, D2, D6, D10). Class 2 genes showed higher expression in intact animals, intermediate levels following Finasteride treatment, but were absent in castrated animals (mRNA D8). Two classes of gene were particularly intriguing: class 3 showed gene expression only in the intact animal (mRNA D7, D9) and class 4 showed increased gene expression following Finasteride treatment (mRNA D3). While the patterns observed for some of these genes (e.g. D8) suggest that the different biological effects of testosterone and DHT may be due to the lower affinity of the AR for testosterone and limiting tissue concentrations of androgen, our results also suggest that some genes expressed in the rat prostate may be regulated in fundamentally different ways in response to testosterone and DHT.     

Intercellular communication within the rat anterior pituitary gland: VI. Development of gap junctions between folliculo-stellate cells under the influence of ovariectomy and sex steroids in the female rat

BACKGROUND: Farquhar (1957) initially described the folliculo-stellate cells in the rat anterior pituitary gland and found them to be located in groups around follicles throughout the anterior lobe. Soji and his co-workers have published a series of reports concerning cell-to-cell communication in the male rat hypophysis involving folliculo-stellate cells as mediated through a gap junctional network and recorded a reduction in junctional number following castration that was reversed by the administration of testosterone (Soji and Herbert, 1990, Anat. Rec., 226:342-346; Soji et al., 1990, Anat. Rec., 226:337-341). METHODS: Animals were ovariectomized at 10 days of age and separated into three groups: (1) intact control, (2) ovariectomized controls, (3) ovariectomized and given either estradiol, testosterone, or progesterone. On days 10, 20, 30, 40, and 45, the pituitary gland from animals in each group was removed and processed for ultrastructural examination. RESULTS: Gap junctions in the intact control female rats initially appeared between adjacent folliculo-stellate cells on day 20. Their numbers linearly increased until the animals reached the age of 45 days. In contrast, there was a suppression in the number of gap junctions present in the ovariectomized groups and a marked enhancement of the junctions in each of the three steroid-treated groups. CONCLUSIONS: These findings support the observations made in the male rat in which it was found that the development of gap junctions in the anterior pituitary gland of the rat is dependent in part on the presence of sex steroid hormones.     

Connexins and E-cadherin are differentially expressed during trophoblast invasion and placenta differentiation in the rat

We have characterized the spatial and temporal expression pattern of six different connexin genes and E-cadherin during trophectoderm development in the rat. During the initial phase of trophoblast invasion at 6 days postcoitum (dpc), the trophoblast expressed E-cadherin but no connexin expression could be observed. With progressing invasion of the polar trophoblast into the maternal decidua, from 7 dpc onwards E-cadherin expression in the ectoplacental cone cells was lost and was now restricted to the extraembryonic ectoderm. In the ectoplacental cone and extraembryonic ectoderm instead connexin31 mRNA and protein could be found. This pattern was maintained up to day 10 postcoitum. The start of labyrinthine trophoblast differentiation from day 11 postcoitum onwards was characterized by persisting expression of E-cadherin in the extraembryonic ectoderm and its derivative, the chorionic plate. In addition to E-cadherin, from 10 dpc onwards, connexin26 started to be expressed in the chorionic plate, and both molecules remained coexpressed in the labyrinthine trophoblast of the mature placenta. During this differentiation process connexin31 remained expressed mainly in the proliferating spongiotrophoblast. From day 14 postcoitum onwards, the expression of connexin31 in the spongiotrophoblastic cells decreased, and in parallel they started to express connexin43. The trophoblastic giant cells, first characterized by connexin31, lost all of the investigated connexins during midgestation on day 12 postcoitum but started to express connexin43 from day 18 postcoitum onwards. Our studies suggest that loss of E-cadherin and induction of connexin31 expression is correlated with the proliferative and invasive stages of the ectoplacental cone, whereas appearance of connexin26, E-cadherin and connexin43 reflects the switch to the differentiated phenotypes of the mature placenta.     

Massive liver enlargement accompanied by decreased drug metabolism. Effect of anterior pituitary extract on hepatic ultrastructure, zoxazolamine paralysis, and metabolism in the rat

The relationship between liver enlargement and drug metabolism was investigated in female rats. Hepatomegaly (e.g., 31% increase in liver weight in a 17-day experiment) was induced by injection of lyophylized anterior pituitary (LAP) extract. The liver enlargement seemed to be due to an increase in the number and the size (enhanced water content and PAS-positive material) of hepatocytes. Electron microscopic examination of the liver revealed slight proliferation of the smooth endoplasmic reticulum and pronounced fragmentation and dilation of the rough endoplasmic reticulum. Zoxazolamine paralysis time was significantly prolonged (+55% and +102%) after 4 and 17 days, respectively, of treatment with LAP. Metabolism of zoxazolamine by the 9000 g supernatant fraction of the liver of rats given LAP for 17 days was reduced by 73%. Thus, the marked hepatomegaly induced by LAP was associated with a prolonged action of the drug which may result from a decrease in hepatic drug metabolism.     

Transforming growth factor-beta receptor types I and II are expressed in renal tubules and are increased after chronic unilateral ureteral obstruction

Transforming growth factor-beta (TGF-beta) is a profibrotic cytokine which has been implicated in the renal fibrosis which follows unilateral ureteral obstruction (UUO) in the rat. TGF-beta receptor type I (TGF-RI) and TGF-beta receptor type II (TGF-RII) are part of the complex which mediates the response to TGF-beta. We sought to determine if TGF-RI and TGF-RII are found in the kidney, and if their expression is changed as a result of UUO. Polymerase chain reaction (PCR) was used to determine expression of mRNA for TGF-RI and TGF-RII in the kidney. Immunoperoxidase was used to localize and quantify the expression of these receptors at 3, 7, 14, 21 and 28 days after UUO, and in sham-operated animals. Expression of mRNA for TGF-RI and TGF-RII was demonstrated in sham operated, obstructed and contralateral unobstructed kidneys using PCR. Using immunoperoxidase, a uniform distribution of TGF-RI and TGF-RII was found in cortical tubules of sham operated kidneys, whereas medullary tubules showed a patchy TGF-RI distribution and no TGF-RII staining. After UUO, an increased tubular expression of TGF-RI and TGF-RII was noted in both obstructed and contralateral kidneys compared to sham operated kidneys. No staining for either TGF-RI or TGF-RII was noted in glomeruli, vasculature or interstitial cells. TGF-beta receptors I and II were found exclusively in renal tubules and were shown to increase in both the obstructed and contralateral kidneys relative to sham operated animals. Upregulation of TGF-beta receptors in both kidneys suggests that TGF-beta may contribute to the fibrotic response in the obstructed kidney and the hypertrophic response of the contralateral kidney.