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See related article, pp 238–244 In the decade since the publication of the Randomized Aldactone Evaluation Study (RALES),1 interest in the role of aldosterone and the mineralocorticoid receptor (MR) has increased exponentially. The comfortable notion that mineralocorticoid hypertension was just a matter of salt and water has been supplanted by a recognition that multiple tissues and systems are impacted.2,3 It is clear that the adverse cardiovascular consequences of mineralocorticoid-induced hypertension are far in excess of what might be expected for the magnitude and duration of the blood pressure rise.4 These observations have focused research on the consequences of MR activation in nonepithelial tissues.5 This represents somewhat of a renaissance as the consequences for the cardiovascular system of exposure to mineralocorticoids was first noted by Hans Seyle in 1946 and rediscovered by Brilla and Weber with further characterization by Young et al.6 These investigators established a robust rodent model of mineralocorticoid-induced hypertension, cardiac hypertrophy, and cardiac fibrosis resulting from mineralocorticoid/salt administration over a period of weeks. The cardiac fibrosis is preceded by vascular inflammation and is independent of the hypertension, cardiac hypertrophy, angiotensin II, and potassium status. It is blocked by coadministration of MR antagonists (spironolactone or eplerenone) and, more importantly, once established, as in RALES, …

Interest in inhibin as a marker of ovarian malignancy was stimulated by the description of elevated immunoreactive inhibin levels in the sera of patients with granulosa cell tumours. Several groups have confirmed the value of serum inhibin in the diagnosis and follow-up of patients with this uncommon malignancy. Immunoreactive inhibin levels are also frequently elevated in patients with mucinous cystadenocarcinoma and less frequently in other forms of ovarian tumour. Assay of sera using the specific dimeric inhibin assays has shown that ovarian tumours are able to secrete dimeric inhibin particularly inhibin B. The less specific α-subunit directed assays, however, most frequently show elevated concentrations. Used in combination with CA125 as a dual tumour marker, it appears in principle that inhibin can be a useful diagnostic agent. Immunohistochemistry for the inhibin subunits has been reported with increasing frequency as a helpful method to assess suspected ovarian stromal cell tumours. Its diagnostic accuracy for other types of ovarian adenocarcinoma appears less reliable. Expression of the inhibin subunit mRNAs has been demonstrated in a variety of ovarian malignancies. The observation that inhibin levels are elevated in ovarian cancer has stimulated studies of their relevance to the molecular pathogenesis of these malignancies. Findings to date have been largely negative with no evidence for activating mutations of the FSH receptor or of the post-receptor signalling pathway proteins.

The molecular mechanisms by which corticosteroids affect fluid and electrolyte balance have yet to be fully elucidated. The apical amiloride-sensitive electrogenic epithelial sodium channel (ENaC) has been shown to have a central role in corticosteroid-mediated sodium transport in the distal colon. The acute response of the alpha-, beta- and gammaENaC subunit genes to a single parenteral dose of aldosterone or dexamethasone was examined in the rat distal colon in vivo. The response of the Nedd4 gene, whose product is involved in channel turnover, was also examined. Whilst the alphaENaC and Nedd4 genes showed no significant response to either steroid, both the beta- and gammaENaC mRNA levels were increased acutely by both aldosterone and dexamethasone. The gammaENaC mRNA appears to have a very short half-life. Use of the highly selective glucocorticoid receptor agonist RU28362 confirmed that the response was mediated by both the mineralocorticoid and glucocorticoid receptors.

A case is presented of secondary infertility due to the concurrence of sperm autoimmunity and hyperprolactinemia. Treatment of his hyperprolactinemia with bromocriptine did not improve his fertility; however, concurrent treatment of both conditions resulted in improved semen quality, conception, and the birth of a healthy baby girl.

The small bowel has a remarkable ability to adapt after injury, inflammation or resection. It has long been suggested that humoral factors, particularly enteroglucagon, epidermal growth factor, neurotensin and growth hormone/insulin-like growth factor I, might stimulate bowel growth. Of particular interest is the recent finding that glucagon-like peptide 2 (GLP-2), a product of the gene encoding proglucagon, exerts a trophic effect on the intestinal epithelium via a specific G-protein-coupled receptor. GLP-2 and/or these other trophic peptides might prove to have a role in the treatment of bowel diseases associated with structural or functional loss of the small bowel.

We have used in situ hybridization to compare the distributions of estrogen receptor α (ERα) and ERβ messenger RNA (mRNA)-containing cells in the preoptic area and hypothalamus of ewes and rams. Perfusion-fixed brain tissue was collected from luteal phase ewes and intact rams (n = 4) during the breeding season. Matched pairs of sections were hybridized with sheep-specific, 35S-labeled riboprobes, and semiquantitative image analysis was performed on emulsion-dipped slides. A number of sex differences were observed, with females having a greater density of labeled cells than males (P < 0.001) and a greater number of silver grains per cell (P < 0.01) in the ventromedial nucleus for both ER subtypes. In addition, in the retrochiasmatic area, males had a greater (P < 0.05) cell density for ERα mRNA-containing cells than females, whereas in the paraventricular nucleus, females had a greater density (P < 0.05) of ERα mRNA-containing cells than males. There was a trend (P = 0.068) in the arcuate nucleus for males to have a greater number of silver grains per cell labeled for ERα mRNA. In both sexes, there was considerable overlap in the distributions of ERα and ERβ mRNA-containing cells, but the density of labeled cells within each nucleus differed in a number of instances. Nuclei that contained a higher (P < 0.001) density of ERα than ERβ mRNA-containing cells included the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus, whereas the subfornical organ (P < 0.001), paraventricular nucleus (males only, P < 0.05), and retrochiasmatic nucleus (females only, P < 0.05) had a greater density of ERα than ERβ mRNA-containing cells. The anterior hypothalamic area and supraoptic nucleus had similar densities of cells containing both ER subtypes. The lateral septum and arcuate nucleus contained only ERα, whereas only ERβ mRNA-containing cells were seen in the zona incerta. The sex differences in the populations of ER mRNA-containing cells in the ventromedial and arcuate nuclei may explain in part the sex differences in the neuroendocrine and behavioral responses to localized estrogen treatment in these nuclei. Within sexes, the differences between the distributions of ERα and ERβ mRNA-containing cells may reflect differential regulation of the actions of estrogen in the sheep hypothalamus. Low levels of ERβ mRNA in the preoptic area and ventromedial and arcuate nuclei, regions known to be important for the regulation of reproduction, suggest that ERβ may not be involved in these functions.

Previous observations from our laboratory have demonstrated that the levels of immunoreactive inhibin (ir-inh) are elevated in almost all patients with granulosa cell tumours and in the majority of postmenopausal women with mucinous ovarian cancers. The present manuscript confirms these findings in a larger group of postmenopausal women. Immunohistochemistry for the inhibin α , β A and β B subunits shows predominantly epithelial staining in granulosa cell tumours and in the majority of mucinous cancers. Serous cystadenocarcinomas also frequently show positive staining. Studies seeking to identify G α i-2 or FSH receptor mutations have provided negative results in contrast to other reports. Further studies of the roles of the inhibin-related family of peptides in ovarian cancer diagnosis and monitoring are clearly indicated.

Objective(s). Granulosa cell tumors (GCT) and mucinous cystadenocarcinoma of the ovary are associated with elevated circulating levels of immunoreactive inhibin. Measurement of serum inhibin levels provides a useful tumor marker in the management of ovarian tumors. Inhibin is a dimeric ovarian glycoprotein hormone consisting of one α and one of two β subunits. The β subunits can dimerize to form activin. Activin is bound and its action modulated by another gonadal peptide, follistatin. In this study the patterns of expression of the three inhibin subunit genes, the follistatin gene, and the activin receptor type II gene have been determined. Methods. Gene expression was analyzed in RNA prepared from 16 primary ovarian tumors using reverse transcriptase-polymerase chain reaction (RT-PCR). Gene-specific primes were used for RT-PCR; the products were analyzed by Southern blot analysis with gene-specific 32 P-labeled probes. Results. Widespread expression of these genes was found in all of the tumor types examined. Abundant expression of the inhibin α subunit gene was observed in the GCT and to a lesser extent in the mucinous and serous tumors. β subunit expression was also present in the GCT and to a lesser extent in the other tumors. Widespread expression of both the activin receptor type II and the follistatin genes was also observed. Conclusions. Expression of the inhibin subunit genes in GCT and some epithelial tumors confirms that these tumors are the source of the increased immunoreactive inhibin seen in the circulation of patients with ovarian tumors. Expression of the activin receptor type II and follistatin genes suggests a paracrine role for activin in these tumors which may be modulated by follistatin, particularly in the GCT.

The molecular mechanisms by which corticosteroids affect fluid and electrolyte balance are unclear. Though glucocorticoid-responsive genes have been identified, genes regulated by aldosterone have not. CHIF (channel-inducing factor gene) is a recently identified gene that is up-regulated in the distal colon by chronic corticosteroid exposure, is expressed in the kidney, and induces a K+-specific current in Xenopus oocytes. The predicted protein shows similarity to gammaNa.K-ATPase, phospholemman, and Mat-8; all seem to be involved in ion transport. CHIF thus presents as a potential aldosterone target gene. In this study, CHIF expression was examined in rats in the acute timeframe of 0.5-4 h after corticosteroid administration. CHIF messenger RNA showed up-regulation by both mineralocorticoid and glucocorticoid receptor agonists in the distal colon, which was not diminished by cycloheximide. Corticosteroid regulation was not observed in the kidney. Basal and induced expression was absent in the lung and in all gastrointestinal tissues except colon, with expression increasing proximal to distal. CHIF is the first gene to show acute regulation by aldosterone and thus encodes a candidate aldosterone-induced protein. In addition, gammaNa.K-ATPase gene expression was found to be very low in colon and significantly higher in kidney. Regulation by corticosteroids was not evident in either tissue.

Although hype and hyperbole may sometimes seem synonymous with stem cell research, the fundamental technology offers very exciting opportunities for endocrinology research. In this issue of Endocrinology, you will find four minireviews, each demonstrating how this emerging technology may be applied to addressing questions in endocrine biology. Stem cells are defined as cells that undergo so-called asymmetric cell division, yielding both a self-renewing population of cells and giving rise to differentiated cells (1). The notion of stem cells, pluripotent cells, and multipotent cells emerged from the hematopoietic field, initially through histological characterization. These concepts are now widely accepted for most tissues, including those of interest to endocrinologists. In her review of pancreatic progenitor cells, Ku (2) emphasizes the concept, derived from the hematopoietic system, that the stem cells and the multipotent progenitor cells are minor populations with each subsequent level being more abundant. The actual identification of a tissue stem cell, be it in the breast (3), ovary (4), prostate (5), or pancreas (2), was originally through morphological criteria; in each minireview the authors emphasize the importance of more rigorous criteria for identification and characterization, including molecular markers and functional assays. Debates about stemness although arguably arcane, certainly serve to emphasize the difficulty of identifying stem cell populations and the need for rigorous criteria. The issues of reproductive cloning, therapeutic cloning, regenerative medicine, and the ethics of stem cell technology are of undeniable importance; they have, however, been extensively canvassed elsewhere (6, 7). In selecting this series of minireviews, we sought to highlight the potential that this technology has to enhance research in endocrinology. The difficulty of treating estrogen receptor-negative breast cancer and the relapses observed in the face of apparently effective endocrine ablation therapies provide a major impetus for understanding growth and development in the mammary epithelium. LaMarca and Rosen (3) outline their work and that of others, which has important insights into the development of the mammary epithelium. The notion that cancer may be a disease of stem cells has achieved considerable currency, making the search for a breast stem cell particularly pertinent (3). Although not the central focus of their work, LaMarca and Rosen (3) describe work in which stem cells/pluripotent cells have been used to develop a mammary ductal tree; such a system may provide ex vivo models for the study of this complex epithelium with the facility of an in vitro system, which allows an array of analyses and retains the intrinsic complexity of the tissue. A major driver in the study of pancreatic progenitors is the possibilities associated with regeneration of pancreatic islets/ -cells and the consequent implications for diabetes treatment. The full molecular characterization of the pancreatic developmental program also provides unique insights and a critical platform for studies of the biology of islet cell function (2). The reviews of both Risbridger and Taylor (5) on prostatic stem cells and Tilly and Rueda (4) emphasize the importance of stromal interactions, although from a very different perspective. Tilly and Rueda (4) emphasized the ability of the stem cell, in this case the oocyte, to dictate the phenotype of the surrounding stroma, whereas Risbridger and Taylor (5) create tissue chimeras in which the choice of mesenchyme dictates the fate of the stem cells, with respect to both tissue type and malignant status. The ability to induce differentiation of stem cells into a complex epithelium must represent an opportunity for research with other endocrine tissues, as seen with the breast (3), such an in vitro model, provides exciting possibilities. Thus, for instance, the study of aldosterone action, a subject of some interest to my group (8), has suffered from a lack of stable, tractable in vitro systems, which express the mineralocorticoid receptor. The construction of complex polarized sodium-transporting epithelium in vitro using stem cell technology would be a major advance. Similar examples must apply across endocrine physiology. The history of modern endocrinology is characterized by technology-driven advances that can be related to specific decades. Bioassays were followed by RIA and then radioreceptor assays, which were followed by the full molecular characterization of both the hormone and the receptor using recombinant DNA technology; this technology then expanded into functional analyses using transgenic mice and homologous recombination. One cannot help feeling that we are entering the stem cell decade in endocrine research. These four minireviews therefore will hopefully provide both insights and impetus for the further application of this rapidly maturing technology to endocrine research. First Published Online May 15, 2008

Summary The gastrointestinal epithelum undergoes a continual process of cell renewal which is partly regulated by apoptosis. The process of growth and differentiation is greatly enhanced in the terminal ileum after massive small bowel resection, a well-established model of intestinal adaptation. We have applied the terminal deoxynucleotidyl transferase (TdT)-mediated UTP nick end-labelling (TUNEL) method to establish the role of apoptosis in intestinal adaption in terminal ileum of control animals after small bowel resection. Healing skin wound and lymph node were used as positive control tissue for apoptotic cells. We report that considerable inter-animal variation was observed in the intestinal tissue. We believe that caution is required in the interpretation of TUNEL staining in intestinal tissue and in its use as a specific marker for apoptosis in this setting.

Recently a second estrogen receptor termed estrogen receptor beta (ERbeta) has been cloned and characterized, and shown to be expressed at the highest levels in ovarian granulosa cells and prostatic epithelium. In the course of amplifying a region of the ligand-binding domain of the rat ERbeta cDNA we identified a second, larger transcript which appears to arise through differential splicing. The second isoform has 54 nucleotides inserted after position 1372 encoding 18 additional amino acids. Both isoforms are expressed at similar relative abundance in a range of tissues.

Introduction Though this chapter will deal in detail with the clinical assessment of the male partner in an infertile couple, it is necessary to emphasize (as described in Chapter 2) the importance of treating the couple rather than just the individual. There is no doubt that subfertility frequently occurs in both partners and the effects tend, on the basis of a number of studies, to be synergistic rather than additive. Therefore both partners in any couple with subfertility must be assessed fully and a strategy of investigation and management planned that takes account of them both. Thus the assessment of the male should occur in parallel with the assessment outlined in Chapter 3 for the female partner. With the recent exciting developments in assisted reproductive technologies, particularly as applied to male subfertility (as outlined in Chapter 10), there is a temptation to take a minimalistic approach to the assessment of the male partner and to rely simply on the semen analysis followed by a decision as to whether to proceed to in-vitro fertilization, perhaps with sperm microinjection. It is important to recognize that there are uncommon but potentially treatable and/or preventable causes of male subfertility which should be identified before proceeding to more invasive and expensive technologies. The couple must be made fully aware of the options available to them, including the likely success rates, complications and expenses involved.

RU486 acts as a potent anti-progestin in humans but does not antagonise progesterone action in the chicken or hamster reflecting a substitution in the ligand binding domain (LBD) of cysteine for glycine in both the chicken and the hamster progesterone receptor (PR), at the position corresponding to codon 722 of the human PR. The tammar wallaby, Macropus eugenii , is also resistant to the effects of RU486. Cloning of a partial cDNA of the PR in the tammar wallaby reveals a glycine to alanine substitution (gly 722 in the human PR), as well as a glutamine to histidine substitution two amino acids upstream of this alanine residue. Both the glycine and glutamine residues are substituted in all three resistant species. These substitutions are also found in the mineralocorticoid receptor, which also does not bind RU486, and suggest an important role for these residues in the formation of the 11-β pocket of the receptor, which accommodates the bulky side-chains of 11-β substituted steroids.

The physiology of the steroid hormone, aldosterone is well defined. The molecular events that mediate this response remain to be elucidated. Aldosterone binds to a specific mineralocorticoid receptor (MR) in sodium transporting epithelia. The structural determinants of ligand-binding have been explored through the analysis of steroid resistance syndromes, however, the molecular basis of resistance to aldosterone, pseudo-hypoaldosteronism remains an enigma. Cortisol also binds MR, access is however restricted by the enzyme 11β-hydroxysteroid dehydrogenase. The MR induces specific genes which regulate apical amiloride-sensitive epithelial sodium channels; the finding of activating mutations in Liddles syndrome (pseudoaldosteronism) has emphasised their key role. Such mechanisms may apply not only to the peripheral effects of aldosterone but also to the central regulation of blood pressure.

Sodium transport across high-resistance epithelia involves both an apical amiloride-sensitive sodium channel and the basal Na(+)-K(+)-ATPase pump. Aldosterone regulates sodium transport by increasing the sodium permeability of the sodium channels. To study further the regulation of gene expression in sodium-transporting epithelia by corticosteroids, we have cloned an amiloride-binding protein (ABP) cDNA from rat descending colon and kidney. Identical 311 nucleotide cDNAs were amplified from both rat descending colon and kidney, and the predicted amino acid sequence exhibited 83% homology to the equivalent region of the human peptide sequence. Use of this cDNA as a probe resulted in detection of a transcript in both the small and large bowel, thymus, and seminal vesicle. The latter tissue exhibited the highest level of rat ABP expression. Low to undetectable levels of rat ABP were expressed in the descending colon and kidney. No regulation of rat ABP by either class of corticosteroids was observed. Levels of ABP were low at birth and increased gradually to adult levels just before weaning in the bowel. The distribution of rat ABP is not as would be predicted for an aldosterone-induced gene and is thus unlikely to be a component of the amiloride-sensitive electrogenic sodium channel.

Granulosa cell tumors (GCT) of the ovary are rare, hormonally-active neoplasms characterized by endocrine manifestations, an indolent course and late relapse. Chemotherapy and hormonal therapy have proved to be of limited efficacy. Nuclear receptors (NR) have a central pathogenic role in endocrine malignancy and present as potential targets for therapeutic intervention. NR have established roles in granulosa cell biology but their roles in GCT remain largely unexplored. In order to systematically examine the NR family in GCT, we used ABI Taqman® Low Density Array microfluidic cards to analyse 14 GCT and two GCT-derived cell lines for expression of the 48 NR. The levels of expression were remarkably consistent across the GCT. We found that peroxisome proliferator-activated receptor gamma (PPARγ) had greater than ten-fold absolute expression when compared with either the NCBI tumour or brain reference RNA pools. PPARγ agonists are regarded as potential therapeutic agents in the treatment of inflammatory diseases and certain cancers. Given the high expression levels of PPARγ in GCT, we investigated whether the use of PPARγ and/or retinoid × receptor (RXR) agonists or antagonists have an effect on the GCT-derived cell lines, KGN and COV434. We used real-time cell analysis (RTCA) to investigate the cytotoxicity of the PPARγ agonist, troglitazone, and the RXR agonist, 9-cis-retinoic acid, by continuous monitoring of cell growth, proliferation and viability in the GCT-derived cell lines in real-time. We observed that a combination of troglitazone and 9-cis-retinoic acid significantly inhibited cell proliferation in the cell lines (56% compared to control). Because we have previously reported that steroid receptors are transrepressed in these GCT cells due to constitutive activation of the NF-κB signalling pathway, we tested the effect of troglitazone and 9-cis-retinoic acid in conjunction with a NF-κB inhibitor, BAY-117082. We observed a further decrease in cell proliferation (85% compared to control). This was a PPARγ-mediated effect as it was reversed by co-administration with the PPARγ antagonist, GW9662. The decrease in proliferation was due to cellular apoptosis. We also investigated whether PPARγ is transcriptionally active in these cells using a reporter construct, specific for PPARγ, and observed that GCT cells were not responsive to either PPARγ agonists or antagonists in vitro. This was found to be due to NF-κB transrepression; when NF-κB was inactivated, PPARγ signaling was restored. We conclude that, while the use of PPARγ agonists may have potential as therapeutic agents for treating GCT, a combination of therapies involving the abrogation of NF-κB signaling may be of greater efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4536. doi:10.1158/1538-7445.AM2011-4536

Background: Proglucagon-derived peptides are potential mediators of the adaptive response of the terminal ileum to massive small bowel resection. Ileal proglucagon messenger RNA (mRNA) levels increase during ileal adaptation. The present study explored the cellular basis of this response. Methods: Sections of control ileum, ileum 4 days after resection, and pancreas were analyzed by in situ hybridization with 35 S-labeled complementary RNA (cRNA) probes. Results: Both the proglucagon and the peptide YY cRNA probes hybridized to discrete cells in the ileal mucosa, the disposition of which corresponds to that reported for intestinal L cells. Four days after resection there was a marked increase in the intensity of the signal for both probes without an increase in cell number. Insulin and histone H3 probes were used as controls to confirm the specificity of the hybridization seen with the L-cell specific, proglucagon, and peptide YY probes. Conclusions: The increase in proglucagon mRNA levels after massive small bowel resection is caused by an increase in the cellular content. The parallel increase in PYY mRNA levels implies an L cell- rather than a proglucagon gene-specific response.

Nuclear factor-κB (NFκB) designates a family of transcription factors that has been shown to modulate antiviral, inflammatory and immune responses and promote tumorigenesis. Activation of NFκB is dependent on IKKβ, a component of the IκB kinase (IKK) complex which promotes degradation of IκB inhibitory proteins and allows nuclear translocation of NFκB. Little is known about the role of NFκB signalling in the ovary. We created a gonadal specific IKKβ conditional knockout mouse to explore NFκB action in the ovary. A transgenic mouse line containing floxed IKKβ alleles (M Karin, UCSD) was crossed with a transgenic mouse line expressing cre-recombinase under the control of the anti-Müllerian hormone receptor (AMHR) promoter (M Matzuk, BCM). Female mice arising from this breeding regime will not express IKKβ in granulosa cells and cannot activate NFκB signalling. Ovaries and serum were collected from mice at 7 and 15 weeks of age. Histological analyses were undertaken and the gonadotrophic hormones, follicle stimulating hormone (FSH) and luteinising hormone (LH) were measured. The ovaries of 7 week old IKKβ null mice contained follicles of all developmental stages although corpora lutea were absent indicating that these mice were infertile. Some follicle subtypes may be under-represented and apoptosis may be enhanced; these studies are ongoing. Serum FSH and LH levels were elevated compared to littermate controls. By 15 weeks of age corpora lutea were present. The fertility of the IKKβ conditional knockout is currently being assessed. In summary, IKKβ conditional knockout mice exhibit a reproductive phenotype which includes delayed ovulation. These results validate the hypothesis that ovulation is an inflammatory-like response. This model will be a valuable tool for reproductive research; the subtlety of the phenotype allowing us to tease out the underlying mechanisms and role of NFκB signalling in ovarian function.

Changes in the levels of ileal insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) mRNA in the rat following massive small bowel resection (MSBR) have been investigated with a sensitive S1 nuclease assay. IGF-I mRNA levels vary little over 7 days; in contrast IGFBP-3 mRNA levels decreased to one-third 7 h post-MSBR, and remained suppressed for the length of this study. We postulate that decreased ileal synthesis of IGFBP-3 enhances the ability of IGF-I to stimulate the adaptive response.

Na-K-ATPase is a heterodimer of α- and β-subunits often referred to as the ‘sodium pump’ responsible for the maintenance of cell volume and electric potential across cell membranes. In the present study we have used antisense RNA probes to localize these subunits in the rat kidney by in situ hybridization. α1-Subunit and β-subunit gene expression are highest in cortical distal convoluted tubules, and the thick ascending limb of the loops of Henle; high in some proximal convoluted tubules, and low in the collecting ducts and blood vessels. Expression of the α11 and α111 isoforms is very low or absent thoughout the kidney. These results confirm and extend studies of Na-K-ATPase enzyme activity levels and immunocytochemical localization in the kidney.

We have used reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and insitu hybridization to further define the tissue and cell-specific pattern of expression of the rat kallikrein gene family. rKlk1 gene expression has been unequivocally demonstrated in the rat heart, ovary and testis. Similarly, rKlk3, 4 and 9 gene expression has been demonstrated in the testis/ovary, heart and testis respectively. Insitu hybridization has identified the expression of kallikrein gene family members to specific cell-types in the rat ovary (granulosa cell) and testis (germ cell).

Nuclear factor-κB (NF-κB) designates a family of transcription factors that have been shown to modulate antiviral, inflammatory and immune responses. Activation of NF-κB is dependent on IKKβ a component of the IκB kinase (IKK) complex which promotes degradation of IκB inhibitory proteins and allows nuclear translocation of NF-κB. Our studies in ovarian granulosa cell tumour cell lines (COV434 and KGN) indicate that NF-κB signalling is constitutively activated. FSH has been reported to increase XIAP expression through NFκB activity in granulosa cells, but beyond that the role of NFκB signalling in folliculogenesis has not been elucidated. To establish the significance of NF-κB signalling in the ovary (and testis), we have generated a gonadal specific IKKbeta conditional knockout mouse. A transgenic mouse line containing floxed IKKβ alleles (gift of M Karin, UCSD) was crossed with a cre mouse line (gift of M Matzuk, BCM) expressing the recombinase in anti-Müllerian hormone receptor expressing cells (granulosa cells or Sertoli cells). The resulting mice will not express IKKβ in granulosa cells or Sertoli cells and thus cannot activate the classical NFκB signalling pathway. On histological assessment, the ovaries and testes from flox x cre (heterogenous) mice appear normal with follicles of all developmental stages and corpora lutea. Preliminary data suggests that breeding with the heterogenous females resulted in increased litter sizes. The histology of the testes is also unremarkable. The mice homozygous for the deletion of IKKβ in the granulosa cells appear healthy and a preliminary assessment does not reveal gross morphological abnormalities of the ovaries. The results of detailed histological and overall assessment of these mice will be presented. These IKK conditional knockout mice should provide insights into the role of NFκB signalling in gonadal function.

The steroid receptor superfamily of ligand-dependent transcription factors is characterized by marked conservation of both structure and function between the various receptors. Despite their well-documented extensive similarities, these receptors respond to a diverse range of ligands, which results in an even more impressive diversity of function. A variety of strategies is used at each point in the pathway from ligand binding to gene expression to achieve this diversity. The nature of the ligand is important as are the tissue-specific patterns of receptor gene expression, the presence of binding proteins, and the effects of cell- or tissue-specific ligand-modifying enzymes. Once bound to the receptor, the nature of which may vary as a result of either differential splicing or gene duplication yielding multiple isoforms, the activated receptor may form hetero- or homodimers. A complex interplay then occurs between the receptor dimer, other nuclear proteins, the response element, and the promoter complex to regulate gene expression. These elements may vary as a function of the cell type, other stimuli, and the context and sequence of the response element (or elements) in a given gene. By these mechanisms diversity may even be achieved for a given ligand, receptor subtype, gene, or cell. The observations may help to explain certain phenomena in hormone biology that are difficult to reconcile with the previous, simple, univariant model of steroid hormone action.

The guinea-pig has high levels of circulating cortisol. Though adrenocorticotropin (ACTH) levels are similar to those in other mammals, guinea-pig ACTH has been reported to have a single amino-acid substitution which results in increased bioactivity of the peptide. Pro-opiomelanocortin (POMC) is the precursor for ACTH, gamma-melanocyte-stimulating hormone (gamma-MSH) and the endogenous opioid peptide beta-endorphin. Both to confirm this substitution in guinea-pig ACTH and to establish whether other non-conservative substitutions occur elsewhere in the precursor we cloned guinea-pig POMC. The guinea-pig alanine for proline substitution at position 24 of ACTH was confirmed. Potentially significant mutations were also identified in gamma-MSH and beta-endorphin. A similar pattern of POMC mRNA expression was obtained for guinea-pig and rat as determined by Northern analysis and in situ hybridization. Southern blot analysis indicated that guinea-pig POMC is a single-copy gene. Cloning and sequencing of guinea-pig POMC thus further demonstrate the divergence of the New World hystricomorph peptides from those in New World primates, and underscore the differences observed in other endocrine axes in the guinea-pig.

Betaglycan is a type III TGF-β receptor that binds to both inhibin and TGF-β with high affinity and determines cellular sensitivity to these ligands. Previous studies have suggested that betaglycan acts as a tumour suppressor in certain human epithelial cancers. However, the roles of betaglycan in ovarian granulosa cell tumours (GCTs) are poorly understood. The objective of this study was to determine whether human GCTs exhibit betaglycan expression, and if so, what impact this receptor has on tumour biology. Real-time PCR was used to quantify betaglycan transcripts in human GCTs (n = 18) and normal premenopausal ovaries (n = 11). This analysis established that GCTs exhibited a significant 2-fold reduction in mean betaglycan mRNA levels as compared with the normal ovary (P < 0.05). Similarly, two human GCT cell lines, the KGN and COV434, exhibited low betaglycan expression and poor responsiveness to TGF-β and inhibin in luciferase reporter assays. Stable transfection of GCT cell lines with a wildtype betaglycan (WT-BG) expression plasmid conveyed ligand responsiveness. FACS analysis was used to examine cell cycle progression and cell death in the GCT cell lines. This analysis revealed that WT-BG had no effect on the number of cells cycling or undergoing apoptosis. However, WT-BG significantly increased the adhesion of COV434 (P < 0.05) and KGN (P < 0.0001) cells to collagen IV and fibronectin, decreased cellular invasion through Matrigel COV434 (50%) and KGN (75%), and inhibited wound healing COV434 (70%) and KGN (80%). Collectively, the data establish that betaglycan is an important regulator of granulosa cell biology and suggest that a deficiency in betaglycan contributes to the pathogenesis of granulosa cell cancer. The work further suggests a role for betaglycan in the prevention of tumour invasion, possibly by increasing the adhesion of granulosa cells to matrix components. Supported by: the NHMRC of Australia (RegKeys 338516; 241000; 441101; 388904).

Massive small bowel resection with re-anastomosis of the residual jejunum and terminal ileum results in marked adaptive responses. Various luminal and humoral factors have been implicated in the adaptive response, which may be analogous to the changes occurring in the ileum in the postnatal growth phase. Enteroglucagon, which is synthesized in the L cells of the intestinal mucosa, is thought to be an important humoral factor in this response. In this study, the levels of glucagon gene expression in the rat ileum both after massive small bowel resection and during development are examined. Glucagon messenger RNA levels are increased threefold as part of the adaptive response; the increase is maximal at 2 days and is at least partly dependent on luminal nutrition. Levels of glucagon messenger RNA in the developing ileum increase in the postnatal period until weaning when they decrease somewhat before gradually reaching adult levels.

The gastrointestinal tract is a well characterized target tissue for aldosterone, where it regulates electrolyte transport, particularly in the descending colon. Previous studies have demonstrated the presence of aldosterone receptors in gastrointestinal tissues. We have used specific cRNA probes for the rat mineralocorticoid receptor to explore both the distribution and ontogeny of mineralocorticoid receptor gene expression in the gastrointestinal tract. Mineralocorticoid receptor gene expression is found throughout the small and large intestine, but is absent from the stomach. The highest levels are observed in the distal colon, and significant expression is found in the duodenum; in both tissues levels of expression are higher than those in kidney. In both the developing duodenum and colon, mineralocorticoid receptor gene expression precedes the development of the full physiological response to aldosterone. These findings emphasise the colon as an important target tissue for aldosterone, and raise the question of potential roles for aldosterone in the duodenum.

The neurohypophysial hormones oxytocin and arginine vasopressin (AVP) have been identified on immunological criteria in the ovary. Confirmation of extraneuronal synthesis requires the demonstration in the tissue of the specific messenger RNA (mRNA) for the preprohormone. Using a synthetic pentadecamer nucleotide probe, highly specific for the 5′ region of rat neurophysin II (NPII), we have demonstrated the presence of AVP-NPII mRNA in the ovary of Sprague–Dawley, Long–Evans and Brattleboro rats, with an apparent molecular weight identical to that seen for hypothalamus. These findings, together with the presence of immunoreactive AVP in the ovaries but not hypothalami of Brattleboro rats, suggest that tissue-specific differences in AVP-NP II gene expression occur at the translational as well as transcriptional level. J. Endocr. (1985) 105, 317–321

The report of 'kallikrein-like' activity in the rat neuro-intermediate lobe (N-IL) and its possible involvement in pro-opiomelanocortin processing led us to explore the expression of the kallikrein gene(s) in the pituitary. Using 32P-labelled rat pancreatic kallikrein cDNA, we have shown positive hybridization for rat anterior pituitary poly(A)+ RNA, of identical size on Northern blots (approximately 1.0 kb) to rat kidney poly(A)+ RNA run in parallel. Prior adrenalectomy or ovariectomy decreased the level of kallikrein mRNA seen in the anterior pituitary; total RNA from rat N-IL showed no significant hybridization. On hybridization histochemistry the anterior pituitary was strongly positive, and the neural and intermediate lobes negative. The previously reported kallikrein-like activity in the N-IL is therefore probably due to a non-kallikrein kininogenase; in the anterior pituitary, kallikrein may have a physiological role in limited precursor proteolysis, but lack kininogen activity.

Hypersecretion of 18-hydroxydeoxycorticosterone (18-OHDOC) may be implicated in some cases of low renin hypertension and in salt-sensitive hypertension in Dahl rats. To investigate the mechanism of action of the steroid, the binding of tritiated 18-OHDOC was studied in cytoplasmic fractions of kidney, liver and heart. Binding was displaceable by excess unlabelled 18-OHDOC, but to sites with affinity too low for a physiological role as 18-OHDOC receptors. Similarly, no specific high affinity plasma binding protein could be demonstrated ; by equilibrium dialysis, high capacity albumin binding was shown to be ~65%. The weak mineralocorticoid effects of 18-OHDOC would therefore appear to be mediated by occupation of physiological aldosterone receptors, and not by occupation of physiological receptors specific for 18-OHDOC itself.

Using a rat pancreatic kallikrein cDNA probe (pcXP39), previously shown to hybridize to kallikrein mRNA in a variety of tissues, we have explored the control of kallikrein gene expression in rat anterior pituitary. Intact female rats have substantially higher levels of AP kallikrein mRNA than intact males; male levels are unaffected by castration, whereas female levels fall markedly postovariectomy. Administration of estradiol benzoate to intact male or ovariectomized female rats causes an increase in anterior pituitary levels of kallikrein mRNA. Since the pattern of responsiveness parallels that of PRL, we have studied GH3 cells grown in the presence and absence of estradiol; in neither instance was kallikrein mRNA above detection limits. Parallel changes were seen on Northern blots and by hybridization histochemistry; on emulsion autoradiography of pituitary sections, scattered positive cells were seen, but precise definition was not possible. We conclude that whereas in the submaxillary gland kallikrein gene expression appears androgen dependent and in the kidney is postulated to be mineralocorticoid regulated, in the anterior pituitary expression of the gene is under estrogen control; and that the local role(s) of pituitary kallikrein, whether precursor processing, control of blood flow, or other effects, would, in turn, appear to be modulated by estrogen in vivo.

Mineralocorticoid and glucocorticoid receptors in human kidney.Currently, studies on renal mineralocorticoid and glucocorticoid receptors have been confined to experimental animals. In experiments made upon human kidneys removed at operation, tritiated aldosterone has been shown to bind specifically to a single class of cytoplasmic sites which show steroid specificity requisite for physiological mineralocorticoid receptors. Tritiated dexamethasone, similarly, binds specifically to a single class of cytoplasmic sites with affinity characteristics appropriate for glucocorticoid receptors. The similarity between the human renal receptor profile for adrenocorticoids and that found in experimental animals may validate cautious extrapolation from animal models of adrenocorticoid effector mechanisms to man. Recepteurs aux mineralocorticoides et aux glucocorticoides au niveau du rein humain. Habituellement, les etudes des recepteurs renaux aux glucocorticoides et aux mineralocorticoides furent realisees seulement chez l'animal d'experience. Dans des experiences factes sur des reins humains preleves lors d'operation, l'aldosterone tritiee se fixe specifiquement au niveu d'une seule type de site cytoplasmique qui presente exactement les caracteristiques d'un recepteur physiologique aux mineralocorticoides. De meme la dexamethasone tritiee se fixe specifiquement a une seule classe de site cytoplasmique qui montre une affinite caracteristique et appropriee aux recepteurs aux glucocorticoides. Cet aspect similaire des recepteurs renaux humains et ceux des animaux d'experience permet une extrapolation circonspecte des mecanismes effecteurs surrenaliens du modele animal a l'homme.

Gastrin gene expression exhibits a complex pattern of tissue-specific expression during the neonatal development of the gastrointestinal tract and pancreas. In the rat fetus, the pancreas is the major site of gastrin expression; very little gastrin is found in the antrum which is the major site of adult expression. Pancreatic gastrin mRNA is identical to the antral species having the same transcriptional initiation site. The post-translational processing of the pancreatic gastrin peptide, however, differs in being fully tyrosine sulfated, and thus it resembles gastrin's homologue, cholecystokinin. Pancreatic gastrin is maximally expressed during late fetal gestation before the marked rise in somatostatin, insulin, and glucagon mRNA levels which occurs during the last 4 days of gestation. After birth, gastrin mRNA levels rapidly disappear as the other islet mRNAs achieve stable maximal levels. Unlike the pancreas, gastrin gene expression in the duodenum and antrum is similar to the development profiles of somatostatin and glucagon gene expression. In the duodenum, however, expression of cholecystokinin differs from the common developmental pattern in being expressed only after birth. Thus, pancreatic gastrin gene expression shows a reciprocal relationship to the developmental expression of cholecystokinin in the duodenum.

The tissue-specific processing of proopiomelanocortin (POMC), the precursor of ACTH, beta-endorphin, and their related peptides, is currently of considerable interest. We report a patient with a large aggressive pituitary tumor, Cushing's syndrome, and hyperpigmentation managed by transsphenoidal hypophysectomy, bilateral adrenalectomy, and sellar irradiation. Preoperatively, plasma levels of immunoreactive ACTH (ir-ACTH; 280 ng/liter) and beta-endorphin (ir-beta EP; 520 ng/liter) were moderately elevated. Chromatography of the plasma showed two peaks of ACTH immunoreactivity, with the major peak eluting in the void volume, and two major peaks of ir-beta EP, corresponding to the elution positions of beta-lipotropin and beta-endorphin standards. Plasma ir-ACTH and ir-beta EP were not suppressed by high doses of glucocorticoid or bromocriptine, a degree of autonomy more commonly found with POMC production from ectopic sources than that from pituitary tumors. Tissue removed at operation was enzymatically dispersed, and the cells were cultured in suspension, propagated, and passaged sequentially for over 20 passages. Using this cell line, we demonstrated that the biosynthesis of POMC, its pattern of processing, and the release of POMC/ir-beta EP/ir-ACTH in vitro were consistent with the in vivo evidence of autonomous secretion and abnormal processing of POMC by this pituitary tumor.