ACTH-producing tumors of nonpituitary origin characteristically exhibit insensitivity to the negative feedback effects of glucocorticoids. In the DMS-79 cell line derived from an ACTH-producing small cell lung cancer we have previously identified an aberrantly spliced glucocorticoid receptor (GRDelta) that lacks a ligand-binding domain. We examined the interactions of this truncated form of GR with the proximal human proopiomelanocortin (POMC) promoter. In electrophoretic mobility shift assays GRDelta bound to the negative glucocorticoid response element (nGRE) at position -78 to -50 in the human POMC promoter. Nur77, an orphan nuclear receptor that exerts positive regulatory effects on the POMC gene is also known to bind to this DNA element. The functional properties of GR and GRDelta binding to this DNA element were examined in transient transfection experiments in murine AtT-20 corticotroph tumor cells. Reporter gene expression under the control of proximal POMC promoter elements was stimulated by addition of forskolin to the culture medium or by transfection with expression constructs for human Nak1, the human homologue of Nur77. Treatment of transfected cells with dexamethasone resulted in suppression of forskolin- or Nak1-stimulated POMC-reporter gene expression in the presence of co-transfected GR but not with GRDelta. The experiments indicate that in the human POMC promoter GRDelta is capable of binding to the nGRE but cannot effect trans-repression of POMC-reporter gene expression.
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Sudipta Paul, Sourav Kundu, Kousik Pramanick, Arun Bandyopadhyay and Dilip Mukherjee
Multiple signal transduction pathways mediating gonadotropin-induced testosterone and 17β-estradiol (E2) production were identified in carp ovarian theca and granulosa cells in short-term co-incubation. Inhibitors of voltage-sensitive calcium channels (VSCCs) and calmodulin attenuated human chorionic gonadotropin (HCG)-induced steroid production, whereas modulators of adenylate cyclase and protein kinase A (PKA) increased their production, indicating that both calcium- and PKA-dependent pathways are involved in the regulation of gonadotropin-induced steroidogenesis in carp ovary. Interactions between these two pathways are evident from the positive effect of elevated intracellular calcium on HCG-induced steroid production and the reduction of forskolin (FK)- and dibutyryl cAMP (dbcAMP)-induced steroidogenesis by inhibitors of VSCCs and calmodulin. In this study, we found the involvement of a third signaling pathway, a mitogen-activated protein kinase (MAP kinase), in the regulation of gonadal steroidogenesis in this fish. An antagonist of mitogen-activated protein kinase kinases 1/2 (MEK1/2; also known as MAP2K1/MAP2K2) markedly attenuated HCG-induced steroid production. Cells treated with HCG stimulated MEK1/2-dependent phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERKs1/2) in a concentration and time-dependent manner. Moreover, ERK1/2 activation in cells was mimicked by FK and dbcAMP suggesting that ERK1/2 transduce signal downstream of PKA in HCG-induced ovarian steroidogenesis. Evidence for presence of cross talk between calcium-dependent pathways and this MAP kinase cascade has been shown by demonstrating the inhibitory effects of verapamil and calmodulin on ERK1/2 activation after HCG stimulation. Our results suggest that activation of ERK1/2 by HCG as well as other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis in carp ovary.
Edward F Orlando, Yoshinao Katsu, Shinichi Miyagawa and Taisen Iguchi
The mechanisms underlying sex determination and differentiation in fishes are labile in response to environmental parameters. Sex-specific phenotypes are largely regulated by sex steroids, and the inhibition or the stimulation of aromatase can reverse sex as well as alter secondary sexual characteristics in fishes. Among vertebrates, the mangrove rivulus is the only known self-fertilizing hermaphrodite. Throughout most of its range, rivulus appear to exist as clonally reproducing hermaphrodites. However, outcrossing has been documented in Belize, where up to 25% of rivulus collected are males. The direct development of (primary) males occurs when embryos are incubated at 18 °C and hermaphrodites develop into secondary males when held at 28 °C. Given the importance of sex steroids, their receptors, and aromatase in sex determination and differentiation of fishes, we cloned, sequenced, and quantified the expression of estrogen receptors (ERα, ERβ) and ovarian (AroA) and brain (AroB) aromatase genes. Hermaphrodites had increased ERα, ERβ, AroA, and AroB gene expression in the liver, gonad, gonad, and brain respectively, compared to males. These data are consistent with the gene expression data reported for other species and are reflective of the presence of ovarian tissue in the hermaphrodites. Interestingly, we show the elevated expression of brain aromatase in the hermaphrodite brain. The role of the dimorphic expression of brain aromatase in the regulation of sex-specific characteristics is intriguing and requires further research. Because of the uniqueness of its reproductive biology, rivulus is an excellent model for elucidating the mechanisms regulating vertebrate sex determination and sexual differentiation.
D. A. Rodin, S. D. Abbot, G. Saade and R. N. Clayton
There are significant differences between rats and mice in the gonadal regulation of several aspects of gonadotroph function. To investigate whether these extend to the pretranslational regulation of FSH synthesis by gonadal steroids, we have measured FSH-β mRNA levels following gonadectomy and sex-steroid replacement and have related these to serum and pituitary FSH as a reflection of overall hormone synthesis.
In ovariectomized rats, FSH-β mRNA levels increased by 8 h, decreased, and then rose progressively over the next 28 days. A similar pattern of response was observed in orchidectomized rats. In mice, there were progressive increases in FSH-β mRNA levels in both males and females following gonadectomy, without evidence of the early peaks observed in rats. In both species, the change in FSH-β mRNA levels after gonadectomy was greater in females than in males. These changes in FSH-β mRNA following gonadectomy were paralleled by changes in the serum FSH concentration. In ovariectomized female rats and mice, pituitary FSH stores increased by 8 h and 3 days respectively, whereas in male rats, pituitary FSH content did not rise until 10 days after orchidectomy. The most striking species difference was the marked and prolonged reduction of pituitary FSH after orchidectomy of mice.
Treatment of rats and mice from the time of ovariectomy, with a dose of oestradiol that prevents increases in serum LH, only partially attenuated the rises in FSH-β mRNA and serum FSH and did not prevent the increase in pituitary FSH content. Treatment of intact or orchidectomized rats with testosterone suppressed FSH-β mRNA levels to 50% below intact control values without affecting pituitary FSH content. In mice, testosterone treatment for 10 days reduced the post-castration increase in FSH-β mRNA by only 26%, and prevented the fall in pituitary FSH content, although the increased serum concentration of FSH was unaffected.
In conclusion: (1) there is a good correlation between FSH-β mRNA levels and overall FSH biosynthesis in male and female rats and female mice, but this relationship is less obvious in male mice where pituitary FSH stores are not increased; (2) the inability of oestradiol to prevent completely the post-ovariectomy increase in FSH-β mRNA and FSH synthesis in female rats and mice indicates either that other gonadal products are necessary or that higher doses of oestradiol are required than for complete suppression of LH synthesis; (3) whilst the post-gonadectomy increases in FSH-β mRNA are larger in the female of both species, there are no major differences between rats and mice in the regulation of FSH-β gene expression by sex steroids.
T Kitano, K Takamune, T Kobayashi, Y Nagahama and SI Abe
The phenotypic sex of many teleost fishes including flounders can be experimentally altered by treating embryos or larvae with varied temperatures or sex-steroid hormones. To analyse the sex determination mechanism, especially the role of cytochrome P450 aromatase (P450arom), an enzyme that catalyses the conversion of androgens to estrogens, in temperature-dependent gonadal sex differentiation in the Japanese flounder, we generated two populations of larvae, both having XX (genetic females) but each growing up to display all phenotypic females or males, by rearing the larvae at normal (18 degrees C) or high (27 degrees C) water temperatures from days 30 to 100 after hatching respectively. The larvae (XX) were produced artificially by mating normal females (XX) with gynogenetic diploid males (XX) which had been sex-reversed to phenotypic males by 17alpha-methyltestosterone. To study the role of P450arom in sex determination in the flounder, we first isolated a P450arom cDNA containing the complete open reading frame from the ovary. RT-PCR showed that P450arom mRNA was highly expressed in the ovary and spleen but weakly in the testis and brain. Semi-quantitative analyses of P450arom mRNA in gonads during sex differentiation showed that there was no difference in the levels of P450arom mRNA between the female and male groups when the gonad was sexually indifferent (day 50 after hatching). However, after the initiation of sex differentiation (day 60), the mRNA levels increased rapidly in the female group, whereas they decreased slightly in the male group. Similarly, estradiol-17beta levels rose remarkably in the female group, yet remained constant in the male group. These results suggest that induction of sex reversal of genetically female larvae to phenotypic males by rearing them at a high water temperature caused a suppression of P450arom gene expression. Furthermore, we suggest that the maintenance of P450arom mRNA at very low levels is a prerequisite for testicular differentiation, while the increased levels are indispensable for ovarian differentiation.
JT Dickey and P Swanson
The effect of steroid hormone treatment on coho salmon (Oncorhynchus kisutch) was examined. The cDNAs for coho salmon FSH beta and LH beta subunits were cloned and sequenced using reverse transcriptase PCR. Northern blot analysis revealed that a single transcript of 1 kb for each of these subunits was present in the pituitaries of vitellogenic and spermiating coho salmon. RNase protection assays (RPAs) were developed to quantify FSH beta and LH beta subunit transcript levels. For the RPAs, antisense RNA probes and sense RNA standards were prepared from a region of the cDNAs which spanned the signal peptide and a portion of the mature protein. These RPAs were used to examine the effects of exogenous steroids including testosterone, estradiol-17beta (E2) and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P) in vivo, in coho salmon at three time points during the spring period of gonadal growth when plasma levels of FSH are increasing. Both testosterone and E2 increased steady state mRNA levels of LH beta, whereas E2 decreased steady state mRNA levels of FSH beta in one experiment. Thus, the RPAs were able to detect changes in steady state mRNA levels in response to exogenous steroid treatment. Plasma and pituitary levels of FSH and LH were also measured using RIA. Throughout the experimental series, FSH plasma levels decreased in response to exogenous testosterone and E2 administration, while 17alpha,20beta-P had no effect on FSH plasma levels. Plasma LH levels were not detected throughout the course of the experiment. Pituitary LH increased in response to testosterone and E2, while pituitary FSH levels did not change. 17alpha,20beta-P had no effect on pituitary FSH or LH content during the experiment. Thus, regulation of the gonadotropins in coho salmon occurs at both the transcriptional as well as the translational level. Testosterone and E2 appear to have negative feedback effects on FSH, but positive feedback on LH.
S. A. Nicholson, B. Gillham and M. T. Jones
Two chemically characterized peptides, arginine vasopressin (AVP) and corticotrophin-releasing factor-41 (CRF-41), known to stimulate ACTH secretion by interaction with their respective specific receptors on the corticotroph, were shown to cause the accumulation of phosphate esters of inositol (IP) and adenosine 3′,5′-monophosphate (cAMP) respectively when added to rat anterior pituitary fragments incubated in vitro. The former 'second messenger' response (IP production) was unaffected in tissues removed from animals treated with prednisolone in the drinking water (1035 μmol/1) for 14 days. On the other hand, the cAMP response, whilst still present, was inhibited by some 50% in tissues taken from such animals. In contrast, pituitary glands from steroid-treated rats failed to respond to challenge with a variety of substances expected to cause the release of ACTH by mimicking or provoking the production of IP or cAMP. Indeed, of the wide range of ACTH secretagogues tested, only the phospholipase A2 activator melittin was able to cause attenuated ACTH release from tissues removed from treated rats. The failure to provoke ACTH release from tissues removed from steroid-treated animals was also seen when submaximal concentrations of CRF-41 or AVP, or hypothalamic extract or 48 mmol K+/1 were used as the stimuli.
The staged recovery of the ACTH secretory response and IP and cAMP accumulation in vitro following the withdrawal of prednisolone treatment was also investigated. A cAMP response that did not differ significantly from that of control tissue and a normal ACTH response to K+ and to melittin were all recovered by 3 days after withdrawal, and the response to cholera toxin showed a partial recovery. Responses to all stimuli of ACTH secretion which cause their effect by entering the corticotrophs were normal by 5 days after withdrawal, when the response to CRF-41 was still significantly, and that to AVP still slightly, reduced compared with controls. Surprisingly, restoration of the ACTH response was most delayed when the expectedly most potent extracellular stimulus (hypothalamic extract) was used. In this case, release was still significantly impaired 7 days after steroid withdrawal.
The results show that the glucocorticoid acts to compromise several distinct steps in the process whereby extracellular signals such as CRF-41 and AVP cause the secretion of ACTH. The only step that appears to be spared is the generation of IP by AVP. The staging of the recovery of the ACTH response following steroid withdrawal suggests that adenylate cyclase activation and release mechanisms recover first (by 3 days), then the coupling of individual second messenger production to ACTH synthesis and release (by day 5) and finally the integrated response to extracellular stimuli, which requires 7 or more days. From the present data, however, it is not clear why the recovery of the normal responses to intra- and extracellular signals stimulating ACTH secretion are temporally dissociated.
P. Pakarinen and I. Huhtaniemi
Serum and pituitary LH and FSH, and their pituitary mRNA levels, were measured in neonatal male and female rats after gonadectomy and after gonadectomy with sex steroid replacement. The animals were gonadectomized on day 3 of life, and those given sex steroid replacement were implanted with silicone elastomer capsules containing testosterone for males and diethylstilboestrol for females. Shamoperated rats served as controls. The animals were killed 4 or 8 days later and the sera and pituitaries collected. Pituitary contents of mRNAs for the α subunit, FSH-β and LH-β were determined by blot hybridization using corresponding cDNAs. Distinct sex differences were found in the mRNA responses to gonadectomy and steroid replacement. In the males, gonadectomy increased all mRNA levels at 7 days of age. In the females, a rise on day 7 was detected only for FSH-β; the other mRNAs were increased on day 11 of age. The steroid replacements reversed all the post-gonadectomy increases of mRNAs in both sexes. Moreover, the common α and LH-β mRNAs of the male animals were consistently suppressed below control levels. The serum concentrations of gonadotrophins increased after gonadectomy on day 7 in the males but only on day 11 in the females. The steroid replacements also suppressed the post-gonadectomy increases in serum gonadotrophins, but only the serum concentration of FSH in the females was reduced below controls. Pituitary gonadotrophin concentrations were not affected by gonadectomy, but the steroids suppressed LH in the males and FSH in the females.
It is concluded that the onset of negative-feedback regulation of gonadotrophin synthesis by gonads and/or gonadal steroids starts earlier in male rats, before 7 days of age. In female rats these responses appear between 7 and 11 days of age. Clear sex differences were observed in how gonadotrophin mRNAs and pituitary and serum hormone levels responded to gonadectomy and steroid replacement in the neonatal period. Some of the responses differed from those previously reported in adult animals.
B S Nunez, P M Piermarini, A N Evans and S L Applebaum
The steroidogenic acute regulatory protein (StAR) is critical to the regulated synthesis of steroids in vertebrates. We have isolated cDNA sequences encoding StAR in the freshwater stingrays Potamotrygon hystrix and P. motoro. A single P. hystrix StAR transcript (3376 bp) and two overlapping P. motoro StAR transcripts (1272 and 3365 bp) were isolated. The P. hystrix and P. motoro StAR transcripts contain open reading frames encoding proteins of 284 amino acids that are 99% identical to each other and 56–64% identical to other StAR proteins. Pregnenolone synthesis by green monkey kidney (COS-1) cells transfected with an expression construct encoding a human cholesterol side chain cleavage/adrenodoxin reductase/adrenodoxin fusion protein was increased 16-fold by coexpression with a pCMV5/P. motoro StAR expression construct. Northern blot analysis revealed a single 4000 bp StAR transcript in the P. motoro interrenal gland, but RT-PCR indicates StAR mRNA is also expressed in the brain, gonads, atria, ventricle, gill (female only) and muscle (female only). Expression in extragonadal and extraadrenocortical tissues is an indication that StAR may be critical to processes other than steroidogenesis. The longest P. motoro StAR transcript contains a sequence with great similarity to short interspersed repetitive elements found in other elasmobranchs. This study is the first to isolate and characterize elasmobranch StAR cDNA sequences and to demonstrate the activity of a nonmammalian StAR protein in a heterologous expression system.
J. U. Weaver, G. A. Hitman and P. G. Kopelman
Obesity is likely to be a multifactorial disease with an important genetic component. Animal models of genetic and experimentally induced obesity suggest that glucocorticoid receptor (GR) activity plays a role in the aetiology and maintenance of the obese state. Glucocorticoid activity appears to be essential for the development of hyperinsulinaemia and subsequent fat deposition. In humans, glucocorticoid excess is associated with central fat distribution. We have therefore investigated the restriction fragment length polymorphisms of the human GR gene locus (GRL) and have sought associations of specific alleles with anthropometric measurements and indices of insulin secretion and resistance in obesity.
Fifty-six extremely obese, unrelated, nondiabetic premenopausal British Caucasian females and 43 age-matched, normal weight controls were studied. The obese subjects were characterized by fat distribution (waist to hip ratio), insulin secretion and insulin resistance (fasting insulin (FI)), an index of insulin resistance (HOMA), stimulated insulin secretion during an oral glucose tolerance test and insulin-mediated glucose disposal, steady-state plasma glucose). A BclI polymorphism (fragments of 4·5 and 2·3 kb) demonstrated significant association with indices of glucose metabolism in obesity; those subjects homozygous for the 4·5 kb fragment had elevated FI (Pc=0·012) and HOMA (Pc=0·012) values. The genotypic and allelic frequencies of the GRL BclI polymorphism were otherwise similar in obese and normal weight subjects. We postulate that the GRL BclI polymorphism may directly affect GR gene expression, or be in linkage disequilibrium with a possible mutation within one of three exons of the GR gene, and thereby modulate GR transcriptional activity on target genes involved in glucose and insulin homeostasis.