Loss of BMAL1 in ovarian steroidogenic cells results in implantation failure in female mice.

The circadian clock plays a significant role in many aspects of female reproductive biology, including estrous cycling, ovulation, embryonic implantation, onset of puberty, and parturition. In an effort to link cell-specific circadian clocks to their specific roles in female reproduction, we used the promoter that controls expression of Steroidogenic Factor-1 (SF1) to drive Cre-recombinase-mediated deletion of the brain muscle arnt-like 1 (Bmal1) gene, known to encode an essential component of the circadian clock (SF1-Bmal1(-/-)). The resultant SF1-Bmal1(-/-) females display embryonic implantation failure, which is rescued by progesterone supplementation, or bilateral or unilateral transplantation of wild-type ovaries into SF1-Bmal1(-/-) dams. The observation that the central clock, and many other peripheral clocks, are fully functional in this model allows the assignment of the implantation phenotype to the clock in ovarian steroidogenic cells and distinguishes it from more general circadian related systemic pathology (e.g., early onset arthropathy, premature aging, ovulation, late onset of puberty, and abnormal estrous cycle). Our ovarian transcriptome analysis reveals that deletion of ovarian Bmal1 disrupts expression of transcripts associated with the circadian machinery and also genes critical for regulation of progesterone production, such as steroidogenic acute regulatory factor (Star). Overall, these data provide a powerful model to probe the interlocking and synergistic network of the circadian clock and reproductive systems.

Follicular thyroid cancers demonstrate dual activation of PKA and mTOR as modeled by thyroid-specific deletion of Prkar1a and Pten in mice.

CONTEXT: Thyroid cancer is the most common form of endocrine cancer, and it is a disease whose incidence is rapidly rising. Well-differentiated epithelial thyroid cancer can be divided into papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Although FTC is less common, patients with this condition have more frequent metastasis and a poorer prognosis than those with PTC. OBJECTIVE: The objective of this study was to characterize the molecular mechanisms contributing to the development and metastasis of FTC. DESIGN: We developed and characterized mice carrying thyroid-specific double knockout of the Prkar1a and Pten tumor suppressor genes and compared signaling alterations observed in the mouse FTC to the corresponding human tumors. SETTING: The study was conducted at an academic research laboratory. Human samples were obtained from academic hospitals. PATIENTS: Deidentified, formalin-fixed, paraffin-embedded (FFPE) samples were analyzed from 10 control thyroids, 30 PTC cases, five follicular variant PTC cases, and 10 FTC cases. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Mouse and patient samples were analyzed for expression of activated cAMP response element binding protein, AKT, ERK, and mammalian target of rapamycin (mTOR). Murine FTCs were analyzed for differential gene expression to identify genes associated with metastatic progression. RESULTS: Double Prkar1a-Pten thyroid knockout mice develop FTC and recapitulate the histology and metastatic phenotype of the human disease. Analysis of signaling pathways in FTC showed that both human and mouse tumors exhibited strong activation of protein kinase A and mTOR. The development of metastatic disease was associated with the overexpression of genes required for cell movement. CONCLUSIONS: These data imply that the protein kinase A and mTOR signaling cascades are important for the development of follicular thyroid carcinogenesis and may suggest new targets for therapeutic intervention. Mouse models paralleling the development of the stages of human FTC should provide important new tools for understanding the mechanisms of FTC development and progression and for evaluating new therapeutics.

Hypoglycemia, hyperglucagonemia, and fetoplacental defects in glucagon receptor knockout mice: a role for glucagon action in pregnancy maintenance.

Alterations in insulin signaling as well as insulin action predispose to infertility as well as adverse pregnancy outcomes; however, little is known about the role of glucagon signaling in reproduction. The glucagon receptor knockout (Gcgr(-/-)) mouse created by our laboratory was used to define the role of glucagon signaling in maintaining normal reproduction. In this mouse model, lack of glucagon signaling did not alter the hypothalamic-pituitary-ovarian axis. Pregnant Gcgr(-/-) female mice displayed persistent hypoglycemia and hyperglucagonemia. Gcgr(-/-) pregnancies were associated with decreased fetal weight, increased late-gestation fetal demise, and significant abnormalities of placentation. Gcgr(-/-) placentas contained areas of extensive mineralization, fibrinoid necrosis, narrowing of the vascular channels, and a thickened interstitium associated with trophoblast hyperplasia. Absent glucagon signaling did not alter glycogen content in Gcgr(-/-) placentas but significantly downregulated genes that control growth, adrenergic signaling, vascularization, oxidative stress, and G protein-coupled receptors. Our data suggest that, similarly to insulin, glucagon action contributes to normal female reproductive function.

Pcsk6 mutant mice exhibit progressive loss of ovarian function, altered gene expression, and formation of ovarian pathology.

Bioactivation of precursor proteins by members of the proprotein convertase (PC) family is essential for normal reproduction. The Pcsk6 gene is a member of the PC family that is expressed in numerous ovarian cell types including granulosa cells and oocytes. We hypothesized that loss of PCSK6 would produce adverse effects in the mouse ovary. Mice incapable of expressing PCSK6 (Pcsk6(tm1Rob)) were obtained, and reproductive parameters (serum hormones, whelping interval, estrus cyclicity, and fertility) were compared to Pcsk6(+/+) mice. While Pcsk6(tm1Rob) female mice are fertile, they manifest reduced reproductive capacity at an accelerated rate relative to Pcsk6(+/+) mice. Reproductive senescence is typically reached by 9 months of age and is correlated with loss of estrus cyclicity, elevated serum FSH levels, and gross alterations in ovarian morphology. A wide range of ovarian morphologies were identified encompassing mild, such as an apparent reduction in follicle number, to moderate--ovarian atrophy with a complete absence of follicles--to severe, manifesting as normal ovarian structures replaced by benign ovarian tumors, including tubulostromal adenomas. Targeted gene expression profiling highlighted changes in RNA expression of molecules involved in processes such as steroidogenesis, gonadotropin signaling, transcriptional regulation, autocrine/paracrine signaling, cholesterol handling, and proprotein bioactivation. These results show that PCSK6 activity plays a role in maintaining normal cellular and tissue homeostasis in the ovary.

Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth.

The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M(3) muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M(3) receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M(3) receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M(3) receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.

Expression of a gonadotropin-releasing hormone receptor-simian virus 40 T-antigen transgene has sex-specific effects on the reproductive axis.

The GnRH receptor (GnRHR) responds to pulsatile GnRH signals to coordinate pituitary gonadotropin synthesis and secretion. Previously, a 1.2-kb fragment of the 5'-flanking region isolated from the mouse GnRHR gene was shown to target expression to pituitary gonadotropes in vivo. The 1.2-kb gene promoter fused to the simian virus 40 large T antigen (TAg) was used to generate transgenic mice that form gonadotrope-derived pituitary tumors at 4-5 months of age. Transgenic female mice have hypogonadotropic hypogonadism, infantile gonads, and are infertile throughout their life span, whereas males remain reproductively intact until their tumors become large. We hypothesized that the targeted TAg expression causes a sex-specific disruption of the reproductive axis at the level of the pituitary gland. To test this hypothesis, we characterized the pituitary gonadotropin beta-subunit and TAg expression patterns, and measured plasma gonadotropin and gonadal steroid levels in female and male mice before and after pituitary tumor development. TAg expression was observed in transgenic females and males 15 d of age, before tumor development. Interestingly, and in contrast to the transgenic males, pituitary LH beta and FSH beta subunit protein levels, and plasma LH and FSH levels, were reduced in transgenic females. Reproductive organs in transgenic female mice remained underdeveloped but were normal in transgenic males. We conclude that the expression of the TAg transgene driven by the GnRHR gene promoter results in female-specific infertility due to disruption of gonadotropin production and secretion even before tumor development.

Insensitivity of human prolactin receptors to nonhuman prolactins: relevance for experimental modeling of prolactin receptor-expressing human cells.

Prolactin (PRL) receptors are expressed in a broad range of human cell types and in a majority of human breast and prostate cancers. Experimentally, normal and malignant human cells are typically cultured in vitro in media containing bovine PRL (bPRL) from fetal bovine serum or as xenotransplants in vivo in the presence of murine PRL (mPRL). The biological efficacy of bPRL toward hPRL receptors (hPRLR) is controversial, and hPRLR are insensitive to mPRL, but the mechanism is not known. To clarify limitations of current in vitro and in vivo experimental model systems for studies of hPRLR-expressing cells, we tested human and relevant subprimate prolactins in multiple hPRLR bioassays. bPRL and ovine PRL were 10-fold less potent hPRLR agonists than hPRL, although maximal responses at high ligand concentrations (efficacies) equaled that of hPRL. mPRL and rat PRL had greater than 50-fold lower potencies toward hPRLR than hPRL and had 50% reduced efficacies. In fact, mPRL and rat PRL were less effective hPRLR agonists than murine GH. Unexpectedly, mPRL was an effective competitive inhibitor of hPRL binding to hPRLR with an inhibitory constant of 1.3 nm and showed partial antagonist activity, suggesting reduced site-2 binding. Collectively, low bioactivities of bPRL and mPRL toward hPRLR suggest that existing laboratory cancer cell lines grown in 10% bovine serum-supplemented media or in mice are selected for growth under lactogen-depleted conditions. The biology and drug responsiveness of existing human cell lines may therefore not be representative of clinical cancers that are sensitive to circulating PRL.

Steroid receptor coactivator 3 maintains circulating insulin-like growth factor I (IGF-I) by controlling IGF-binding protein 3 expression.

Steroid receptor coactivator 3 (SRC-3/AIB1/ACTR/NCoA-3) is a transcriptional coactivator for nuclear receptors including vitamin D receptor (VDR). Growth hormone (GH) regulates insulin-like growth factor I (IGF-I) expression, and IGF-I forms complexes with acid-labile subunit (ALS) and IGF-binding protein 3 (IGFBP-3) to maintain its circulating concentration and endocrine function. This study demonstrated that the circulating IGF-I was significantly reduced in SRC-3(-/-) mice with the C57BL/6J background. However, SRC-3 deficiency affected neither GH nor ALS expression. The low IGF-I level in SRC-3(-/-) mice was not due to the failure of IGF-I mRNA and protein synthesis but was a consequence of rapid degradation. The rapid IGF-I degradation was associated with drastically reduced IGFBP-3 levels. Because IGF-I and IGFBP-3 stabilize each other, SRC-3(-/-) mice were crossbred with the liver-specific transthyretin (TTR)-IGF-I transgenic mice to assess the relationship between reduced IGF-I and IGFBP-3. In SRC-3(-/-)/TTR-IGF-I mice, the IGF-I level was significantly increased over that in SRC-3(-/-) mice, but the IGFBP-3 level failed to increase proportionally, indicating that the low IGFBP-3 level is a responsible factor that limits the IGF-I level in SRC-3(-/-) mice. Furthermore, IGFBP-3 mRNA was reduced in SRC-3(-/-) mice. The IGFBP-3 promoter activity induced by vitamin D, through VDR, was diminished in SRC-3(-/-) cells, suggesting an important role of SRC-3 in VDR-mediated transactivation of the IGFBP-3 gene. In agreement with the role of SRC-3 in VDR function, the expression of several VDR target genes was also reduced in SRC-3(-/-) mice. Therefore, SRC-3 maintains IGF-I in the circulation through enhancing VDR-regulated IGFBP-3 expression.

Ovarian granulosa cell survival and proliferation requires the gonad-selective TFIID subunit TAF4b.

Oocyte development in the mammalian ovary requires productive interactions with somatic granulosa cells of the ovarian follicle. Proliferating granulosa cells support the progression of follicular growth and maturation, multiplying dramatically as it unfolds. The cell cycle recruitment of granulosa cells is regulated at least in part by hormones such as follicle-stimulating hormone (FSH) and estrogen. Follicles recruited into the growth phase following formation of multiple layers of granulosa cells have two major fates: either to continue proliferation followed by differentiation, or to die by programmed cell death, or atresia. While many of the signaling pathways orchestrating ovarian follicle development are known, the downstream transcriptional regulators that integrate such signals in the mammalian ovary remain to be defined. Recent experiments in diverse organisms have revealed multiple instances of gonad-selective components of the basal transcriptional machinery. One such protein, TAF4b, is a gonadal-enriched coactivator subunit of the TFIID complex required for normal female fertility in the mouse. To determine the etiology of female infertility of the TAF4b-deficient mice, we have determined multiple functions of TAF4b during postnatal ovarian follicle development. Here we demonstrate that the TAF4b protein is expressed in the granulosa cell compartment of the mammalian ovarian follicle. Furthermore, TAF4b-deficient mouse ovaries contain reduced numbers of primordial as well as growing follicles and a concomitant increased proportion of apoptotic follicles in comparison to wild type counterparts. Importantly, TAF4b-null follicles are largely resistant to induction of proliferation in response to multiple hormonal stimuli including estrogen and FSH and demonstrate compromised granulosa cell survival. Together, these data suggest that TAF4b integrates a program of granulosa cell gene expression required for normal ovarian follicle survival and proliferation in response to diverse ovarian signaling events.

Meiotic resumption in response to luteinizing hormone is independent of a Gi family G protein or calcium in the mouse oocyte.

The signaling pathway by which luteinizing hormone (LH) acts on the somatic cells of vertebrate ovarian follicles to stimulate meiotic resumption in the oocyte requires a decrease in cAMP in the oocyte, but how cAMP is decreased is unknown. Activation of Gi family G proteins can lower cAMP by inhibiting adenylate cyclase or stimulating a cyclic nucleotide phosphodiesterase, but we show here that inhibition of this class of G proteins by injection of pertussis toxin into follicle-enclosed mouse oocytes does not prevent meiotic resumption in response to LH. Likewise, elevation of Ca2+ can lower cAMP through its action on Ca2+-sensitive adenylate cyclases or phosphodiesterases, but inhibition of a Ca2+ rise by injection of EGTA into follicle-enclosed mouse oocytes does not inhibit the LH response. Thus, neither of these well-known mechanisms of cAMP regulation can account for LH signaling to the oocyte in the mouse ovary.

Mouse as a model of growth retardation in cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease that results in lung failure and premature death. A long recognized symptom of CF is growth failure, which is clinically relevant because it correlates with the severity of lung disease. We describe growth retardation in a mouse model of CF and discuss its potential for modeling certain aspects of human growth retardation. Mice with a null mutation in Cftr (cystic fibrosis transmembrance conductance regulator) were compared with wild-type (WT) mice at 31, 45, and 84 d of age. CF mice were severely growth retarded in weight and length compared with wild-type controls. Serum insulin like growth factor I (Igf-1) was lower in CF mice by 31-55% (depending on age and sex) and it significantly correlated with the size of mice after controlling for gender, age, and Cftr genotype. There was a marginally significant deficiency of serum growth hormone (Gh) in CF females, but not males. Our findings were consistent with models of an energy deficit in rodents. We, therefore, assessed food intake and found no difference between CF and WT mice, suggesting that CF mice had a malabsorption-mediated energy deficit. We argue that CF mice are suited to study the effects of intestinal disease on growth as well as other proposed growth-modulating processes.

Mice null for Frizzled4 (Fzd4-/-) are infertile and exhibit impaired corpora lutea formation and function.

Previous studies showed that transcripts encoding specific Wnt ligands and Frizzled receptors including Wnt4, Frizzled1 (Fzd1), and Frizzled4 (Fzd4) were expressed in a cell-specific manner in the adult mouse ovary. Overlapping expression of Wnt4 and Fzd4 mRNA in small follicles and corpora lutea led us to hypothesize that the infertility of mice null for Fzd4 (Fzd4-/-) might involve impaired follicular growth or corpus luteum formation. Analyses at defined stages of reproductive function indicate that immature Fzd4-/- mouse ovaries contain follicles at many stages of development and respond to exogenous hormone treatments in a manner similar to their wild-type littermates, indicating that the processes controlling follicular development and follicular cell responses to gonadotropins are intact. Adult Fzd4-/- mice also exhibit normal mating behavior and ovulate, indicating that endocrine events controlling these processes occur. However, Fzd4-/- mice fail to become pregnant and do not produce offspring. Histological and functional analyses of ovaries from timed mating pairs at Days 1.5-7.5 postcoitus (p.c.) indicate that the corpora lutea of the Fzd4-/- mice do not develop normally. Expression of luteal cell-specific mRNAs (Lhcgr, Prlr, Cyp11a1 and Sfrp4) is reduced, luteal cell morphology is altered, and markers of angiogenesis and vascular formation (Efnb1, Efnb2, Ephb4, Vegfa, Vegfc) are low in the Fzd4-/- mice. Although a recently identified, high-affinity FZD4 ligand Norrin (Norrie disease pseudoglioma homolog) is expressed in the ovary, adult Ndph-/- mice contain functional corpora lutea and do not phenocopy Fzd4-/- mice. Thus, Fzd4 appears to impact the formation of the corpus luteum by mechanisms that more closely phenocopy Prlr null mice.

Ovarian insufficiency and early pregnancy loss induced by activation of the innate immune system.

We describe a murine model of early pregnancy failure induced by systemic activation of the CD40 immune costimulatory pathway. Although fetal loss involved an NK cell intermediate, it was not due to lymphocyte-mediated destruction of the fetus and placenta. Rather, pregnancy failure resulted from impaired progesterone synthesis by the corpus luteum of the ovary, an endocrine defect in turn associated with ovarian resistance to the gonadotropic effects of prolactin. Pregnancy failure also required the proinflammatory cytokine TNF-alpha and correlated with the luteal induction of the prolactin receptor signaling inhibitors suppressor of cytokine signaling 1 (Socs1) and Socs3. Such links between immune activation and reproductive endocrine dysfunction may be relevant to pregnancy loss and other clinical disorders of reproduction.

Luteinizing hormone, a reproductive regulator that modulates the processing of amyloid-beta precursor protein and amyloid-beta deposition.

Hormonal changes associated with the dysregulation of the hypothalamic-pituitary-gonadal (HPG) axis following menopause/andropause have been implicated in the pathogenesis of Alzheimer's disease (AD). Experimental support for this has come from studies demonstrating an increase in amyloid-beta (Abeta) deposition following ovariectomy/castration. Because sex steroids and gonadotropins are both part of the HPG feedback loop, any loss in sex steroids results in a proportionate increase in gonadotropins. To assess whether Abeta generation was due to the loss of serum 17beta-estradiol or to the up-regulation of serum gonadotropins, we treated C57Bl/6J mice with the anti-gonadotropin leuprolide acetate, which suppresses both sex steroids and gonadotropins. Leuprolide acetate treatment resulted in a 3.5-fold (p < 0.0001) and a 1.5-fold (p < 0.024) reduction in total brain Abeta1-42 and Abeta1-40 concentrations, respectively, after 8 weeks of treatment. To further explore the role of gonadotropins in promoting amyloidogenesis, M17 neuroblastoma cells were treated with the gonadotropin luteinizing hormone (LH) at concentrations equivalent to early adulthood (10 mIU/ml) or post-menopause/andropause (30 mIU/ml). LH did not alter amyloid-beta precursor protein (AbetaPP) expression but did alter AbetaPP processing toward the amyloidogenic pathway as evidenced by increased secretion and insolubility of Abeta, decreased alphaAbetaPP secretion, and increased AbetaPP-C99 levels. These results suggest the marked increases in serum LH following menopause/andropause as a physiologically relevant signal that could promote Abeta secretion and deposition in the aging brain. Suppression of the age-related increase in serum gonadotropins using anti-gonadotropin agents may represent a novel therapeutic strategy for AD.

Expression of pituitary hormones in the Pax8-/- mouse model of congenital hypothyroidism.

Signaling mechanisms in pituitary morphogenesis as well as pituitary cell fate determination during early embryonic development are relatively well characterized. In contrast, the cues that determine the progression of the various anterior pituitary cell types during postnatal periods are poorly defined. Pax8-/- mice, which are born without a thyroid gland, were used to study the influence of thyroid hormones on the expression of pituitary hormones during early postnatal life. Serum pituitary hormones were determined by RIAs, and the pituitaries were analyzed by Northern blotting, in situ hybridization histochemistry, and immunocytochemistry. In 21-d-old Pax8-/- mice, the cellular composition of the anterior pituitary was dramatically distorted. Thyrotropes exhibited hypertrophy and hyperplasia, the number of detectable somatotropes was drastically reduced, and lactotropes were almost undetectable. Expression of LH and FSH was also reduced, but ACTH and proopiomelanocortin expression was not significantly different. Serum pituitary hormone levels were changed correspondingly. T(4) replacement therapy for variable time periods normalized TSH and GH mRNA expression within 3 d but not prolactin expression, not even when T(4) was administered for 6 d in combination with estradiol. These findings reveal the importance of thyroid hormones in developing the appropriate proportions of anterior pituitary cell types, especially with regard to lactotropes.

Diminished milk synthesis in upstream stimulatory factor 2 null mice is associated with decreased circulating oxytocin and decreased mammary gland expression of eukaryotic initiation factors 4E and 4G.

Previous studies have suggested that upstream stimulatory factors (USFs) regulate genes involved with cell cycle progression. Because of the relationship of USFs to an important oncogene in breast cancer, c-myc, we chose to determine the importance of USF to normal mammary gland development in the mouse. Expression of USF in the mammary gland throughout development demonstrated only modest changes. Mutation of the Usf2 gene was associated with reduced fertility in females, but had no effect on prepartum mammary gland development. However, lactation performance in Usf2-/- females was only half of that observed in Usf2+/+ females, and both lactose and nitrogen were decreased in milk from Usf2-/- dams. This decrease was associated with diminished mammary tissue wet weight and luminal area by d 9 of lactation and with a decreased protein-DNA ratio. This decrease was associated with reduced abundance of the eukaryotic initiation factors eIF4E and eIF4G. Blood oxytocin concentrations on d 9 postpartum were also lower in Usf2-/- mice than Usf2+/+ mice. In contrast, the mutation had no effect on blood prolactin concentrations, mammary cell proliferation or apoptosis, mammary tissue oxytocin receptors, or milk protein gene expression. The mutation had only modest effects on maternal behavior. These data support the idea that USF is important to physiological processes necessary for the establishment and maintenance of normal lactation and suggest that USF-2 may impact lactation through both systemic and mammary cell-specific mechanisms.

Genetic rescue of Cdk4 null mice restores pancreatic beta-cell proliferation but not homeostatic cell number.

Lack of Cdk4 expression in mice leads to insulin-deficient diabetes and female infertility owing to a reduced number of pancreatic beta cells and prolactin-producing pituitary lactotrophs, respectively. Cdk4 null mice display also reduced body and organ size. Here, we show that Cdk4 is essential for the postnatal proliferation of pancreatic beta cells but not for embryonic neogenesis from ductal epithelial cells. Re-expression of endogenous Cdk4 in beta cells and in the pituitary gland of Cdk4 null mice restores cell proliferation and results in fertile and normoglycemic animals, thus, demonstrating that the proliferation defects in these cellular populations are cell autonomous because of the lack of Cdk4 expression. However, these mice remain small in size, indicating that this phenotype is not because of pancreatic- or pituitary-mediated endocrine defects. This phenotype is a consequence of reduced cell numbers rather than reduced cell size. Thus, mammalian Cdk4 is not only involved in controlling proliferation of specific cell types but may play a wider role in establishing homeostatic cell numbers.

Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ERbeta in the uterus.

Estrogen is of great importance in the regulation of uterine function. The aim of this study was to examine the individual physiological roles of each of the two receptors for estradiol, estrogen receptor (ER) alpha and ERbeta, and their potential comodulatory effects on gene expression and uterine growth using recently developed ER subtype-selective agonist ligands. The use of ER subtype-selective ligands provides an alternative, complementary approach to the use of receptor knockout mice. Administration of the ERalpha-selective ligand propyl pyrazole triol (PPT) to immature mice resulted in a significant increase in uterine weight, as well as bromodeoxyuridine incorporation and proliferating cell nuclear antigen expression in luminal epithelial cells. PPT also increased complement component 3, lactoferrin, and glucose-6-phosphate dehydrogenase (G6PDH), and decreased androgen receptor (AR) and progesterone receptor (PR) mRNA levels in uterine tissue, as did estradiol (E(2)). However, when compared with E(2), PPT was less effective in stimulating uterine weight, complement component 3, and G6PDH expression but was as effective as E(2) in regulating lactoferrin, AR, and PR expression. In contrast to the action of the ERalpha agonist PPT, the ERbeta agonist diarylpropionitrile (DPN) did not increase uterine weight or luminal epithelial cell proliferation at a dose that reduced G6PDH and elicited a decrease in PR and AR mRNA and protein expression. Interestingly, DPN reduced the uterine weight stimulation by PPT, and enhanced the effect of PPT in decreasing uterine PR and AR mRNA. These findings with ER subtype-selective ligands indicate that ERalpha is the major regulator of estrogen function in the uterus, but that ERbeta does exert effects on some uterine markers of estrogen action. In addition, ERbeta can modulate ERalpha activity in a response-specific and dose-dependent manner.

Activin induces x-zone apoptosis that inhibits luteinizing hormone-dependent adrenocortical tumor formation in inhibin-deficient mice.

Inhibin and activin are members of the transforming growth factor beta (TGF-beta) family of ligands produced and secreted primarily by the gonads and adrenals. Inhibin-null (INH(-/-)) mice develop gonadal tumors and-when gonadectomized-adrenocortical carcinoma. The mechanisms leading to adrenal tumorigenesis have been proposed to involve the lack of a gonadal factor and/or a compensatory increase in gonadotropins. In order to achieve elevation of gonadotropins without the concomitant loss of a gonadal hormone, we crossed INH(-/-) mice with a transgenic mouse strain that has chronically elevated luteinizing hormone (LH) levels (LH-CTP). Compound INH(-/-)-LH-CTP mice die within 6 weeks of age from severe cancer cachexia induced by large, activin-secreting ovarian tumors. Unexpectedly, INH(-/-)-LH-CTP mice not only fail to develop adrenal tumors but have smaller adrenals, with a regressed x zone, indicating that elevated LH levels are not sufficient to induce adrenal tumor formation. However, following gonadectomy, INH(-/-)-LH-CTP mice develop large, sex steroid-producing adrenal tumors that arise from the x zone, indicating a growth-promoting effect of high levels of LH on the adrenal cortex in the absence of ovarian tumors. In addition, in vivo and in vitro data indicate that activin induces apoptosis specifically in the adrenal x zone. The restricted expression of activin receptor subunits and Smad2 in cells of the adrenal x zone, together with the elevated activin levels in INH(-/-)-LH-CTP mice, supports the conclusion that activin inhibits adrenal tumor growth by inducing x-zone regression.

Interleukin-6 inhibits hepatic growth hormone signaling via upregulation of Cis and Socs-3.

Cytokines may cause an acquired growth hormone (GH) resistance in patients with inflammatory diseases. Anabolic effects of GH are mediated through activation of STAT5 transcription factors. We have reported that TNF-alpha suppresses hepatic GH receptor (GHR) gene expression, whereas the cytokine-inducible SH2-containing protein 1 (Cis)/suppressors of cytokine signaling (Socs) genes are upregulated by TNF-alpha and IL-6 and inhibit GH activation of STAT5. However, the relative importance of these mechanisms in inflammatory GH resistance was not known. We hypothesized that IL-6 would prevent GH activation of STAT5 and that this would involve Cis/Socs protein upregulation. GH +/- LPS was administered to TNF receptor 1 (TNFR1) or IL-6 null mice and wild-type (WT) controls. STAT5, STAT3, GHR, Socs 1-3, and Cis phosphorylation and abundance were assessed by using immunoblots, EMSA, and/or real time RT-PCR. TNF-alpha and IL-6 abundance were assessed by using ELISA. GH activated STAT5 in WT and TNFR1 or IL-6 null mice. LPS pretreatment prevented STAT5 activation in WT and TNFR1 null mice; however, STAT5 activation was preserved in IL-6 null mice. GHR abundance did not change with LPS administration. Inhibition of STAT5 activation by LPS was temporally associated with phosphorylation of STAT3 and upregulation of Cis and Socs-3 protein in WT and TNFR1 null mice; STAT3, Cis, and Socs-3 were not induced in IL-6 null mice. IL-6 inhibits hepatic GH signaling by upregulating Cis and Socs-3, which may involve activation of STAT3. Therapies that block IL-6 may enhance GH signaling in inflammatory diseases.