Adolescent growth: genes, hormones and the peer group. Proceedings of the 20th Aschauer Soiree, held at Glücksburg castle, Germany, 15th to 17th November 2013.

The association between poverty, malnutrition, illness and poor socioeconomic conditions on the one side, and poor growth and short adult stature on the other side, is well recognized. Yet, the simple assumption by implication that poor growth and short stature result from poor living conditions, should be questioned. Recent evidence on the impact of the social network on adolescent growth and adult height further challenges the traditional concept of growth being a mirror of health. Twenty-nine scientists met at Glücksburg castle, Northern Germany, November 15th - 17th 2013, to discuss genetic, endocrine, mathematical and psychological aspects and related issues, of child and adolescent growth and final height.

Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2.

The actions of corticotropin-releasing hormone (Crh), a mediator of endocrine and behavioural responses to stress, and the related hormone urocortin (Ucn) are coordinated by two receptors, Crhr1 (encoded by Crhr) and Crhr2. These receptors may exhibit distinct functions due to unique tissue distribution and pharmacology. Crhr-null mice have defined central functions for Crhr1 in anxiety and neuroendocrine stress responses. Here we generate Crhr2-/- mice and show that Crhr2 supplies regulatory features to the hypothalamic-pituitary-adrenal axis (HPA) stress response. Although initiation of the stress response appears to be normal, Crhr2-/- mice show early termination of adrenocorticotropic hormone (Acth) release, suggesting that Crhr2 is involved in maintaining HPA drive. Crhr2 also appears to modify the recovery phase of the HPA response, as corticosterone levels remain elevated 90 minutes after stress in Crhr2-/- mice. In addition, stress-coping behaviours associated with dearousal are reduced in Crhr2-/- mice. We also demonstrate that Crhr2 is essential for sustained feeding suppression (hypophagia) induced by Ucn. Feeding is initially suppressed in Crhr2-/- mice following Ucn, but Crhr2-/- mice recover more rapidly and completely than do wild-type mice. In addition to central nervous system effects, we found that, in contrast to wild-type mice, Crhr2-/- mice fail to show the enhanced cardiac performance or reduced blood pressure associated with systemic Ucn, suggesting that Crhr2 mediates these peripheral haemodynamic effects. Moreover, Crhr2-/- mice have elevated basal blood pressure, demonstrating that Crhr2 participates in cardiovascular homeostasis. Our results identify specific responses in the brain and periphery that involve Crhr2.

Gonadotroph-specific expression of metallothionein fusion genes in pituitaries of transgenic mice.

Transgenic mice expressing a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin in the anterior pituitary gland, a tissue that does not normally produce somatostatin. Immunoreactive somatostatin within the anterior pituitaries was found exclusively within gonadotrophs. Similarly, a metallothionein-human growth-hormone fusion gene was also expressed selectively in gonadotrophs. It is proposed that sequences common to the two fusion genes are responsible for the gonadotroph-specific expression.

Pituitary lactotroph hyperplasia and chronic hyperprolactinemia in dopamine D2 receptor-deficient mice.

Dopamine secreted from hypophysial hypothalamic neurons is a principal inhibitory regulator of pituitary hormone secretion. Mice with a disrupted D2 dopamine receptor gene had chronic hyperprolactinemia and developed anterior lobe lactotroph hyperplasia without evidence of adenomatous transformation. Unexpectedly, the mutant mice had no hyperplasia of the intermediate lobe melanotrophs. Aged female D2 receptor -/- mice developed uterine adenomyosis in response to prolonged prolactin exposure. These data reveal a critical role of hypothalamic dopamine in controlling pituitary growth and support a multistep mechanism for the induction and perpetuation of lactotroph hyperplasia, involving the lack of dopamine signaling, a low androgen/estrogen ratio, and a final autocrine or paracrine "feed-forward" stimulation of mitogenesis, probably by prolactin itself.

Alcohol preference and sensitivity are markedly reduced in mice lacking dopamine D2 receptors.

Although dopaminergic transmission has been strongly implicated in alcohol self-administration, the involvement of specific dopamine receptor subtypes has not been well established. We studied the ethanol preference and sensitivity of D2-receptor-deficient mice to directly evaluate whether dopamine D2 receptors contribute to alcohol (ethanol) consumption. We report a marked aversion to ethanol in these mice, relative to the high preference and consumption exhibited by wild-type littermates. Sensitivity to ethanol-induced locomotor impairment was also reduced in these mutant mice, although they showed a normal locomotor depressant response to the dopamine D1 antagonist SCH-23390. These data demonstrate that dopamine signaling via D2 receptors is an essential component of the molecular pathway determining ethanol self-administration and sensitivity.

Somatostatin is required for masculinization of growth hormone-regulated hepatic gene expression but not of somatic growth.

Pulsatile growth hormone (GH) secretion differs between males and females and regulates the sex-specific expression of cytochrome P450s in liver. Sex steroids influence the secretory dynamics of GH, but the neuroendocrine mechanisms have not been conclusively established. Because periventricular hypothalamic somatostatin (SST) expression is greater in males than in females, we generated knockout (Smst(-/-)) mice to investigate whether SST peptides are necessary for sexually differentiated GH secretion and action. Despite marked increases in nadir and median plasma GH levels in both sexes of Smst(-/-) compared with Smst(+/+) mice, the mutant mice had growth curves identical to their sibling controls and retained a normal sexual dimorphism in weight and length. In contrast, the liver of male Smst(-/-) mice was feminized, resulting in an identical profile of GH-regulated hepatic mRNAs between male and female mutants. Male Smst(-/-) mice show higher expression of two SST receptors in the hypothalamus and pituitary than do females. These data indicate that SST is required to masculinize the ultradian GH rhythm by suppressing interpulse GH levels. In the absence of SST, male and female mice exhibit similarly altered plasma GH profiles that eliminate sexually dimorphic liver function but do not affect dimorphic growth.

The contribution of endogenous opioids to food reward is dependent on sex and background strain.

Complex behaviors such as those associated with reward to unconditioned positive reinforcers are polygenic processes. In studies using genetically modified mice specific for the endogenous opioid systems an observed phenotype in a complex behavior is likely to be dependent on interacting genes which, in inbred mouse lines, influence that phenotype. To address this issue we examined operant responding for palatable food reinforcers in mice lacking the expression of beta-endorphin, enkephalin or both peptides congenic to two different genetic backgrounds; C57BL/6J and DBA/2J. These two inbred strains were chosen because their endogenous opioid states differ and they respond differently to exogenous opioids in many behavioral assays. We found that wildtype and mutant C57BL/6J mice acquired operant responding for food reinforcers faster than DBA/2J mice, regardless of their opioid genotype. Although wildtype DBA/2J mice had a significant deficit in acquisition of bar-pressing behavior to reach a pre-established performance criterion, no subsequent deficit was observed under two different schedules of reinforcement. Additionally, we found that mice lacking enkephalin had decreased motivation to bar press for palatable food reinforcers under a progressive ratio regardless of sex or background strain. In contrast, the only subset of beta-endorphin-deficient mice that had decreased motivation to bar press under a progressive ratio was males on the C57BL/6J background. Of the two classical endogenous opioid peptides with preferential activation of the mu opioid receptor, the knockout models would suggest that enkephalins play a more consistent role than beta-endorphin in mediating the motivation for food reward when tested under a progressive ratio.

The absence of endogenous beta-endorphin selectively blocks phosphorylation and desensitization of mu opioid receptors following partial sciatic nerve ligation.

Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the beta-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid beta-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice.

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

Hypothalamic-specific proopiomelanocortin deficiency reduces alcohol drinking in male and female mice.

Opioid receptor antagonist naltrexone reduces alcohol consumption and relapse in both humans and rodents. This study investigated whether hypothalamic proopiomelanocortin (POMC) neurons (producing beta-endorphin and melanocortins) play a role in alcohol drinking behaviors. Both male and female mice with targeted deletion of two neuronal Pomc enhancers nPE1 and nPE2 (nPE-/-), resulting in hypothalamic-specific POMC deficiency, were studied in short-access (4-h/day) drinking-in-the-dark (DID, alcohol in one bottle, intermittent access (IA, 24-h cycles of alcohol access every other day, alcohol vs. water in a two-bottle choice) and alcohol deprivation effect (ADE) models. Wild-type nPE+/+ exposed to 1-week DID rapidly established stable alcohol drinking behavior with more intake in females, whereas nPE-/- mice of both sexes had less intake and less preference. Although nPE-/- showed less saccharin intake and preference than nPE+/+, there was no genotype difference in sucrose intake or preference in the DID paradigm. After 3-week IA, nPE+/+ gradually escalated to high alcohol intake and preference, with more intake in females, whereas nPE-/- showed less escalation. Pharmacological blockade of mu-opioid receptors with naltrexone reduced intake in nPE+/+ in a dose-dependent manner, but had blunted effects in nPE-/- of both sexes. When alcohol was presented again after 1-week abstinence from IA, nPE+/+ of both sexes displayed significant increases in alcohol intake (ADE or relapse-like drinking), with more pronounced ADE in females, whereas nPE-/- did not show ADE in either sex. Our results suggest that neuronal POMC is involved in modulation of alcohol 'binge' drinking, escalation and 'relapse', probably via hypothalamic-mediated mechanisms, with sex differences.

Somatostatin receptors in wildtype and somatostatin deficient mice and their involvement in nitric oxide physiology in the retina.

The present study investigated the localization and density of somatostatin (SRIF) receptor subtypes (sst(1-5)) and SRIF-nitric oxide (NO()) interactions in the retina of wildtype [WT, (+/+)] and somatostatin deficient mice [SRIF (-/-)]. Immunohistochemistry and radioligand binding studies with subsequent autoradiography were performed. Monoclonal antibodies [SRIF, protein kinase C (rod bipolar cells marker), microtubule associated protein 1A (ganglion cell marker)] and polyclonal antibodies (anti-sst(1), sst(2A), sst(4) receptor) were applied to 10-14 microm sections of retinas fixed in paraformaldehyde. NADPH-diaphorase reactivity was assessed histochemically. [(125)I]LTT SRIF-28 alone or in the presence of MK678 (sst(2) agonist) and [(125)I]Tyr(3)-octreotide were employed to quantify sst(1-5), sst(1/4)and sst(2/5) receptor densities, respectively. sst(1), sst(2A), and sst(4) receptor immunoreactivities were observed in processes of the inner plexiform layer (IPL), rod bipolar, and in ganglion cells and processes, respectively, in WT and SRIF (-/-) mice. Specific [(125)I]LTT SRIF-28 and [(125)I]Tyr(3)-octreotide binding was increased significantly in SRIF (-/-) mice. NADPH-diaphorase staining was localized in photoreceptors and amacrine cells, but not rod bipolar and ganglion cells. Also, NADPH-diaphorase staining was not colocalized with sst(1), sst(2A) or sst(4) receptor immunoreactivity. These results demonstrate an upregulation of SRIF receptors in mice lacking SRIF, but no evident SRIF-NO(*) interaction was observed in the mouse retina.

Dopamine D4 receptor-knock-out mice exhibit reduced exploration of novel stimuli.

The involvement of dopamine neurotransmission in behavioral responses to novelty is suggested by reports that reward is related to increased dopamine activity, that dopamine modulates exploratory behavior in animals, and that Parkinson's disease patients report diminished responses to novelty. Some studies have reported that polymorphisms of the human dopamine D4 receptor (D4R) gene are associated with personality inventory measures of the trait called "novelty-seeking". To explore a potential role for the D4R in behavioral responses to novelty, we evaluated D4R-knock-out (D4R-/-) and wild-type (D4R+/+) mice in three approach-avoidance paradigms: the open field, emergence, and novel object tests. These three paradigms differ in the degree to which they elicit approach, or exploratory behavior, and avoidance, or anxiety-related behavior. Thus, we used these three tests to determine whether the D4R primarily influences the exploratory or the anxious component of responses to approach-avoidance conflicts. D4R-/- mice were significantly less behaviorally responsive to novelty than D4R+/+ mice in all three tests. The largest phenotypic differences were observed in the novel object test, which maximizes approach behavior, and the smallest phenotypic differences were found in the open field test, which maximizes avoidance behavior. Hence, D4R-/- mice exhibit reductions in behavioral responses to novelty, reflecting a decrease in novelty-related exploration.

The dopamine D2, but not D3 or D4, receptor subtype is essential for the disruption of prepulse inhibition produced by amphetamine in mice.

Brain dopamine (DA) systems are involved in the modulation of the sensorimotor gating phenomenon known as prepulse inhibition (PPI). The class of D2-like receptors, including the D2, D3, and D4 receptor subtypes, have all been implicated in the control of PPI via studies of DA agonists and antagonists in rats. Nevertheless, the functional relevance of each receptor subtype remains unclear because these ligands are not specific. To determine the relevance of each receptor subtype, we used genetically altered strains of "knock-out" mice lacking the DA D2, D3, or D4 receptors. We tested the effects of each knock-out on both the phenotypic expression of PPI and the disruption of PPI produced by the indirect DA agonist d-amphetamine (AMPH). No phenotypic differences in PPI were observed at baseline. AMPH significantly disrupted PPI in the D2 (+/+) mice but had no effect in the D2 (-/-) mice. After AMPH treatment, both DA D3 and D4 receptor (+/+) and (-/-) mice had significant disruptions in PPI. These findings indicate that the AMPH-induced disruption of PPI is mediated via the DA D2 receptor and not the D3 or D4 receptor subtypes. Uncovering the neural mechanisms involved in PPI will further our understanding of the substrates of sensorimotor gating and could lead to better therapeutics to treat gating disorders, such as schizophrenia.

Dopamine D4 receptor-deficient mice display cortical hyperexcitability.

The dopamine D(4) receptor (D(4)R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this dopamine receptor subtype has been difficult to explore because of the slow development of D(4)R agonists and antagonists the selectivity and efficacy of which have been rigorously demonstrated in vivo. We have attempted to overcome this limitation by taking a multidimensional approach to the characterization of mice completely deficient in this receptor subtype. Electrophysiological current and voltage-clamp recordings were performed in cortical pyramidal neurons from wild-type and D(4)R-deficient mice. The frequency of spontaneous synaptic activity and the frequency and duration of paroxysmal discharges induced by epileptogenic agents were increased in mutant mice. Enhanced synaptic activity was also observed in brain slices of wild-type mice incubated in the presence of the selective D(4)R antagonist PNU-101387G. Consistent with greater electrophysiological activity, nerve terminal glutamate density associated with asymmetrical synaptic contacts within layer VI of the motor cortex was reduced in mutant neurons. Taken together, these results suggest that the D(4)R can function as an inhibitory modulator of glutamate activity in the FC.

Increased pituitary vascular endothelial growth factor-a in dopaminergic D2 receptor knockout female mice.

Vascular endothelial growth factor (VEGF)-A is an important angiogenic cytokine in cancer and pathological angiogenesis and has been related to the antiangiogenic activity of dopamine in endothelial cells. We investigated VEGF expression, localization, and function in pituitary hyperplasia of dopamine D2 receptor (D2R)-knockout female mice. Pituitaries from knockout mice showed increased protein and mRNA VEGF-A expression when compared with wild-type mice. In wild-type mice, prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF expression and prolactin release, suggesting that dopamine inhibits pituitary VEGF expression. VEGF expression was also increased in pituitary cells from knockout mice, even though these cells proliferated less in vitro when compared with wild-type cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium proliferation assay, proliferating cell nuclear antigen expression, and [(3)H]thymidine incorporation. In contrast to other animal models, estrogen did not increase pituitary VEGF protein and mRNA expression and lowered serum prolactin secretion in vivo and in vitro in both genotypes. VEGF (10 and 30 ng/ml) did not modify pituitary cell proliferation in either genotype and increased prolactin secretion in vitro in estrogen-pretreated cells of both genotypes. But conditioned media from D2R(-/-) cells enhanced human umbilical vein cell proliferation, and this effect could be partially inhibited by an anti-VEGF antiserum. Finally, using dual-labeling immunofluorescence and confocal laser microscopy, we found that in the hyperplastic pituitaries, VEGF-A was mostly present in follicle-stellate cells. In conclusion, pituitary VEGF expression is under dopaminergic control, and even though VEGF does not promote pituitary cellular proliferation in vitro, it may be critical for pituitary angiogenesis through paracrine actions in the D2R knockout female mice.

Brain stimulation and morphine reward deficits in dopamine D2 receptor-deficient mice.

RATIONALE: The rewarding effects of lateral hypothalamic brain stimulation, various natural rewards, and several drugs of abuse are attenuated by D1 or D2 dopamine receptor (D1R or D2R) antagonists. Much of the evidence for dopaminergic involvement in rewards is based on pharmacological agents with limited or "relative" selectivity for dopamine receptor subtypes. Genetically engineered animal models provide a complementary approach to pharmacological investigations. OBJECTIVES: In the present study, we explored the contribution of dopamine D2Rs to (1) brain stimulation reward (BSR) and (2) the potentiation of this behavior by morphine and amphetamine using D2R-deficient mice. METHODS: Wild-type (D2Rwt), heterozygous (D2Rhet), and D2R knockout (D2Rko) mice were trained to turn a wheel for rewarding brain stimulation. Once equivalent rate-frequency curves were established, morphine-induced (0, 1.0, 3.0, and 5.6 mg/kg s.c.) and amphetamine-induced (0, 1.0, 2.0, and 4.0 mg/kg i.p.) potentiations of BSR were determined. RESULTS: The D2Rko mice required approximately 50% more stimulation than the D2Rwt mice did. With the equi-rewarding levels of stimulation current, amphetamine potentiated BSR equally across the three genotypes. In contrast, morphine potentiated rewarding stimulation in the D2Rwt, had no effect in the D2Rhet, and antagonized rewarding stimulation in the D2Rko mice. CONCLUSIONS: D2R elimination decreases, but does not eliminate, the rewarding effects of lateral hypothalamic stimulation. After compensation for this deficit, amphetamine continues to potentiate BSR, while morphine does not.

Gonadal steroid hormone regulation of human and mouse follicle stimulating hormone beta-subunit gene expression in vivo.

Transgenic mice that express a 10-kilobase human FSH beta (hFSH beta) gene exclusively in pituitary gonadotropes were used to study the regulation of hFSH beta gene expression by gonadal steroids. For comparison, the mouse FSH beta (mFSH beta) gene was studied in parallel in nontransgenic sibling (normal) mice. The hFSH beta gene showed a sexually dimorphic expression pattern, identical to mFSH beta, in the mouse environment. Intact normal and transgenic male mice had elevated (P < 0.05) levels of serum [16 +/- 2 ng/ml (normal); 38 +/- 6 (transgenic)] and pituitary FSH content [2 +/- 0.3 micrograms/mg protein (normal); 36 +/- 6 (transgenic)] and FSH beta mRNA [1.47 +/- 0.10 arbitrary density units (normal); 1.00 +/- 0.23 (transgenic)] compared to the corresponding female mice ([< 2.0 ng/ml (normal and transgenic)] [0.1 +/- 0.01 microgram/mg protein (normal); 0.2 +/- 0.03 (transgenic)] [< 0.03 arbitrary density units (normal and transgenic)]). Serum FSH levels were increased (P < 0.05) 2 weeks after castration of normal (22 +/- 2 ng/ml) and transgenic males (135 +/- 19 ng/ml) and were suppressed (P < 0.05) by testosterone [7 +/- 0.8 ng/ml (normal); 12 +/- 2 (transgenic)] or estradiol [14 +/- 1 ng/ml (normal); 16 +/- 1 (transgenic)] replacement. The increased serum FSH levels were associated with an inverse drop (P < 0.05) in pituitary FSH content to 1 +/- 0.1 microgram/mg protein in normal and 16 +/- 2 in transgenic males. Testosterone replacement further suppressed (P < 0.05) pituitary FSH content in transgenic (3 +/- 0.5 micrograms/mg protein) but not normal (1 +/- 0.1) males.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotrope-specific expression of the human follicle-stimulating hormone beta-subunit gene in pituitaries of transgenic mice.

The molecular basis for the cell-specific and hormonal regulation of the pituitary gonadotropin beta-subunit genes is unknown. To identify the essential DNA regulatory elements of the human FSH beta (hFSH beta) gene, we introduced a 10-kilobase cloned fragment encompassing the gene into the germline of transgenic mice. We showed by a combination of Northern blot hybridization, RIA, and immunofluorescence histochemistry that the hFSH beta gene was expressed exclusively in mouse pituitary gonadotropes. Human FSH beta was actively secreted, presumably as an interspecies heterodimer, with mouse alpha-subunit, and no free hFSH beta was detected in serum. Male transgenic mice consistently had higher basal pituitary expression and secretion of hFSH beta than the female transgenic mice. Castration combined with testosterone replacement caused a marked reduction in hFSH beta gene expression in transgenic males, but had no effect on the pituitary levels of FSH in normal male mice. These experiments demonstrate that the essential DNA regulatory elements and gonadotrope transcription factors required for cell-specific expression of the FSH beta gene must be conserved between human and mouse. There appear to be important differences in the hormonal regulation of the gene between species, however.

Pituitary-specific and hormonally regulated gene expression directed by the rat proopiomelanocortin promoter in transgenic mice.

All aspects of POMC biosynthesis exhibit tissue-specific regulation. The single copy gene is highly expressed in anterior lobe (AL) corticotrophs and intermediate lobe (IL) melanotrophs of the pituitary gland and in the arcuate nucleus of the hypothalamus. POMC gene transcription in corticotrophs is induced by hypothalamic CRH and vasopressin and inhibited by adrenal glucocorticoids, while in melanotrophs it is predominantly regulated by beta-adrenergic neural input and dopamine. To identify the rat POMC (rPOMC) gene sequences necessary and sufficient to target expression and hormonal regulation in corticotrophs and melanotrophs, we generated 13 transgenic mice carrying rPOMC fusion genes. The genes consisted of 706 or 480 basepairs of rPOMC 5' flanking sequences ligated to either the E. coli LacZ gene encoding beta-galactosidase or the K1 mutant of the SV40 large T-antigen gene. Overall, half of the transgenic lines had reporter gene expression in their AL and IL in a pattern indistinguishable from ACTH immunohistochemistry. In three of these lines, beta-galactosidase or K1 T-antigen was localized by double immunofluorescence exclusively to ACTH-positive corticotrophs and melanotrophs. Transcriptional regulation of the rPOMC-LacZ fusion gene in response to hormonal manipulation was quantified by a fluorescence assay for beta-galactosidase enzyme activity in pituitary extracts. There was a 15-fold increase in AL enzyme activity after adrenalectomy and a 3-fold increase in IL activity after haloperidol treatment. X-gal histochemistry of pituitaries from hormonally treated mice confirmed the cellular specificity of these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryptic human growth hormone gene sequences direct gonadotroph-specific expression in transgenic mice.

Our laboratory reported previously that chimeric genes encoding either rat somatostatin (SS) or human GH (hGH), but containing the identical mouse metallothionein-I (MT) promoter/enhancer sequences and hGH 3'-flanking sequences, were selectively expressed in the gonadotrophs of transgenic mice. The experiments reported here were designed to identify the DNA sequences responsible for this unexpected cell-specific expression within the anterior pituitary. We produced new transgenic mice expressing fusion genes that tested separately the requirement of the MT or 3'-hGH sequences for gonadotroph expression. A fusion gene that retained the original MT and SS sequences, with a simian virus 40 polyadenylation signal exchanged for the 3'-hGH sequences, no longer directed strong pituitary expression, but was active in the liver. In contrast, a cytomegalovirus promoter/enhancer-SS-hGH fusion gene was expressed at the same high level in the anterior pituitaries of transgenic mice as the originally studied MT-SS-hGH gene. Immunohistochemical analysis indicated that pituitary expression of the cytomegalovirus promoter/enhancer-SS-hGH fusion gene was also restricted to gonadotroph cells in adult mice. These studies indicate that sequences within the 3'-flanking region of the hGH gene can direct expression of chimeric genes to pituitary cells that do not normally produce growth hormone.