Regulation Of Hormonal Responses Research Articles

Oocyte-specific disruption of adrenomedullin 2 gene enhances ovarian follicle growth after superovulation.

Adrenomedullin 2 (ADM2), adrenomedullin (ADM), and calcitonin gene-related peptides (α- and β-CGRPs) signal through heterodimeric calcitonin receptor-like receptor/receptor activity-modifying protein 1, 2 and 3 (CLR/RAMP1, 2 and 3) complexes. These peptides are important regulators of neurotransmission, vasotone, cardiovascular development, and metabolic homeostasis. In rodents, ADM is essential for regulating embryo implantation, fetal-placental development, and hemodynamic adaptation during pregnancy. On the other hand, ADM2 was shown to affect vascular lumen enlargement, and cumulus cell-oocyte complex (COC) communication in rodent and bovine ovarian follicles. To investigate whether oocyte-derived ADM2 plays a physiological role in regulating ovarian folliculogenesis, we generated mice with oocyte-specific disruption of the Adm2 gene using a LoxP-flanked Adm2 transgene (Adm2 loxP/loxP) and crossed them with Zp3-Cre mice which carry a zona pellucida 3 (Zp3) promoter-Cre recombinase transgene. While heterozygous Adm2 +/-/Zp3-Cre and homozygous Adm2 -/-/Zp3-Cre mice were fertile, Adm2 disruption in oocytes significantly increased the number of ovulated oocytes following a superovulation treatment. Oocyte-specific Adm2 disruption also significantly impaired the developmental capacity of fertilized eggs and decreased the size of the corpus luteum following superovulation, perhaps due to a reduction of ovarian cyclin D2-associated signaling. The disruption of intrafollicular ADM2 signaling leads to follicular dysfunction. These data suggested that oocyte-derived ADM2 plays a facilitative role in the regulation of hormonal response and follicle growth independent of the closely related ADM and CGRP peptides, albeit in a subtle manner.

Identification, localization and expression analysis of beta-carotene isomerase gene family in sugarcane

<p indent=0mm>Strigolactones (SLs), a class of plant hormones, exists widely in plants and involves in the regulation of plant tillering and environmental adaptation. β-carotene isomerase (D27) is a key enzyme for SLs synthesis, but there are few reports about the identification and analysis of <italic>D27</italic> gene family in sugarcane. In this study, five members of <italic>D27</italic> gene family from one of the original parent of modern sugarcane cultivars, <italic>Saccharum spontaneum</italic>, were analyzed. Phylogenetic tree analysis showed that these D27s from <italic>S</italic>.<italic> spontaneum</italic> was clustered into 3 different branches and were highly homologous to sorghum D27s. Conserved domain prediction revealed that D27s contained a typical domain of β-carotene isomerase, Pfam: DUF4033. The results of cis-element analysis showed that<italic> D27s</italic> mainly involved in the regulation of hormone response, plant growth and development, and stress response. Based on the transcriptomic data of the modern cultivated varieties, the expression analysis of the homologous transcripts of <italic>Saccharum spontaneum D27s</italic> showed that <italic>Sspon</italic>.<italic>06G0012830-1A</italic> was regulated at tillering stage and under <italic>Sporisorium scitamineum </italic>infection. Besides, the cDNA sequence of <italic>Sspon</italic>.<italic>06G0012830-1A</italic> was cloned from sugarcane cultivar ROC22 and named as <italic>ScD27</italic>.<italic>1 </italic>(GenBank accession number: MT499895). Bioinformatics analysis indicated that <italic>ScD27</italic>.<italic>1</italic> encoded an unstable protein of 266 amino acids with an isoelectric point of 8.91 and a molecular weight of 30.00 kD, and might be located in the chloroplast, containing chloroplast transit peptides, 4 ubiquitination sites and 18 phosphorylation sites. Its secondary structure mainly included alpha helix and random coils. qRT-PCR analysis demonstrated that <italic>ScD27</italic>.<italic>1</italic> was significantly induced by ABA and H₂O₂ and did not respond to MeJA and SA. Subcellular localization revealed that ScD27.1 might be located in cell membrane and chloroplast and involved in the vesicle sorting or transported by sorting vesicle in plant cells. The results suggested that <italic>ScD27</italic>.<italic>1</italic> may involve in the tillering induced by <italic>S</italic>.<italic> scitamineum </italic>and signaling pathway of ABA and H₂O₂. The present study provides a basic understanding of intracellular transport and tillering in sugarcane and the involvement of <italic>ScD27</italic>.<italic>1 </italic>during sugarcane-<italic>S</italic>.<italic> scitamineum</italic> interaction.

Identification and Analysis of microRNAs in the SAM and Leaves of Populus tomentosa

The shoot apical meristem (SAM) is a crucial tissue located at the tops of plants which can continually grow and differentiate to develop into all aboveground parts. SAM development is controlled by a series of complicated molecular regulation networks, among which microRNAs (miRNAs) and their target genes play key roles. However, little is known about these miRNAs in woody plants. In this study, we used small RNA (sRNA) sequencing to build four libraries derived from shoot tips and mature leaf tissues of Populus tomentosa, and identified 99 known miRNA families. In addition, 193 known miRNAs, including phytohormone-, developmental-, and cellular process-related miRNAs, showed significant differential expression. Interestingly, quantitative real-time reverse transcription polymerase chain reaction (PCR) analysis of miR172, miR164, and miR393 expression showed marked changes in expression patterns during the development of shoot tips. The target genes of these miRNAs were involved in the regulation of hormone responses and stem cell function. In particular, the miR172 target APETALA2 (AP2), involved in the maintenance of stem cells in the shoot apex, was expressed specifically during the initial active stage of development. These findings provide new insights into the regulatory mechanisms of miRNAs involved in SAM development and differentiation in tree species.

Major Histocompatibility Complex Class I Core Promoter Elements Are Not Essential for Transcription in vivo

The role of core promoter elements in regulating transcription initiation is largely unknown for genes subject to complex regulation. Major histocompatibility complex class I genes are ubiquitously expressed and governed by tissue-specific and hormonal signals. Transcription initiates at multiple sites within the core promoter, which contains elements homologous to the canonical elements CCAAT, TATAA, Sp1 binding site (Sp1BS), and Initiator (Inr). To determine their functions, expression of class I transgenes with individually mutated elements was assessed. Surprisingly, all mutant promoters supported transcription. However, each mutated core promoter element had a distinct effect on expression: CAAT box mutations modulated constitutive expression in nonlymphoid tissues, whereas TATAA-like element mutations dysregulated transcription in lymphoid tissues. Inr mutations aberrantly elevated expression. Sp1BS element mutations resulted in variegated transgene expression. RNA polymerase II binding and histone H3K4me3 patterns correlated with transgene expression; H3K9me3 marks partially correlated. Whereas the wild-type, TATAA-like, and CAAT mutant promoters were activated by gamma interferon, the Sp1 and Inr mutants were repressed, implicating these elements in regulation of hormonal responses. These results lead to the surprising conclusion that no single element is required for promoter activity. Rather, each plays a distinct role in promoter activity, chromatin structure, tissue-specific expression, and extracellular signaling.

Hormonal regulation of the NF-κB signaling pathway

The NF-κB transcription factor modulates a number of gene responses to hormonal stimuli. NF-κB can be induced by growth promoting hormones and cytokines, has been shown to counteract the effectiveness of steroid hormones and has recently been found to be regulated during mammalian spermatogenesis. Recent advances in the characterization of the NF-κB signaling pathway offer new opportunities to examine how hormonal stimuli regulate NF-κB mediated gene expression. In this mini-review we outline the signal pathways responsible for activating NF-κB, discuss the hormonal regulation of NF-κB and the regulation of hormonal responses by NF-κB, as well as summarize new studies characterizing NF-κB expression and activity in the mammalian testis.

Progressive cytoplasmic tail truncations of the lutropin-choriogonadotropin receptor prevent agonist- or phorbol ester-induced phosphorylation, impair agonist- or phorbol ester-induced desensitization, and enhance agonist-induced receptor down-regulation.

Stably transfected human kidney 293 cells expressing the wild type rat LH/CG receptor (rLHR) or receptors with C-terminal tails truncated at residues 653, 631, or 628 (designated rLHR-t653, rLHR-t631, and rLHR-t628) were used to probe the importance of this region on the regulation of hormonal responsiveness. The chosen cells line express comparable densities of cell surface rLHR, bind human CG (hCG) with high affinity, and respond to hCG with increases in cAMP and inositol phosphate accumulation. Cells expressing rLHR-wt or rLHR-t653 responded to hCG, or phorbol 12-myristate-13-acetate (PMA) stimulation with a similar increase in rLHR phosphorylation. Neither of these two stimuli increased rLHR phosphorylation in cells expressing rLHR-t631 or rLHR-t628, however. The cell line expressing rLHR-t653, the phosphorylation-positive receptor mutant, desensitized normally in response to PMA or hCG stimulation. This truncated form of rLHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing rLHR-t631 or rLHR-t628, the two phosphorylation-negative receptor mutants, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the rLHR are involved in PMA-induced desensitization, hCG-induced de-sensitization, and hCG-induced down-regulation. These results also establish a positive correlation between rLHR phosphorylation and desensitization and a negative correlation between rLHR phosphorylation and down-regulation.

Effect of hepatic vagotomy on hormonal response to exercise in gluconeogenesis-inhibited rats

The liver, through the afferent pathway of the hepatic vagus nerve, has been reported to influence the hormonal response to exercise in adrenodemedullated rats. The present investigation was designed to evaluate the role played by gluconeogenesis in this hepatic influence. To this end, we studied the effects of a selective hepatic vagotomy on the hormonal response to a 30-min treadmill run (26 m/min, 0% grade) in adrenodemedullated rats injected with 3-mercaptopicolinic acid, a gluconeogenic inhibitor. Hepatic vagotomy was associated with small but significant higher (P less than 0.05) elevations of peripheral blood glucose levels at rest and after exercise. No significant differences were observed between hepatic-vagotomized and sham-operated rats in either resting or exercising levels of liver glycogen, portal glucose, portal and peripheral insulin and glucagon, and peripheral epinephrine and norepinephrine. All hormonal responses, with the exception of epinephrine, were either decreased or increased similarly in the two experimental conditions after exercise. These data indicate that contrary to what has been reported in rats with an intact capacity for gluconeogenesis, hepatic-vagotomized and sham-operated rats with an inhibited gluconeogenesis had similar hormonal responses to exercise. It is concluded that gluconeogenesis plays a role in the afferent neural influence exerted by the liver via the hepatic vagus nerve in the regulation of hormonal response to exercise.

Tubulin binds specifically to the signal-transducing proteins, Gs alpha and Gi alpha 1.

Participation of cytoskeletal elements in regulation of hormonal response and responsiveness has been suggested by several laboratories. Addition of dimeric tubulin to rat cerebral cortex synaptic membranes causes stable inhibition of adenylyl cyclase, and the molecular basis for this effect appears to require a direct interaction between tubulin and G proteins. To test whether such tubulin-G protein interaction occurred, several purified G proteins were bound to nitrocellulose, and 125I-tubulin overlay studies were performed. 125I-Tubulin bound to the alpha subunits of Gs and Gil with high specificity and an apparent Kd of approximately 130 nM. Other G protein alpha subunits (alpha i2, alpha i3, alpha 0, and transducin) displayed a much lower affinity for tubulin, despite the much closer relationship of those proteins to alpha il than to alpha s. Association of beta gamma subunits with alpha il or alpha s did not alter the binding of tubulin to these G protein heterotrimers, and the binding of a hydrolysis-resistant GTP analog to the alpha subunits was similarly without effect. These results suggest that tubulin forms complexes with specific G proteins and these complexes might provide a locus for the interaction of cytoskeletal components and signal transduction cascades. These results also provide evidence of a functional distinction among the closely related alpha i subtypes.

Bacterial toxins and the role of ADP-ribosylation.

Studies on the pathogenesis of "Whooping Cough" and cholera have resulted in the discovery of important pathways in the regulation of cellular metabolism leading to the realization of a complex family of proteins that appear to play central roles in the regulation of hormonal responses and which utilize guanine nucleotides in their mechanism of action. The fact that these bacterial toxins interfere so precisely with the complex regulation of eukaryotic cellular metabolism and the discovery of analogous enzymes within the cytosol of eukaryotic cells suggests that ADP-ribosylation may be an important pathway through which the cell can establish its responsiveness to its environment. Clearly, future work directed towards the role of ADP-ribosylation and towards the mechanisms of the regulation of these endogenous ADP-ribosyltransferases and lyases may provide great insights into the mechanisms of hormone action.

Regulation of hormonal responsiveness in LLC-PK1L cells grown in defined medium.

LLC-PK1L cells, a kidney-derived cell line, were able to grow in a chemically defined medium. Growth of the cells in the presence of retinol, ergocalciferol, d-alpha-tocopherol, 3,3',5-triiodothyronine, hydrocortisone, l-carnitine, d-l-methionine-S-methylsulfonium chloride, insulin, transferrin, cholesterol, and sodium linoleate increased the number of vasopressin receptors by 20- to 40-fold. All the newly detectable vasopressin receptors were coupled to the adenylate cyclase activity with similar efficiency. The same growth conditions did not alter the basal adenylate cyclase activity or the responses to calcitonin, parathyroid hormone, prostaglandin, adenosine, and GTP. In contrast, the increased responsiveness of the adenylate cyclase to vasopressin was associated with a reduced response to isoproterenol. Such an inverse correlation was also found when the time course of vasopressin receptor induction was studied. The supplemented medium permitted the growth of cells for several weeks. The effects of the enriched medium were fully reversible when we returned to the original cell growth medium. Thus such a cellular system appears as a useful tool for further work in cellular and kidney endocrinology and for detailing the molecular mechanisms of receptor-adenylate cyclase regulations.

Properties of gonadotropin-stimulable adenylyl cyclase of the human corpus luteum: regulation of hormonal responsiveness by guanyl nucleotide and magnesium ion.

We studied the properties of a gonadotropin-responsive adenylyl cyclase in membrane preparations obtained from human corpus luteum and explored the nature of guanyl nucleotide and Mg ion involvement in activation of the enzyme. Maximal adenylyl cyclase activity required ATP concentrations of 1-2.5 mM, total MgCl2 concentrations of 8-10 mM, and 1 mM EDTA. Optimal hCG responsiveness, however, required lower (approximately 5 mM) MgCl2 concentrations. Both GTP and its hydrolysis-resistant analog guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] increased enzyme activity, but the response to each nucleotide had distinct characteristics. The rate and the extent of activation were greater in the presence of GMP-P(NH)P than in that of GTP. Moreover, enzyme activation by GMP-P(NH)P was hysteretic in nature, requiring about 8 min to reach steady state velocity in the absence of hormonal stimuli. The slow rate of activation by GMP-P(NH)P was accelerated by either hCG or increases (3-10 mM) in the concentration of MgCl2. Thus, both gonadotropin and Mg ion are inherently antihysteretic in the human luteal adenylyl cyclase system. Basal and hCG stimulation were under the control of guanine nucleotides. Dose-response curves showed that the apparent activation constants for GTP and GMP-P(NH)P were 0.30 and 0.51 microM, respectively; these values did not shift after the addition of hCG. At a higher concentration of guanyl nucleotides (1000 microM), basal and hCG-stimulated activities were markedly reduced, suggesting bimodal regulation of the enzyme by the nucleotides. We also found that enzyme responsiveness to prostaglandin E2 was small and that, in contrast to a number of other nonprimate species, adenylyl cyclase from the human corpus luteum was not stimulated by isoproterenol. Taken together, these data support the usefulness of the cell-free model for studying the role of adenylyl cyclase in the regulation of luteal function in the human.