Mark A. Lawson

Society for Cardiovascular Magnetic Resonance guidelines for reporting cardiovascular magnetic resonance examinations

These reporting guidelines are recommended by the Society for Cardiovascular Magnetic Resonance (SCMR) to provide a framework for healthcare delivery systems to disseminate cardiac and vascular imaging findings related to the performance of cardiovascular magnetic resonance (CMR) examinations.

GATA factors are essential for activity of the neuron-specific enhancer of the gonadotropin-releasing hormone gene.

The multicomponent neuron-specific enhancer of the gonadotropin-releasing hormone (GnRH) gene specifically targets expression to the GnRH-secreting neurons of the hypothalamus, a small population of specialized cells which play a central role in regulating reproductive function. Utilizing the GnRH-secreting hypothalamic neuronal cell line, GT1, as a model system, we show that members of the GATA family of transcription factors regulate GnRH transcription through two GATA factor-binding motifs that occur in a tandem repeat within the GnRH neuron-specific enhancer. Although GT1 cells contain GATA-2 and GATA-4 mRNAs, only GATA-4 was detected in a GnRH enhancer GATA site-specific complex. Cotransfection experiments with wild-type and mutant GnRH enhancer reporter plasmids with wild-type and dominant negative GATA factor expression vectors demonstrated that both GATA-binding elements are functional in the context of the enhancer. We conclude that GATA-binding proteins are important factors in regulating the neuron-specific expression of the GnRH gene in hypothalamic cells. Although the presence of GATA-2 in a neuronal cell type is not unusual, the presence of GATA-4 in GT1 cells is novel for a neuronal cell type. However, the presence of GATA-4 is consistent with the unique developmental origin of GnRH neurons and may provide insight into the transcriptional mechanisms mediating the differentiation of this limited population of GnRH-secreting neurons.

Evidence for Insulin Suppression of Baseline Luteinizing Hormone in Women with Polycystic Ovarian Syndrome and Normal Women

CONTEXT In women with polycystic ovarian syndrome (PCOS), the relationship of insulin to LH secretion and responses to GnRH remains unresolved. A rigorous analytical examination of this relationship has not been performed. OBJECTIVE Our objective was to determine the relationship of basal LH secretion and responses to GnRH, insulin, and other endocrine variables in normal and PCOS women. DESIGN In PCOS and normal women, mean composite 12-h LH secretion was analyzed for correlating factors. LH responses to varying doses of GnRH during a fixed rate of insulin infusion and LH responses to a fixed dose of GnRH during varying doses of insulin infusion were analyzed for contributing factors. PATIENTS AND SETTING Eighteen PCOS and 21 normal women underwent studies of frequent blood sampling and GnRH stimulation before and during insulin infusion at the General Clinical Research Center, University of California, San Diego. MAIN OUTCOME MEASURES Group mean composite 12-h LH levels were assessed with respect to other endocrine variables. In addition, LH responses to GnRH with or without insulin infusion were assessed. RESULTS In normal women, insulin negatively predicted mean LH. In PCOS, the combined effect of body mass index (negative) and testosterone (positive) predicted LH. The best predictor of LH was body mass index and insulin combined. Basal LH and LH responses to GnRH were unaltered by insulin infusion in normal women. These measures were reduced during insulin infusion in PCOS women. CONCLUSIONS In PCOS, insulin infusion suppresses pituitary response to GnRH. In normal women, insulin negatively correlates with mean LH and suppresses GnRH response at a high infusion rate.

Serum anti-mullerian hormone concentrations are elevated in oligomenorrheic girls without evidence of hyperandrogenism.

CONTEXT Serum anti-Müllerian hormone (AMH) levels are significantly elevated in adolescents with polycystic ovary syndrome (PCOS) compared to normal controls. Whether adolescents with oligomenorrhea have elevated AMH levels is unknown. OBJECTIVE This study was performed to assess serum AMH levels in oligomenorrheic (OLIGO) girls without evidence of hyperandrogenism. DESIGN This was a prospective study comparing AMH levels in OLIGO, PCOS, and normal control adolescents. SETTING The study was conducted through four tertiary academic medical centers. PARTICIPANTS The study groups were comprised of OLIGO (n = 24), PCOS (n = 153), and normal adolescent girls (n = 39), as well as PCOS (n = 73) and normal adult women (n = 36). INTERVENTIONS In each subject, serum AMH levels were assessed in the early to midfollicular phases for regularly menstruating subjects and on an arbitrary day for OLIGO or PCOS subjects. MAIN OUTCOME MEASURE(S) Basal serum AMH levels were assessed among OLIGO, PCOS, and normal girls, in addition to PCOS and normal women. RESULTS OLIGO girls had serum AMH levels (5.33 +/- 0.47 ng/ml) that were significantly greater than the normal adolescents (3.05 +/- 0.31 ng/ml) and adults (2.33 +/- 0.22 ng/ml), but similar to values seen in the PCOS adolescents (5.28 +/- 0.26 ng/ml) and adults (6.36 +/- 0.47 ng/ml). Obese adolescents and PCOS women had significantly lower AMH levels compared to lean controls (P < 0.02). CONCLUSION In OLIGO adolescents, elevated serum AMH levels suggest increased antral follicle number similar to that observed in girls with PCOS.

hCG-induced endoplasmic reticulum stress triggers apoptosis and reduces steroidogenic enzyme expression through activating transcription factor 6 in Leydig cells of the testis.

Endoplasmic reticulum (ER) stress generally occurs in secretory cell types. It has been reported that Leydig cells, which produce testosterone in response to human chorionic gonadotropin (hCG), express key steroidogenic enzymes for the regulation of testosterone synthesis. In this study, we analyzed whether hCG induces ER stress via three unfolded protein response (UPR) pathways in mouse Leydig tumor (mLTC-1) cells and the testis. Treatment with hCG induced ER stress in mLTC-1 cells via the ATF6, IRE1a/XBP1, and eIF2α/GADD34/ATF4 UPR pathways, and transient expression of 50 kDa protein activating transcription factor 6 (p50ATF6) reduced the expression level of steroidogenic 3β-hydroxysteroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD) enzyme. In an in vivo model, high-level hCG treatment induced expression of p50ATF6 while that of steroidogenic enzymes, especially 3β-HSD, 17α-hydroxylase/C17-20 lyase (CYP17), and 17β-hydrozysteroid dehydrogenase (17β-HSD), was reduced. Expression levels of steroidogenic enzymes were restored by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Furthermore, lentivirus-mediated transient expression of p50ATF6 reduced the expression level of 3β-HSD in the testis. Protein expression levels of phospho-JNK, CHOP, and cleaved caspases-12 and -3 as markers of ER stress-mediated apoptosis markedly increased in response to high-level hCG treatment in mLTC-1 cells and the testis. Based on transmission electron microscopy and H&E staining of the testis, it was shown that abnormal ER morphology and destruction of testicular histology induced by high-level hCG treatment were reversed by the addition of TUDCA. These findings suggest that hCG-induced ER stress plays important roles in steroidogenic enzyme expression via modulation of the ATF6 pathway as well as ER stress-mediated apoptosis in Leydig cells.

EXPRESSION OF GATA-4 IN MIGRATING GONADOTROPIN-RELEASING NEURONS OF THE DEVELOPING MOUSE

The hypothalamic gonadotropin-releasing hormone (GnRH) neurons are important regulators of reproductive function. During development, these cells arise in the olfactory placode and migrate to the central nervous system, where they form a diffuse population of neurosecretory cells that mediate central nervous system control of reproduction. Little is known of the mechanisms regulating the differentiation of these cells. Studies of the transcriptional regulation of the GnRH gene have demonstrated the importance of the GATA family of zinc-finger transcription factors in gene expression. Although GATA factors are not highly expressed in mature GnRH-secreting neurons, we report that GATA-4 is highly expressed in migrating GnRH neurons in the developing mouse. We also report that a second DNA binding activity regulating GnRH gene expression at the site of GATA-factor action persists in mature hypothalamus and may also play a role in gene expression in the differentiated GnRH neuron.

Activin A regulation of gonadotropin-releasing hormone synthesis and release in vitro.

Activin is essential for the regulation of normal mammalian reproductive function at both the pituitary and gonadal levels. However, its central actions in the control of the hypothalamic-pituitary-gonadal axis remain largely unexplored. The present study aims to determine whether activin could regulate the reproductive axis at the level of the hypothalamus, through control of the GnRH neuroendocrine system. Using the GnRH-secreting GT1-7 neuronal cell line as a model system, we demonstrate expression of mRNAs encoding activin receptor types I, IB, and II. We examined the effects of activin A on GnRH protein secretion and mRNA levels in GT1-7 cells. Treatment with rh-activin A regulated both GnRH protein secretion and GnRH mRNA expression in the GT1-7 cells in a time-dependent fashion. Using transient transfection assays, we explored a potential transcriptional basis for these changes. Activin A increased reporter gene activity driven by minimal GnRH enhancer and promoter elements, suggesting that activin may regulate GnRH gene expression at the level of transcription. Lastly, activin A treatment of male rat hypothalami, in vitro, increased GnRH protein secretion. Collectively, molecular and physiological evidence support the presence of an activin system which might act at a hypothalamic site to regulate mammalian reproduction via activation of GnRH synthesis and release.

Gonadotropin-Releasing Hormone Pulse Sensitivity of Follicle-Stimulating Hormone-β Gene Is Mediated by Differential Expression of Positive Regulatory Activator Protein 1 Factors and Corepressors SKIL and TGIF1

Gonadotropin synthesis and release is dependent on pulsatile stimulation by the hypothalamic neuropeptide GnRH. Generally, slow GnRH pulses promote FSH production, whereas rapid pulses favor LH, but the molecular mechanism underlying this pulse sensitivity is poorly understood. In this study, we developed and tested a model for FSHβ regulation in mouse LβT2 gonadotropes. By mining a previous microarray data set, we found that mRNA for positive regulators of Fshb expression, such as Fos and Jun, were up-regulated at slower pulse frequencies than a number of potential negative regulators, such as the corepressors Skil, Crem, and Tgif1. These latter corepressors reduced Fshb promoter activity whether driven by transfection of individual transcription factors or by treatment with GnRH and activin. Overexpression of binding or phosphorylation-defective ski-oncogene-like protein (SKIL) and TG interacting factor (TGIF1) mutants, however, failed to repress Fshb promoter activity. Knockdown of the endogenous repressors SKIL and TGIF1, but not cAMP response element-modulator, increased Fshb promoter activity driven by constant GnRH or activin. Chromatin immunoprecipitation analysis showed that FOS, SKIL, and TGIF1 occupy the FSHβ promoter in a cyclical manner after GnRH stimulation. Overexpression of corepressors SKIL or TGIF1 repressed induction of the Fshb promoter at the slow GnRH pulse frequency but had little effect at the fast pulse frequency. In contrast, knockdown of endogenous SKIL or TGIF1 selectively increased Fshb mRNA at the fast GnRH pulse frequency. Therefore, we propose a potential mechanism by which production of gonadotropin Fshb is modulated by positive transcription factors and negative corepressors with different pulse sensitivities.

Insulin Augments Gonadotropin-Releasing Hormone Induction of Translation in LβT2 Cells

The integrated signaling of insulin and gonadotropin-releasing hormone in the pituitary gonadotropes may have a profound bearing on reproductive function, although the cross-receptor signaling mechanisms are unclear. We demonstrate that the insulin receptor is constitutively localized to non-caveolar lipid raft microdomains in the pituitary gonadotrope cell line LbetaT2. The localization to rafts is consistent with similar localization of the GnRH receptor. Insulin receptor phosphorylation occurs in raft domains and activates the downstream signaling targets Insulin Receptor Substrate1 and Akt/Protein Kinase B. Although insulin alone does not strongly activate the extracellular signal-regulated kinase second messenger cascade, co-stimulation potentiates the phosphorylation of the extracellular signal-regulated kinase by gonadotropin-releasing hormone. The co-stimulatory effect of insulin and gonadotropin-releasing hormone is also evident in increased activation of cap-dependent translation. In contrast, co-stimulation attenuates Akt/Protein Kinase B activation. Our results show that both gonadotropin-releasing hormone and insulin are capable of mutually altering their respective regulatory signaling cascades. We suggest that this provides a mechanism to integrate neuropeptide and energy homeostatic signals to modulate reproductive function.

A Proteomic Comparison of Immature and Mature Mouse Gonadotrophs Reveals Novel Differentially Expressed Nuclear Proteins that Regulate Gonadotropin Gene Transcription and RNA Splicing

Abstract The alphaT3–1 and LbetaT2 gonadotroph cell lines contain all the known factors required for expression of gonadotropin genes, yet only the LbetaT2 cells express the beta subunits. We hypothesized that comparison of their nuclear proteomes would reveal novel proteins and/or modifications that regulate expression of these genes. We identified nine proteins with different expression profiles in the two cell lines, of which several were chosen for further functional studies. Of those found at higher levels in alphaT3–1 nuclei, 1110005A23RIK was found associated with the Fshb gene promoter and repressed its expression. Transgelin 3 overexpression reduced transcript levels of Fshb, and its knockdown elevated Lhb and Cga transcript levels, indicating an ongoing repressive effect on these more highly expressed genes, possibly through altering levels of phosphorylated mitogen-activated protein kinase. Heterogeneous nuclear ribonucleoprotein A2/B1 repressed splicing of the Fshb primary transcript, which it binds in the first intron. Proteins at higher levels in LbetaT2 nuclei included prohibitin, the overexpression of which reduced promoter activity of all three gonadotropin subunits, and appeared to mediate the differential effect of GnRH on proliferation of the two cell lines; its knockdown also altered cell morphology. Two other splicing factors were also found at higher levels in LbetaT2 nuclei: the knockdown of PRPF19 or EIF4A3 decreased splicing of Lhb, or of both beta subunit transcripts, respectively. The levels of Eif4a3 mRNA were increased by activin, and both factors increased Fshb splicing. This study has revealed a number of novel factors that alter gonadotropin expression and gonadotroph function, and likely mediate or moderate effects of the regulatory hormones.