Modulation of Clock Gene Expression by the Transcriptional Coregulator Receptor Interacting Protein 140 (RIP140)

Circadian rhythms are generated in central and peripheral tissues by an intracellular oscillating timing mechanism known as the circadian clock. Several lines of evidence show a strong and bidirectional interplay between metabolism and circadian rhythms. Receptor interacting protein 140 (RIP140) is a coregulator for nuclear receptors and other transcription factors that represses catabolic pathways in metabolic tissues. Although RIP140 functions as a corepressor for most nuclear receptors, mounting evidence points to RIP140 as a dual coregulator that can repress or activate different sets of genes. Here, we demonstrate that RIP140 mRNA and protein levels are under circadian regulation and identify RIP140 as a modulator of clock gene expression, suggesting that RIP140 can participate in a feedback mechanism affecting the circadian clock. We show that the absence of RIP140 disturbs the basal levels of BMAL1 and other clock genes, reducing the amplitude of their oscillations. In addition, we demonstrate that RIP140 is recruited to retinoid-related orphan receptor (ROR) binding sites on the BMAL1 promoter, directly interacts with RORα, and increases transcription from the BMAL1 promoter in a RORα-dependent manner. These results indicate that RIP140 is not only involved in metabolic control but also acts as a coactivator for RORα, influencing clock gene expression

HS6ST1 Insufficiency Causes Self-Limited Delayed Puberty in Contrast With Other GnRH Deficiency Genes

Context: Self-limited delayed puberty (DP) segregates in an autosomal-dominant pattern, but the genetic basis is largely unknown. Although DP is sometimes seen in relatives of patients with hypogonadotropic hypogonadism (HH), mutations in genes known to cause HH that segregate with the trait of familial self-limited DP have not yet been identified. Objective: To assess the contribution of mutations in genes known to cause HH to the phenotype of self-limited DP. Design, Patients, and Setting: We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited DP, validated the pathogenicity of the identified gene variant in vitro, and examined the tissue expression and functional requirement of the mouse homolog in vivo. Results: A potentially pathogenic gene variant segregating with DP was identified in 1 of 28 known HH genes examined. This pathogenic variant occurred in HS6ST1 in one pedigree and segregated with the trait in the six affected members with heterozygous transmission (P = 3.01 x 10 -5 ). Biochemical analysis showed that this mutation reduced sulfotransferase activity in vitro. Hs6st1 mRNA was expressed in peripubertal wild-type mouse hypothalamus. GnRH neuron counts were similar in Hs6st1 (+/-) and Hs6st1(+/+) mice, but vaginal opening was delayed in Hs6st1(+/-) mice despite normal postnatal growth. Conclusions: We have linked a deleterious mutation in HS6ST1 to familial self-limited DP and show that heterozygous Hs6st1 loss causes DP in mice. In this study, the observed overlap in potentially pathogenic mutations contributing to the phenotypes of self-limited DP and HH was limited to this one gene.Peer reviewe

Contributions of Function-Altering Variants in Genes Implicated in Pubertal Timing and Body Mass for Self-Limited Delayed Puberty

Context: Self-limited delayed puberty (DP) is often associated with a delay in physical maturation, but although highly heritable the causal genetic factors remain elusive. Genome-wide association studies of the timing of puberty have identified multiple loci for age at menarche in females and voice break in males, particularly in pathways controlling energy balance. Objective/Main Outcome Measures: We sought to assess the contribution of rare variants in such genes to the phenotype of familial DP. Design/Patients: We performed whole-exome sequencing in 67 pedigrees (125 individuals with DP and 35 unaffected controls) from our unique cohort of familial self-limited DP. Using a whole-exome sequencing filtering pipeline one candidate gene [fat mass and obesity-associated gene (FTO)] was identified. In silico, in vitro, and mouse model studies were performed to investigate the pathogenicity of FTO variants and timing of puberty in FTO+/- mice. Results: We identified potentially pathogenic, rare variants in genes in linkage disequilibrium with genome-wide association studies of age at menarche loci in 283 genes. Of these, five genes were implicated in the control of body mass. After filtering for segregation with trait, one candidate, FTO, was retained. Two FTO variants, found in 14 affected individuals from three families, were also associated with leanness in these patients with DP. One variant (p. Leu44Val) demonstrated altered demethylation activity of the mutant protein in vitro. Fto(+/-) mice displayed a significantly delayed timing of pubertal onset (P <0.05). Conclusions: Mutations in genes implicated in body mass and timing of puberty in the general population may contribute to the pathogenesis of self-limited DP.Peer reviewe

Follicle-stimulating hormone promotes growth of human prostate cancer cell line-derived tumor xenografts

Chemical castration in prostate cancer can be achieved with gonadotropin-releasing hormone (GnRH) agonists or antagonists. Their effects differ by the initial flare of gonadotropin and testosterone secretion with agonists and the immediate pituitary-testicular suppression by antagonists. While both suppress luteinizing hormone (LH) and follicle-stimulating hormone (FSH) initially, a rebound in FSH levels occurs during agonist treatment. This rebound is potentially harmful, taken the expression of FSH receptors (R) in prostate cancer tissue. We herein assessed the role of FSH in promoting the growth of androgen-independent (PC-3, DU145) and androgen-dependent (VCaP) human prostate cancer cell line xenografts in nude mice. Gonadotropins were suppressed with the GnRH antagonist degarelix, and effects of add-back human recombinant FSH were assessed on tumor growth. All tumors expressed GnRHR and FSHR, and degarelix treatment suppressed their growth. FSH supplementation reversed the degarelix-evoked suppression of PC-3 tumors, both in preventive (degarelix and FSH treatment started upon cell inoculation) and therapeutic (treatments initiated 3 weeks after cell inoculation) setting. A less marked, though significant FSH effect occurred in DU145, but not in VCaP xenografts. FSHR expression in the xenografts supports direct FSH stimulation of tumor growth. Testosterone supplementation, to maintain the VCaP xenografts, apparently masked the FSH effect on their growth. Treatment with the LH analogue hCG did not affect PC-3 tumor growth despite their expression of luteinizing hormone/choriongonadotropin receptor. In conclusion, FSH, but not LH, may directly stimulate the growth of androgen-independent prostate cancer, suggesting that persistent FSH suppression upon GnRH antagonist treatment offers a therapeutic advantage over agonist

Ajmaline blocks INa and IKr without eliciting differences between Brugada syndrome patient and control human pluripotent stem cell-derived cardiac clusters

© 2017 The Authors The class Ia anti-arrhythmic drug ajmaline is used clinically to unmask latent type I ECG in Brugada syndrome (BrS) patients, although its mode of action is poorly characterised. Our aims were to identify ajmaline's mode of action in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), and establish a simple BrS hiPSC platform to test whether differences in ajmaline response could be determined between BrS patients and controls. Control hiPSCs were differentiated into spontaneously contracting cardiac clusters. It was found using multi electrode array (MEA) that ajmaline treatment significantly lengthened cluster activation-recovery interval. Patch clamping of single CMs isolated from clusters revealed that ajmaline can block both I Na and I Kr . Following generation of hiPSC lines from BrS patients (absent of pathogenic SCN5A sodium channel mutations), analysis of hiPSC-CMs from patients and controls revealed that differentiation and action potential parameters were similar. Comparison of cardiac clusters by MEA showed that ajmaline lengthened activation-recovery interval consistently across all lines. We conclude that ajmaline can block both depolarisation and repolarisation of hiPSC-CMs at the cellular level, but that a more refined integrated tissue model may be necessary to elicit differences in its effect between BrS patients and controls

Effects of Cd2+ on anterior pituitary cell viability and hormone release, mechanisms of action

Antecedentes: El Cd2+ es un importante contaminante ambiental. Desde el punto de vista endocrinológico se conoce que afecta los niveles séricos de hormonas adenohipofisarias en humanos, pero su mecanismo de acción permanece desconocido. Objetivos: Estudiar los mecanismos de acción del Cd2+ sobre la adenohipófisis utilizando un modelo de células en cultivo. Validar el modelo in vitro realizando estudios in vivo. Evaluar posibles estrategias terapéuticas. Resultados: El Cd2+ afectó de manera directa la viabilidad y la liberación de prolactina de las células adenohipofisarias. El Cd2+ produjo estrés oxidativo y aumento de los niveles citosólicos de Ca2+. Distintos antioxidante redujeron su efecto, tanto sobre la viabilidad como sobre la liberación hormonal. El Cd2+ aumentó la producción de NO, el cual fue citoprotector. El tratamiento in vivo con Cd2+ causó estrés oxidativo en la hipófisis y el hipotálamo y también aumentó la expresión de las óxido nítrico sintasas (NOS)1 y 2. La melatonina previno el daño oxidativo pero no los cambios a nivel hormonal. Conclusiones: El estrés oxidativo parece ser el principal mediador de los efectos citotóxicos del Cd2+ in vitro. El modelo in vitro parece reproducir los efectos in vivo del Cd2+. Aunque la melatonina previno el daño oxidativo no hizo lo mismo con los cambios a nivel hormonal. Esto sugiere que otros mecanismos estarían involucrados en el efecto del Cd2+ sobre la liberación hormonal in vivo. Aun así, los antioxidantes podrían servir, potencialmente, para el tratamiento de las intoxicaciones con Cd2+.Background: Cd2+ is an important environmental contaminant. Regarding the endocrine system, it is known that this metal affects serum levels of pituitary hormones in humans, but its mechanisms of action remain unknown. Objectives: To study, in a cell culture model, the mechanisms of action of Cd2+ in the anterior pituitary. To validate the in vitro model performing in vivo studies. To evaluate possible therapeutic strategies. Results: Cd2+ affected, in a direct way, the viability and prolactin release of anterior pituitary cells. Cd2+ caused oxidative stress and increased cytosolic levels of Ca2+. Different antioxidants were able to reduce the effect of the metal both on cell viability and prolactin release. Cd2+ increased NO production and this molecule reduced the cytotoxicity caused by the metal. In vivo treatment with Cd2+ caused oxidative stress and increased the expression of nitric oxide synthase (NOS)1 and 2. Melatonin treatment effectively prevented Cd2+-induced oxidative stress but not the hormonal changes. Conclusions: Oxidative stress seems to be a mediator of Cd2+ cytotoxicity in vitro. The in vitro model seems correlates withthe effects of Cd2+ in vivo. Even when melatonin prevented in vivo Cd2+-induced oxidative stress it did not prevented changes in hormonal levels. This may suggest that factors other than oxidative stressmight influence the effects of Cd2+ on hormones release in vivo. Nonetheless, antioxidants may well be useful to reduce treat Cd2+ intoxications.Fil: Benitez Poliandri, Ariel H.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Epididymal protein Rnase10 is required for post-testicular sperm maturation and male fertility

Eutherian spermatozoa are dependent on the environment of the proximal epididymis to complete their maturation; however, no specific epididymal factors that mediate this process have so far been identified. Here, we show that targeted disruption of the novel gene Rnase10 encoding a secreted proximal epididymal protein in the mouse results in a binding defect in spermatozoa and their inability to pass through the uterotubal junction in the female. The failure to gain the site of fertilization in the knockout spermatozoa is associated with a gradual loss of ADAM3 and ADAM6 proteins during epididymal transit. In the distal epididymis, these spermatozoa appear to lack calcium-dependent associations with the immobilizing glutinous extracellular material and are released as single, vigorously motile cells that display no tendency for head-to-head agglutination and lack affinity to the oviductal epithelium. In sperm-egg binding assay, they are unable to establish a tenacious association with the zona pellucida, yet they are capable of fertilization. Furthermore, these sperm show accelerated capacitation resulting in an overall in vitro fertilizing ability superior to that of wild-type sperm. We conclude that the physiological role of sperm adhesiveness is in the mechanism of restricted sperm entry into the oviduct rather than in sperm-egg interaction

In vivo and in vitro effects of chromium VI on anterior pituitary hormone release and cell viability

Hexavalent chromium (Cr VI) is a highly toxic metal and an environmental pollutant. Different studies indicate that Cr VI exposure adversely affects reproductive functions. This metal has been shown to affect several tissues and organs but Cr VI effects on pituitary gland have not been reported. Anterior pituitary hormones are central for the body homeostasis and have a fundamental role in reproductive physiology. The aim of this study was to evaluate the effect of Cr VI at the pituitary level both in vivo and in vitro. We showed that Cr VI accumulates in the pituitary and hypothalamus, and decreases serum prolactin levels in vivo but observed no effects on LH levels. In anterior pituitary cells in culture, the effect of Cr VI on hormone secretion followed the same differential pattern. Besides, lactotrophs were more sensitive to the toxicity of the metal. As a result of oxidative stress generation, Cr VI induced apoptosis evidenced by nuclear fragmentation and caspase 3 activation. Our results indicate that the anterior pituitary gland can be a target of Cr VI toxicity in vivo and in vitro, thus producing a negative impact on the hypothalamic-pituitary-gonadal axis and affecting the normal endocrine function.Fil: Quinteros, Alnilan Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Benitez Poliandri, Ariel Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Machiavelli, Leticia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Cabilla, Jimena Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Duvilanski, Beatriz Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Constitutively active follicle-stimulating hormone receptor enables androgen-independent spermatogenesis

Spermatogenesis is regulated by the 2 pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This process is considered impossible without the absolute requirement of LH-stimulated testicular testosterone (T) production. The role of FSH remains unclear because men and mice with inactivating FSH receptor (FSHR) mutations are fertile. We revisited the role of FSH in spermatogenesis using transgenic mice expressing a constitutively strongly active FSHR mutant in a LH receptor–null (LHR-null) background. The mutant FSHR reversed the azoospermia and partially restored fertility of Lhr–/– mice. The finding was initially ascribed to the residual Leydig cell T production. However, when T action was completely blocked with the potent antiandrogen flutamide, spermatogenesis persisted. Hence, completely T-independent spermatogenesis is possible through strong FSHR activation, and the dogma of T being a sine qua non for spermatogenesis may need modification. The mechanism for the finding appeared to be that FSHR activation maintained the expression of Sertoli cell genes considered androgen dependent. The translational message of our findings is the possibility of developing a new strategy of high-dose FSH treatment for spermatogenic failure. Our findings also provide an explanation of molecular pathogenesis for Pasqualini syndrome (fertile eunuchs; LH/T deficiency with persistent spermatogenesis) and explain how the hormonal regulation of spermatogenesis has shifted from FSH to T dominance during evolution