The tyrosine kinase inhibitor sunitinib affects ovulation but not ovarian reserve in mouse: A preclinical study

The aim of the study was to evaluate ovarian toxicity of tyrosine kinase inhibitor (TKI) sunitinib, since only scarce data are available on gonadal function after this treatment. Six-weekold female mice received orally, once daily, vehicle or sunitinib (50 mg/kg/d) during 5 weeks. Fertility parameters were analyzed from ovulation to litter assessment. Sunitinib exposure significantly reduced (i) corpora lutea number per ovary (1.1 ± 0.38 in sunitinib group versus 4 ± 0.79 in control group, p<0.01) and (ii) serum Anti Müllerian hormone (AMH) levels in sunitinib treated mice (12.01 ± 1.16) compared to control mice (14.33 ± 0.87 ng/ml, p< 0.05). However, primordial and growing follicles numbers per ovary were not different in both groups. After treatment withdrawal, female mice in both groups were able to obtain litters. These data could be helpful to counsel clinicians and patients, when fertility preservation methods are discussed, before TKI treatment in girls and young women

Prolactin Receptor Signaling Is Essential for Perinatal Brown Adipocyte Function: A Role for Insulin-like Growth Factor-2

BACKGROUND: The lactogenic hormones prolactin (PRL) and placental lactogens (PL) play central roles in reproduction and mammary development. Their actions are mediated via binding to PRL receptor (PRLR), highly expressed in brown adipose tissue (BAT), yet their impact on adipocyte function and metabolism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: PRLR knockout (KO) newborn mice were phenotypically characterized in terms of thermoregulation and their BAT differentiation assayed for gene expression studies. Derived brown preadipocyte cell lines were established to evaluate the molecular mechanisms involved in PRL signaling on BAT function. Here, we report that newborn mice lacking PRLR have hypotrophic BAT depots that express low levels of adipocyte nuclear receptor PPARgamma2, its coactivator PGC-1alpha, uncoupling protein 1 (UCP1) and the beta3 adrenoceptor, reducing mouse viability during cold challenge. Immortalized PRLR KO preadipocytes fail to undergo differentiation into mature adipocytes, a defect reversed by reintroduction of PRLR. That the effects of the lactogens in BAT are at least partly mediated by Insulin-like Growth Factor-2 (IGF-2) is supported by: i) a striking reduction in BAT IGF-2 expression in PRLR KO mice and in PRLR-deficient preadipocytes; ii) induction of cellular IGF-2 expression by PRL through JAK2/STAT5 pathway activation; and iii) reversal of defective differentiation in PRLR KO cells by exogenous IGF-2. CONCLUSIONS: Our findings demonstrate that the lactogens act in concert with IGF-2 to control brown adipocyte differentiation and growth. Given the prominent role of brown adipose tissue during the perinatal period, our results identified prolactin receptor signaling as a major player and a potential therapeutic target in protecting newborn mammals against hypothermia

Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy

AIMS/HYPOTHESIS: Upregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes. METHODS: RNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells. RESULTS: mRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24 h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h). CONCLUSIONS/INTERPRETATION: A very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified

Differential Impact of Tetratricopeptide Repeat Proteins on the Steroid Hormone Receptors

Tetratricopeptide repeat (TPR) motif containing co-chaperones of the chaperone Hsp90 are considered control modules that govern activity and specificity of this central folding platform. Steroid receptors are paradigm clients of Hsp90. The influence of some TPR proteins on selected receptors has been described, but a comprehensive analysis of the effects of TPR proteins on all steroid receptors has not been accomplished yet.We compared the influence of the TPR proteins FK506 binding proteins 51 and 52, protein phosphatase-5, C-terminus of Hsp70 interacting protein, cyclophillin 40, hepatitis-virus-B X-associated protein-2, and tetratricopeptide repeat protein-2 on all six steroid hormone receptors in a homogeneous mammalian cell system. To be able to assess each cofactor's effect on the transcriptional activity of on each steroid receptor we employed transient transfection in a reporter gene assay. In addition, we evaluated the interactions of the TPR proteins with the receptors and components of the Hsp90 chaperone heterocomplex by coimmunoprecipitation. In the functional assays, corticosteroid and progesterone receptors displayed the most sensitive and distinct reaction to the TPR proteins. Androgen receptor's activity was moderately impaired by most cofactors, whereas the Estrogen receptors' activity was impaired by most cofactors only to a minor degree. Second, interaction studies revealed that the strongly receptor-interacting co-chaperones were all among the inhibitory proteins. Intriguingly, the TPR-proteins also differentially co-precipitated the heterochaperone complex components Hsp90, Hsp70, and p23, pointing to differences in their modes of action.The results of this comprehensive study provide important insight into chaperoning of diverse client proteins via the combinatorial action of (co)-chaperones. The differential effects of the TPR proteins on steroid receptors bear on all physiological processes related to steroid hormone activity

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field