Éditeur(s) : HAL CCSD Landes Bioscience
Résumé : International audience
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Autophagy

hal-00735751
https://hal.archives-ouvertes.fr/hal-00735751
PUBMED : 22966490

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.
ISSN: 1553-7390

hal-01063929
https://hal.archives-ouvertes.fr/hal-01063929
DOI : 10.1371/journal.pgen.1004340
PUBMED : 24784881

Éditeur(s) : HAL CCSD Endocrine Society
Résumé : International audience
Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.
ISSN: 0888-8809

hal-01146846
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01146846
DOI : 10.1210/me.2014-1356
PUBMED : 25710594

Éditeur(s) : HAL CCSD Society for the Study of Reproduction
Résumé : International audience
The endonuclease DICER that processes micro-RNAs and small interfering RNAs is essential for normal spermatogenesis and male fertility. We previously showed that the deletion of Dicer1 gene in postnatal spermatogonia in mice using Ngn3 promoter-driven Cre expression caused severe defects in the morphogenesis of haploid spermatid to mature spermatozoon, including problems in cell polarization and nuclear elongation. In this study, we further analyzed the same mouse model and revealed that absence of functional DICER in differentiating male germ cells induces disorganization of the cell-cell junctions in the seminiferous epithelium. We detected discontinuous and irregular apical ectoplasmic specializations between elongating spermatids and Sertoli cells. The defective anchoring of spermatids to Sertoli cells caused a premature release of spermatids into the lumen. Our findings may help also explain the abnormal elongation process of remaining spermatids because these junctions and the correct positioning of germ cells in the epithelium are critically important for the progression of spermiogenesis. Interestingly, cell adhesion-related genes were generally upregulated in Dicer1 knockout germ cells. Claudin 5 ( Cldn5 ) was among the most upregulated genes and we show that the polarized localization of CLAUDIN5 in the apical ectoplasmic specializations was lost in Dicer1 knockout spermatids. Our results suggest that DICER-dependent pathways control the formation and organization of cell-cell junctions in the seminiferous epithelium via the regulation of cell adhesion-related genes
ISSN: 0006-3363

hal-01274239
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01274239
DOI : 10.1095/biolreprod.115.131938
PUBMED : 26510868

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Vertebrate genomes contain around 20,000 protein-encoding genes, of which a large fraction is still not associated with specific functions. A major task in future genomics will thus be to assign physiological roles to all open reading frames revealed by genome sequencing. Here we show that C2orf62, a highly conserved protein with little homology to characterized proteins, is strongly expressed in testis in zebrafish and mammals, and in various types of ciliated cells during zebrafish development. By yeast two hybrid and GST pull-down, C2orf62 was shown to interact with TTC17, another uncharacterized protein. Depletion of either C2orf62 or TTC17 in human ciliated cells interferes with actin polymerization and reduces the number of primary cilia without changing their length. Zebrafish embryos injected with morpholinos against C2orf62 or TTC17, or with mRNA coding for the C2orf62 C-terminal part containing a RII dimerization/docking (R2D2) - like domain show morphological defects consistent with imperfect ciliogenesis. We provide here the first evidence for a C2orf62-TTC17 axis that would regulate actin polymerization and ciliogenesis.
ISSN: 1932-6203

hal-00955242
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00955242
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00955242/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00955242/file/C2orf62_and_TTC17-accepted.pdf
DOI : 10.1371/journal.pone.0086476