Granulosa cells provide elimination of apoptotic oocytes through unconventional autophagy-assisted phagocytosis.

STUDY QUESTION: Do human granulosa cells (GCs) ingest and destroy apoptotic oocytes? SUMMARY ANSWER: Somatic GCs ingest and destroy apoptotic oocytes and other apoptotic substrates through unconventional autophagy-assisted phagocytosis. WHAT IS KNOWN ALREADY: Most (99%) ovarian germ cells undergo apoptosis through follicular atresia. The mode of cleaning of atretic follicles from the ovary is unclear. Ovarian GCs share striking similarities with testicular Sertoli cells with respect to their origin and function. Somatic Sertoli cells are responsible for the elimination of apoptotic spermatogenic cells through unconventional autophagy-assisted phagocytosis. STUDY DESIGN, SIZE, DURATION: Human GCs were tested for the ability to ingest and destroy the apoptotic oocytes and other apoptotic substrates. A systemic study of the main phagocytosis steps has been performed at different time points after loading of apoptotic substrates into the GC. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary cultures of GC retrieved following controlled ovarian stimulation of five women for IVF/ICSI and a human granulosa KGN cell line were incubated with different apoptotic substrates: oocytes which underwent spontaneous apoptosis during the cultivation of immature germ cells for IVF/ICSI; apoptotic KGN cells; and apoptotic membranes from rat retinas. Cultured GC were analyzed for the presence of specific molecular markers characteristic of different steps of phagocytic and autophagy machineries by immunocytochemistry, confocal microscopy, transmission electron microscopy and western blotting, before and after loading with apoptotic substrates. MAIN RESULTS AND THE ROLE OF CHANCE: Incubation of human GC with apoptotic substrates resulted in their translocation in cell cytoplasm, concomitant with activation of the phagocytosis receptor c-mer proto-oncogene tyrosine kinase MERTK (P < 0.001), clumping of motor molecule myosin II, recruitment of autophagy proteins: autophagy-related protein 5 (ATG5), autophagy-related protein 6 (Beclin1) and the rise of a membrane form of microtubule-associated protein 1 light chain 3 (LC3-II) protein. Ingestion of apoptotic substrates was accompanied by increased expression of the lysosomal protease Cathepsin D (P < 0.001), and a rise of lysosomes in the GCs, as assessed by different techniques. The level of autophagy adaptor, sequestosome 1/p62 (p62) protein remained unchanged. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The number of patients described here is limited. Also the dependence of phagocytosis on reproductive hormone status of patients should be analyzed. WIDER IMPLICATIONS OF THE FINDINGS: Removal of apoptotic oocytes by surrounding GC seems likely to be a physiological mechanism involved in follicular atresia. Proper functioning of this mechanism may be a new strategy for the treatment of ovarian dysfunctions associated with an imbalance in content of germ cells in the ovaries, such as premature ovarian failure and polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Rennes Metropole (AIS 2015) and Agence de BioMédecine. This work was supported by funding from Université de Rennes1, Institut National de la Santé et de la Recherche Médicale (INSERM) and CHU de Rennes. A.B. is funded in part by the program Actions Concertées Interpasteuriennes (ACIP) and a research grant from the European Society of Pediatric Endocrinology. This work is supported by the Agence Nationale de la Recherche Grants ANR-17-CE14-0038 and ANR-10-LABX-73. The authors declare no competing interests.

Update on the cellular and molecular aspects of cystic fibrosis transmembrane conductance regulator (CFTR) and male fertility.

CFTR protein regulates electrolyte and fluid transport in almost all tissues with exocrine function, including male reproductive tract. Mutation of CFTR gene causes cystic fibrosis (CF), which affects the function of several organs, and impairs male fertility. The role of CFTR protein in different compartments of male reproductive tract (testis, epididymis, sperm) as well as an impact of CFTR mutation(s) on male fertility phenotype is discussed in relation with the choice of optimal technique for Assisted Reproductive Techniques (ART) management.

Bacteroides fragilis prevents Salmonella Heidelberg translocation in co-culture model mimicking intestinal epithelium.

Salmonella Heidelberg is one of the most common serovar causing foodborne illnesses. To limit the development of digestive bacterial infection, food supplements containing probiotic bacteria can be proposed. Commensal non-toxigenic Bacteroides fragilis has recently been suggested as a next-generation probiotic candidate. By using an original triple co-culture model including Caco-2 cells (representing human enterocytes), HT29-MTX (representing mucus-secreting goblet cells), and M cells differentiated from Caco-2 by addition of Raji B lymphocytes, bacterial translocation was evaluated. The data showed that S. Heidelberg could translocate in the triple co-culture model with high efficiency, whereas for B. fragilis a weak translocation was obtained. When cells were exposed to both bacteria, S. Heidelberg translocation was inhibited. The cell-free supernatant of B. fragilis also inhibited S. Heidelberg translocation without impacting epithelial barrier integrity. This supernatant did not affect the growth of S. Heidelberg. The non-toxigenic B. fragilis confers health benefits to the host by reducting bacterial translocation. These results suggested that the multicellular model provides an efficient in vitro model to evaluate the translocation of pathogens and to screen for probiotics that have a potential inhibitory effect on this translocation.

Characterization of aluminum, aluminum oxide and titanium dioxide nanomaterials using a combination of methods for particle surface and size analysis.

The application of appropriate analytical techniques is essential for nanomaterial (NM) characterization. In this study, we compared different analytical techniques for NM analysis. Regarding possible adverse health effects, ionic and particulate NM effects have to be taken into account. As NMs behave quite differently in physiological media, special attention was paid to techniques which are able to determine the biosolubility and complexation behavior of NMs. Representative NMs of similar size were selected: aluminum (Al0) and aluminum oxide (Al2O3), to compare the behavior of metal and metal oxides. In addition, titanium dioxide (TiO2) was investigated. Characterization techniques such as dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) were evaluated with respect to their suitability for fast characterization of nanoparticle dispersions regarding a particle's hydrodynamic diameter and size distribution. By application of inductively coupled plasma mass spectrometry in the single particle mode (SP-ICP-MS), individual nanoparticles were quantified and characterized regarding their size. SP-ICP-MS measurements were correlated with the information gained using other characterization techniques, i.e. transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS). The particle surface as an important descriptor of NMs was analyzed by X-ray diffraction (XRD). NM impurities and their co-localization with biomolecules were determined by ion beam microscopy (IBM) and confocal Raman microscopy (CRM). We conclude advantages and disadvantages of the different techniques applied and suggest options for their complementation. Thus, this paper may serve as a practical guide to particle characterization techniques.