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.

Myelinosome-like vesicles in human seminal plasma: A cryo-electron microscopy study.

Seminal plasma is particularly rich in extracellular vesicles. Myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. Our aim was to determine the presence of myelinosomes in human seminal plasma. Transmission electron microscopy and cryo electron microscopy analysis of standard myelinosome preparation from TM4 Sertoli cells and human seminal plasma samples. We have specified by cryo-EM the morphological aspect of "standard" myelinosomes isolated from the culture media of TM4 Sertoli cells. Vesicles with the same morphological appearance were revealed in human seminal plasma samples. Human seminal plasma contains a population of large EV (average diameter 200 nm) whose morphological appearance resemble those of myelinosomes. Defining the specific biomarkers and functionalities of myelinosome in human seminal plasma are the concerns to be addressed in our further research.

Analysis of a Mathematical Model for the Glutamate/Glutamine Cycle in the Brain.

Our aim in this article is to study the well-posedness and properties of a system with delay which is related with brain glutamate and glutamine kinetics. In particular, we prove the existence and uniqueness of nonnegative solutions. We also give numerical simulations and compare their order of magnitude with experimental data.

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.

Differential motility of p190bcr-abl- and p210bcr-abl-expressing cells: respective roles of Vav and Bcr-Abl GEFs.

The chimeric oncogene Bcr-Abl is known to induce autonomous motility of leukemic cells. We show here that p210(bcr-abl) responsible for chronic myelogenous leukemia induces an amoeboid type of motility while p190(bcr-abl), associated with acute lymphoid leukemia, induces a rolling type of motility. We previously reported that p210(bcr-abl) activates RhoA and Rac1, while p190(bcr-abl) although devoid of a Dbl-homology (DH) domain activates Rac1, but not RhoA. We investigated the regulation of GDP/GTP exchange factor (GEF) activities in the Bcr-Abl complex. For that purpose, different GEF activity mutants of Vav and of Bcr-Abl were constructed and stably transfected in Ba/F3 cells. Using these mutants, we demonstrate that RhoA is exclusively activated by the DH domain of p210(bcr-abl), while Rac1 activation is mostly due to Vav. Inhibition of Rac1 by Vav GEF mutant leads to immobilization of cells. Vav depletion using shRNA also induces immobilization of cells and suppression of GTP-bound Rac1. RhoA inactivation induces the specific loss of amoeboid movements. These results suggest that Rac1 activation by Vav triggers the motility of Bcr-Abl-expressing Ba/F3 cells, while the specific amoeboid mode of motility induced by p210(bcr-abl) is a consequence of RhoA activation.

ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells.

Angiotensin II (ANG II) has long been known for its pressor and growth-promoting effects, which are both mediated by the AT1 receptor. By contrast, the AT2 receptor has recently been reported to mediate inhibition of proliferation through as yet undefined mechanisms. We report here that in bovine adrenal fasciculata cells ANG II by itself does not affect growth but inhibits basic fibroblast growth factor (bFGF)-induced DNA synthesis and blocks the cells in G1 phase. Consistent with this, ANG II inhibits cyclin D1 expression and cyclin D1-associated kinase activity. The antimitogenic effect of ANG II is partly mimicked by the AT2-selective agonist CGP-42112. It is also blocked partly and in an additive fashion by the AT1- and AT2-selective antagonists losartan and PD-123319, indicating the contribution of both receptor subtypes to this response. AT1-dependent antiproliferation is selectively blocked by the cyclooxygenase inhibitor indomethacin and restored by prostaglandin E2, whereas AT2-receptor-mediated inhibition of growth is suppressed by the tyrosine phosphatase inhibitors orthovanadate and bpV(pic). Both pathways are, however, pertussis toxin sensitive. We hypothesize that, in fasciculata cells, the AT1 receptor inhibits bFGF-induced proliferation by stimulating prostaglandin synthesis, whereas the AT2 receptor mediates its effect through a pathway that requires protein tyrosine phosphatase activation.

Phosphorylation of Rho GDI stabilizes the Rho A-Rho GDI complex in neutrophil cytosol.

The GDP dissociation inhibitor Rho GDI from bovine neutrophil cytosol was purified in association with prenylated Rho A. Upon treatment of this complex with alkaline phosphatase, the Rho A and Rho GDI components were released to their free forms. Following migration in 2D-PAGE and specific immunodetection, the shape of the spot of Rho GDI was found to depend markedly on whether Rho GDI subjected to electrophoresis was present in a Rho A-Rho GDI complex or in a free form. In the first case Rho GDI focused as an elongated spot between pI 5.2 and pI 4.6 whereas in the later case it focused at a pI of 5.0-5.2 as a round spot. Activation of neutrophils by anaphylatoxin C5a in a [32Pi] supplemented medium resulted in radiolabeling of Rho GDI. In vitro incubation of Rho GDI with a neutrophil homogenate in the presence of [gamma 32P] ATP led also to radiolabeling of Rho GDI. Taken together these results suggest that Rho GDI in the Rho A-Rho GDI complex is phosphorylated and that the stability of the complex depends on the phosphorylation state of Rho GDI.

Small G proteins and the neutrophil NADPH oxidase.

The NADPH oxidase of phagocytic cells is a multimeric enzyme complex activated during phagocytosis. It catalyzes the production of the superoxide anion, precursor of many toxic oxygen metabolites involved in the defense against microorganisms. The enzyme becomes active after assembly on a membrane bound flavocytochrome b of cytosolic factors p47 phox, p67 phox and p40 phox and of low molecular mass GTP binding proteins. This paper reviews recent results concerning the role of two small G proteins, Rac and Rap 1A in oxidase activation. Native prenylated small G proteins are either in the form of a complex in which the GDP bound G protein is associated with a guanine nucleotide dissociation inhibitor, GDI, or in an active GTP bound form able to trigger the activity of its effector. Rac and Rho share a common GDI. As chemotaxis, under Rho control, and oxidase activation, under Rac control, show mutually exclusive signalling pathways, we propose a model where the GDI would switch from one pathway to the other by sequestering either Rac or Rho.

Role of bound GDP in the stability of the rho A-rho GDI complex purified from neutrophil cytosol.

The rho A-rho GDI complex purified from bovine neutrophil cytosol was found to contain GDP as the only bound nucleotide at a ratio of 1 mol of GDP per mol of complex. The rho GDI component of the complex (pI 4.8-5.0, apparent molecular mass 28-29 kDa) and the rho A component (pI scattered between 5.0-6.2, apparent molecular mass 24 kDa) were resolved by 2D gel electrophoresis. Upon dephosphorylation of bound GDP by apyrase, the rho A component of the complex was prone to proteolytic cleavage. The integrity of rho A in the presence of apyrase was preserved by addition of excess GTP. These data suggest that rho A liganded by GDP in the rho A-rho GDI complex is maintained in a conformation that escapes action of proteases.

Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum.

The substrate of the C3 exoenzyme from botulinum toxin is a protein which is particularly abundant in the cytosol of neutrophils [Stasia, M. J., Jouan, A., Bourmeyster, N., Boquet, P., & Vignais, P. V. (1991) Biochem. Biophys. Res. Commun. 180, 615-622]. Optimal conditions for the ADP-ribosylation of the C3 substrate have been established in order to follow the course of its purification from bovine neutrophil cytosol. In particular, phosphoinositides at micromolar concentrations were found to enhance the ADP-ribosylation capacity of the C3 substrate in crude neutrophil cytosol and partially purified fractions. A [32P]ADP-ribosylatable protein, migrating on SDS-PAGE with a mass of 24 kDa, was copurified with a 29-kDa protein by a series of chromatographic steps on DEAE-Sephacel, Biogel P60, and Mono Q. In the case of the C3 substrate, isoelectric focusing revealed two major labeled bands with pI values of 6.2 and 5.6; the pI of the 29-kDa protein was 4.8-5.0. On the basis of the amino acid sequence of peptides resolved after proteolytic digestion, the 24-kDa protein and the 29-kDa protein were identified respectively as rho and the GDP dissociation inhibitor (GDI), suggesting that rho and GDI copurify from bovine neutrophil cytosol in the form of a complex. The presence of a number of amino acid residues specific of rho A in the enzymatic digest originating from rho indicates that, among the rho proteins, at least rho A belongs to the GDI-rho complex.

ADP-ribosylation of a small size GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clostridium botulinum and effect on the cell motility.

A 24-kDa G protein, ADP-ribosylable by exoenzyme C3 from Clostridium botulinum and therefore related to the rho family, was found to be abundantly present in human and bovine neutrophils, and preferentially located in cytosol. In human myeloid HL60 cells, the amount of C3 substrate increased during differentiation of the HL60 cells into granulocytes. The effect of exoenzyme C3 on different functions of bovine neutrophils, namely generation of O-2, degranulation and chemotaxis, has been tested, using electropermeabilized cells. Exoenzyme C3 hardly affected the respiratory burst and the degranulation. In contrast, it efficiently inhibited the spontaneous and chemoattractant-induced motility of the cells and disorganized the actin microfilament assembly.