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.

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.

[Neo-oogenesis in the adult ovary: What do we know?] / « Néo-ovogenèse dans l'ovaire adulte : mythe ou réalité ? ¼.

For more than a decade, the existence of ovarian stem cells that can contribute to neo-oogenesis in the adult ovary is reported by some teams, challenging the dogma according to which mammalian females are born with a fixed and non-renewed germinal cell pool. The presence of germinal stem cells with mitotic activity suggests the possibility of potential postnatal oogenesis. These cells have both germ-line and stem cell markers in culture. They have been isolated using different strategies and their ability to differentiate into oocytes has been demonstrated since after reintroduction in an ovarian somatic environment, these cells generate follicles capable of producing healthy offspring in rodents. However, many scientists remain skeptical and question the reliability of the methods used. Despite that there is no consensus on the origin of these ovarian stem cells, private companies are now proposing to use their stem cell potential to treat human infertility.

Iron, ferritin, transferrin, and transferrin receptor in the adult rat retina.

PURPOSE: The retina and other tissues need iron to survive. However, the normal iron metabolism in rodent retinas had not been characterized. This study was intended to investigate iron and iron homeostasis protein (ferritin, transferrin [Tf] and transferrin receptor [Tf-R]) distribution in 20- to 55-day-old rat retinas. METHODS: Iron was revealed on retinal sections directly by proton-induced x-ray emission (PIXE) and indirectly by electron microscopy (EM). Ferritin, Tf, and Tf-R proteins were localized by immunohistochemistry. Transferrin expression was localized by in situ hybridization (ISH). Transferrin and ferritin proteins and mRNA were analyzed by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RESULTS: Iron is widely and unevenly distributed throughout the adult rat retina. The highest concentration was observed by PIXE in the choroid and the retinal pigmented epithelial cell (RPE) layer, and in inner segments of photoreceptors (IS). Outer segments of photoreceptors (OS) also contain iron. EM studies suggested the presence of iron inclusions inside the photoreceptor discs. Choroid, RPE, and IS showed a strong immunoreactivity for ferritin. Transferrin accumulated mainly in the IS and OS areas and in RPE cells but can also be detected slightly in retinal capillaries. Western blot analysis for Tf and ferritin confirmed their presence in the adult neural retina. By RT-PCR, H- and L-chains of ferritin and Tf mRNAs were expressed in neural retina, but the main sites of Tf synthesis observed by ISH were the RPE and choroid cell layers. Tf-R immunoreactivity was detected in the ganglion cell layer, inner nuclear layer, outer plexiform layer, IS, RPE, and choroid. These results were similar for all stages studied. CONCLUSIONS: For the first time, the present study characterized both iron and iron homeostasis proteins in rodent retinas. In the outer retina, iron and ferritin shared the same distribution patterns. In contrast, Tf, mainly synthesized by RPE cells and detected in OS and IS areas, probably helps to transport iron to photoreceptors through their Tf-R. This is a likely pathway for filling iron needs in the outer retina.

Uterine myometrial ferritin and blood plasma transferrin as natural modulators of Ca-calmodulin-independent cyclic nucleotide phosphodiesterase from cow uterine myometrium.

The iron storage protein ferritin was purified to electrophoretic homogeneity from cow uterine myometrium. Its Mr did not exceed 440, 000 and H-chains predominated in the subunit composition; the iron saturation was 43 iron ions per protein molecule. The uterine myometrial ferritin was a potent natural modulator of Ca-calmodulin-independent phosphodiesterase (Ca-CM-independent PDE, EC 3.1.4.17) isolated from the same tissue. Addition of iron-poor ferritin from uterine myometrium and iron-reach liver ferritin caused three- and two-fold inhibition of the enzyme activity, respectively. The iron transport protein transferrin in iron-saturated and iron-depleted forms can also inhibit Ca-CM-independent PDE activity by two-fold. In both cases, the degree of saturation with iron was not crucial for the inhibitory effects of these proteins on the enzyme activity. These data suggest that iron homeostasis proteins can modulate the cyclic nucleotide level in non-nervous tissue via interaction with enzymes involved in cyclic nucleotide hydrolysis.

Transferrin and ferritin modulate the activity of brain calcium-calmodulin-dependent phosphodiesterase.

The effect of the key iron homeostasis proteins transferrin and ferritin on the activity of partially purified brain calcium-calmodulin-dependent phosphodiesterase (CaM-PDE, EC 3.4.1.17) were studied. Transferrin and ferritin were found to be potent natural activators of CaM-PDE. The key factor determining the degree of activation by these proteins is their saturation with iron: apotransferrin activated CaM-PDE 6-7-fold; iron-poor brain ferritin and liver apoferritin (taken for comparison) activated the enzyme 4-5- and 2-fold, respectively. Diferric transferrin and iron-rich liver ferritin had no effects on the enzyme activity. Transferrin and ferritin (both in apo- and iron-saturated forms) did not change the activity of calmodulin-phosphodiesterase complex. The data suggest that apotransferrin and iron-poor transferrin are involved in the regulation of cyclic nucleotide content in nervous tissue.