Zygotic Genome Activation in Vertebrates.

STUDY QUESTION How does biochemical stimulation or inhibition of the PI3K/Akt/mTOR pathway affect the activation and the growth of human primordial follicles in vitro? SUMMARY ANSWER The PI3K/Akt activators act synergistically with the Hippo signaling pathway, disrupted by tissue fragmentation, to accelerate primordial follicles recruitment while mTORC1 inhibitor partially prevents the massive spontaneous activation. WHAT IS KNOWN ALREADY Hippo disruption caused by ovarian fragmentation and exposure to PI3K/Akt activators have been successfully used to activate resting follicles prior to grafting in patients with premature ovarian insufficiency. Outside this indication, massive and premature activation is considered deleterious as it leads to the depletion of the follicular pool and developmental defects in vitro. STUDY DESIGN, SIZE, DURATION One hundred and twelve frozen-thawed ovarian cortex fragments from four patients were exposed to dimethylsulfoxide (DMSO-control group), everolimus (inhibitor group) or bpV (HOpic) and 740Y-P (activators group) during the first 24 and 48 h, respectively, and cultured for additional 5 days. PARTICIPANTS/MATERIALS, SETTING, METHODS The ovarian fragments (4×2×1 mm3) were analyzed at Days 0, 1, 3 and 5 of culture. Cortex were either directly processed for immunohistological or western blot analysis or subjected to follicular isolation for assessment of gene expression. The follicle number and the developmental stage were evaluated in sections of ovarian fragments to assess the early follicular development. Survival and developmental potential were evaluated using TUNEL labeling, GDF9 and Ki67 immunostainings, Kit ligand mRNA relative expression and measurement of 17β-estradiol (E2) levels in the culture medium. qPCR and western blotting were used to explore the expression of PI3K/Akt and Hippo pathways (TSC1, mTOR, LATS1, BIRC1 and CCN2 genes and PTEN/p-PTEN, Akt/p-Akt and rpS6/p-rps6 proteins), and localization of YAP in human ovarian tissue was performed by immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE Around 80% of the follicles spontaneously activated during the culture period, triggered by the activation of the PI3K/Akt/mTOR signaling. Moreover, ovarian fragmentation immediately promoted translocation of the Hippo effector YAP into the nucleus of granulosa cells, leading to upregulation of BIRC1 and CCN2 downstream targets at Day 0 and an increase of follicular growth near the cutting site. Short term exposure of thawed human ovarian fragments to PI3K/Akt activators had significant (P < 0.05 at D1 and D3; P < 0.001 at D6) but limited and potentially negative effects on follicular activation compared to spontaneous follicular growth as suggested by impaired E2 secretion. In contrast, transient incubation with an mTORC1 inhibitor partially prevented spontaneous activation by limiting growing follicle counts (P < 0.01 at D6), granulosa cell proliferation, and Kit ligand expression while ensuring appropriate steroidogenesis. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Impact of in vitro culture might cover up the potential benefit of the everolimus on growing follicles morphology after 6 days. WIDER IMPLICATIONS OF THE FINDINGS Our results demonstrate the synergistic effects of Hippo and PI3K/Akt/mTOR signaling, contributing to the acceleration of early primordial follicle recruitment in thawed human ovarian fragments, and suggest a beneficial effect of the mTORC1 inhibitor. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by a grant from the Fond National de la Recherche Scientifique de Belgique (Grant Televie Nos. 7.6516.16 F and 7.4578.14 F) and the Fonds Erasme. No competing interests declared.

Dynamics of PI3K and Hippo signaling pathways during in vitro human follicle activation.

Ovulation is the appropriately timed release of a mature, developmentally competent oocyte from the ovary into the oviduct, where fertilization occurs Importantly, ovulation is tightly linked with oocyte maturation, demonstrating the interdependency of these two parallel processes, both essential for female fertility Initiated by pituitary gonadotropins, the ovulatory process is mediated by intrafollicular paracrine factors from the theca, mural, and cumulus granulosa cells, as well as the oocyte itself The result is the induction of cumulus expansion, proteolysis, angiogenesis, inflammation, and smooth muscle contraction, which are each required for follicular rupture These complex intercellular communication networks and the essential ovulatory genes have been well defined in mouse models and are highly conserved in primates, including humans Importantly, recent discoveries in regulation of ovulation highlight new areas of investigation

Coordination of Ovulation and Oocyte Maturation: A Good Egg at the Right Time.

1. 1. Da Silva-Buttkus P., 2. et al.
 2008. J. Cell Sci. doi:10.1242/jcs.036400
 [OpenUrl][1][Abstract/FREE Full Text][2]

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Effect of cell shape and packing density on granulosa cell proliferation and formation of multiple layers during early follicle development in the ovary

Background Oocyte growth is accompanied by follicular development in mammalian ovaries. Since the discovery of two oocyte-derived factors, growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15), knowledge of the bidirectional communication between oocytes and granulosa cells for ovarian function and fertility has been accumulated. In addition, the growth culture system of oocytes has been improved, further promoting the studies on the communication between oocytes and granulosa cells in vitro. Methods We provide an overview of the role of granulosa cells in oocyte growth and the role of oocytes in follicular development along with our recent findings in culture experiments of bovine growing oocytes. Main findings Granulosa cells supply nutrients and metabolites through gap junctions to oocytes and secrete paracrine signals to regulate oocytes. Oocytes regulate granulosa cell proliferation and differentiation and induce antrum formation via GDF9 and BMP15. Conclusion Oocytes actively participate in various aspects of follicular development, including antrum formation via the oocyte-derived factors GDF9 and BMP15, whose synthesis is probably regulated by granulosa cells. In vitro studies will reveal the precise communication loop between oocytes and granulosa cells that facilitates the coordinated development of oocytes and granulosa cells in the follicles.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/rmb2.12292

Interaction between growing oocytes and granulosa cells in vitro

Mammalian Oocytes Locally Remodel Follicular Architecture to Provide the Foundation for Germline-Soma Communication

Reproduction depends on the generation of healthy oocytes. Improving therapeutic strategies to prolong or rescue fertility depends on identifying the inter- and intracellular mechanisms that direct oocyte development under physiological conditions. Growth and proliferation of multiple cell types is regulated by the Hippo signaling pathway, whose chief effectors are the transcriptional co-activator YAP and its paralogue WWTR1. To resolve conflicting results concerning the potential role of Hippo in mammalian oocyte development, we systematically investigated the expression and localization of YAP in mouse oocytes. We report that that YAP is expressed in the germ cells beginning as early as Embryonic Day 15.5 and subsequently throughout pre- and postnatal oocyte development. However, YAP is restricted to the cytoplasm at all stages. YAP is phosphorylated at serine-112 in growing and fully grown oocytes, identifying a likely mechanistic basis for its nuclear exclusion, and becomes dephosphorylated at this site during meiotic maturation. Phosphorylation at serine-112 is regulated by a mechanism dependent on cyclic AMP and protein kinase A, which is known to be active in oocytes prior to maturation. Growing oocytes also contain a subpopulation of YAP, likely dephosphorylated, that is able enter the oocyte nucleus, but it is not retained there, implying that oocytes lack the cofactors required to retain YAP in the nucleus. Thus, although YAP is expressed throughout oocyte development, phosphorylation-dependent and -independent mechanisms cooperate to ensure that it does not accumulate in the nucleus. We conclude that nuclear YAP does not play a significant physiological role during oocyte development in mammals.

https://bioone.org/journals/biology-of-reproduction/volume-94/issue-5/biolreprod.115.137968/Multiple-Mechanisms-Cooperate-to-Constitutively-Exclude-the-Transcriptional-Co-Activator/10.1095/biolreprod.115.137968.pdf

Laleh Abbassi

Papers

Mammalian Oocytes Locally Remodel Follicular Architecture to Provide the Foundation for Germline-Soma Communication

Multiple Mechanisms Cooperate to Constitutively Exclude the Transcriptional Co-Activator YAP from the Nucleus During Murine Oogenesis