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A novel regulatory pathway in granulosa cells, the LH/human chorionic gonadotropin-microRNA-125a-3p-Fyn pathway, is required for ovulation. Grossman H et al. (2015) Granulosa cells support the developing oocytes and serve as transducers of the ovulatory stimulus induced by LH surge. Fyn kinase is expressed in granulosa cells, though its role in these cells has not been studied. In human embryonic kidney 293T cells, microRNA (miR)-125a-3p down-regulates Fyn expression, causing a decrease in cells' migratory ability. Our aim was to explore the role of miR-125a-3p and Fyn in granulosa cells toward ovulation, focusing on migration as a possible mechanism. We demonstrate expression of miR-125a-3p and Fyn in mouse mural granulosa cells of preovulatory follicles and miR-125a-3p-induced down-regulation of Fyn expression in a granulosa cell line (rat). Administration of human chorionic gonadotropin (hCG; LH analog) caused a 75% decrease in the in vivo miR-125a-3p:Fyn mRNA ratio, followed by a 2-fold increased migratory ability of mural granulosa cells. In the hCG-treated granulosa cell line, miR-125a-3p expression was decreased, followed by Fyn up-regulation and phosphorylation of focal adhesion kinase and paxillin, enabling cell migration. An in vivo interference with miR-125a-3p:Fyn mRNA ratio in granulosa cells by intrabursal injections of Fyn small interfering RNA or miR-125a-3p mimic caused a 33 or 55% decrease in the number of ovulated oocytes, respectively. These observations reveal a new regulatory pathway in mural granulosa cells under the regulation of LH/hCG. Modulation of cell migration may account for the significance of the LH/hCG-miR-125a-3p-Fyn pathway to ovulation.-Grossman, H., Chuderland, D., Ninio-Many, L., Hasky, N., Kaplan-Kraicer, R., Shalgi, R. A novel regulatory pathway in granulosa cells, the LH/human chorionic gonadotropin-microRNA-125a-3p-Fyn pathway, is required for ovulation.//////////////////
The conformation and activation of Fyn kinase in the oocyte determine its localisation to the spindle poles and cleavage furrow. Levi M et al. Several lines of evidence imply the involvement of Fyn, a Src family kinase, in cell-cycle control and cytoskeleton organisation in somatic cells. By live cell confocal imaging of immunostained or cRNA-microinjected mouse oocytes at metaphase of the second meiotic division, membrane localisation of active and non-active Fyn was demonstrated. However, Fyn with a disrupted membrane-binding domain at its N-terminus was targeted to the cytoplasm and spindle in its non-active form and concentrated at the spindle poles when active. During metaphase exit, the amount of phosphorylated Fyn and of spindle-poles Fyn decreased and it started appearing at the membrane area of the cleavage furrow surrounding the spindle midzone, either asymmetrically during polar body II extrusion or symmetrically during mitosis. These results demonstrate that post-translational modifications of Fyn, probably palmitoylation, determine its localisation and function; localisation of de-palmitoylated active Fyn to the spindle poles is involved in spindle pole integrity during metaphase, whereas the localisation of N-terminus palmitoylated Fyn at the membrane near the cleavage furrow indicates its participation in furrow ingression during cytokinesis.
Fyn kinase activity is required for normal organization and functional polarity of the mouse oocyte cortex. Luo J et al. The objective of the present study was to determine whether Fyn kinase participated in signaling events during sperm-egg interactions, sperm incorporation, and meiosis II. The functional requirement of Fyn kinase activity in these events was tested through the use of the protein kinase inhibitor SKI-606 (Bosutinib) and by analysis of Fyn-null oocytes. Suppression of Fyn kinase signaling prior to fertilization caused disruption of the functional polarity of the oocyte with the result that sperm were able to fuse with the oocyte in the immediate vicinity of the meiotic spindle, a region that normally does not allow sperm fusion. The loss of functional polarity was accompanied by disruption of the microvilli and cortical granule-free zone that normally overlie the meiotic spindle. Changes in the distribution of cortical granules and filamentous actin provided further evidence of disorganization of the oocyte cortex. Rho B, a molecular marker for oocyte polarity, was unaffected by suppression of Fyn activity; however, the polarized association of Par-3 with the cortex overlying the meiotic spindle was completely disrupted. The defects in oocyte polarity in Fyn-null oocytes correlated with a failure of the MII chromosomes to maintain a position close to the oocyte cortex which seemed to underlie the above defects in oocyte polarity. This was associated with a delay in completion of meiosis II. Pronuclei, however, eventually formed and subsequent mitotic cleavages and blastocyst formation occurred normally. Mol. Reprod. Dev. 2009. (c) 2009 Wiley-Liss, Inc.
Steele RE et al reported the structure and expression of fyn genes in Xenopus laev and suggested that multiple protein-tyrosine kinases of the src family are required for
processes in oogenesis or early development.
Localized activation of Src-family protein kinases in the mouse egg. McGinnis LK et al. Recent studies in species that fertilize externally have demonstrated that fertilization triggers localized activation of Src-family protein kinases in the egg cortex. However, the requirement for Src-family kinases in activation of the mammalian egg is different from lower species and the objective of this study was to characterize changes in the distribution and activity of Src-family protein tyrosine kinases (PTKs) during zygotic development in the mouse. Immunofluorescence analysis of mouse oocytes and zygotes with an anti-phosphotyrosine antibody revealed that fertilization stimulated accumulation of P-Tyr-containing proteins in the egg cortex and that their abundance was elevated in the region overlying the MII spindle. In addition, the poles of the MII spindle exhibited elevated P-Tyr levels. As polar body extrusion progressed, P-Tyr-containing proteins were especially concentrated in the region of cortex adjacent to the maternal chromatin and the forming polar body. In contrast, P-Tyr labeling of the spindle poles eventually disappeared as meiosis II progressed to anaphase II. In approximately 24% of cases, the fertilizing sperm nucleus was associated with increased P-Tyr labeling in the overlying cortex and oolemma. To determine whether Src-family protein tyrosine kinases could be responsible for the observed changes in the distribution of P-Tyr containing proteins, an antibody to the activated form of Src-family PTKs was used to localize activated Src, Fyn or Yes. Activated Src-family kinases were found to be strongly associated with the meiotic spindle at all stages of meiosis II; however, no concentration of labeling was evident at the egg cortex. The absence of cortical Src-family PTK activity continued until the blastocyst stage when strong cortical activity became evident. At the pronuclear stage, activated Src-family PTKs became concentrated around the pronuclei in close association with the nuclear envelope. This pattern was unique to the earliest stages of development and disappeared by the eight cell stage. Functional studies using chemical inhibitors and a dominant-negative Fyn construct demonstrated that Src-family PTKs play an essential role in completion of meiosis II following fertilization and progression from the pronuclear stage into mitosis. These data suggest that while Src-family PTKs are not required for fertilization-induced calcium oscillations, they do play a critical role in development of the zygote. Furthermore, activation of these kinases in the mouse egg is limited to distinct regions and occurs at specific times after fertilization.
The role of fyn kinase in the release from metaphase in mammalian oocytes. Levi M et al. Meiosis in mammalian oocytes starts during embryonic life and arrests for the first time before birth, at prophase of the first meiotic division. The second meiotic arrest occurs after spindle formation at metaphase of the second meiotic division (MII) in selected oocytes designated for ovulation. The fertilizing spermatozoon induces the release from MII arrest only after the oocyte's spindle assembly checkpoint (SAC) was deactivated. Src family kinases (SFKs) are nine non-receptor protein tyrosine kinases that regulate many key cellular functions. Fyn is an SFK expressed in many cell types, including oocytes. Recent studies, including ours, imply a role for Fyn in exit from meiotic and mitotic metaphases. Other studies demonstrate that SFKs, particularly Fyn, are required for regulation of microtubules polymerization and spindle stabilization. Altogether, Fyn is suggested to play an essential role in signaling events that implicate SAC pathway and hence in regulating the exit from metaphase in oocytes and zygote.
The involvement of Fyn kinase in resumption of the first meiotic division in mouse oocytes. Levi M et al. The process of resumption of the first meiotic division (RMI) in mammalian oocytes includes germinal vesicle breakdown (GVBD), spindle formation during first metaphase (MI), segregation of homologous chromosomes, extrusion of the first polar body (PBI) and an arrest at metaphase of the second meiotic division (MII). Previous studies suggest a role for Fyn, a non-receptor Src family tyrosine kinase, in the exit from MII arrest. In the current study we characterized the involvement of Fyn in RMI. Western blot analysis demonstrated a significant, proteasome independent, degradation of Fyn during GVBD. Immunostaining of fixed oocytes and confocal imaging of live oocytes microinjected with Fyn complementary RNA (cRNA) demonstrated Fyn localization to the oocyte cortex and to the spindle poles. Fyn was recruited during telophase to the cortical area surrounding the midzone of the spindle and was then translocated to the contractile ring during extrusion of PBI. GVBD, exit from MI and PBI extrusion were inhibited in oocytes exposed to the chemical inhibitor SU6656 or microinjected with dominant negative Fyn cRNA. None of the microinjected oocytes showed misaligned or lagging chromosomes during chromosomes segregation and the spindle migration and anchoring were not affected. However, the extruded PBI was of large size. Altogether, a role for Fyn in regulating several key pathways during the first meiotic division in mammalian oocytes is suggested, particularly at the GV and metaphase checkpoints and in signaling the ingression of the cleavage furrow.
Fyn kinase is involved in cleavage furrow ingression during meiosis and mitosis. Levi M et al. Fertilization of mammalian oocytes triggers their exit from the second meiotic division metaphase arrest. The extrusion of the second polar body that marks the completion of meiosis is followed by the first mitotic cleavage of the zygote. Several lines of evidence in somatic cells imply the involvement of Fyn, a Src family kinase, in cell cycle control and actin functions. In the current study we demonstrated, using live cell confocal imaging and microinjection of Fyn cRNAs, recruitment of Fyn to the oocyte's cortical area overlaying the chromosomes and its colocalization with filamentous actin during exit from the meiotic metaphase. Fyn concentrated asymmetrically at the cortical site designated for ingression of the second polar body cleavage furrow, where filamentous actin had already been accumulated, and then redispersed throughout the entire cortex only to be recruited again to the cleavage furrow during the first mitotic division. Although microinjection of dominant negative Fyn did not affect initiation of the cleavage furrow, it prolonged the average duration of ingression, decreased the rates of polar body extrusion and of the first cleavage, and led to the formation of bigger polar bodies and longer spindles. Extrusion of the second polar body was blocked in oocytes exposed to SU6656, an SFKs inhibitor. Our results demonstrate, for the first time, a continuous co-localization of Fyn and F-actin during meiosis and imply a role for SFKs in general, and for Fyn in particular, in regulating pathways that involve actin cytoskeleton, during ingression of the meiotic and mitotic cleavage furrows.
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Regulation of GVBD in mouse oocytes by miR-125a-3p and Fyn kinase through modulation of actin filaments. Grossman H et al. (2017) Meiotically arrested oocytes are characterized by the presence of the nuclear structure known as germinal-vesicle (GV), the breakdown of which (GVBD) is associated with resumption of meiosis. Fyn is a pivotal factor in resumption of the first meiotic division; its inhibition markedly decreases the fraction of oocytes undergoing GVBD. Here, we reveal that in mouse oocytes Fyn is post-transcriptionally regulated by miR-125a-3p. We demonstrate that in oocytes resuming meiosis miR-125a-3p and Fyn exhibit a reciprocal expression pattern; miR-125a-3p decreases alongside with an increase in Fyn expression. Microinjection of miR-125a-3p inhibits GVBD, an effect that is markedly reduced by Fyn over-expression, and impairs the organization of the actin rim surrounding the nucleus. Lower rate of GVBD is also observed in oocytes exposed to cytochalasin-D or blebbistatin, which interfere with actin polymerization and contractility of actin bundles, respectively. By down-regulating Fyn in HEK-293T cells, miR-125a-3p reduces the interaction between actin and A-type lamins, which constitute the nuclear-lamina. Our findings suggest a mechanism, by which a decrease in miR-125a-3p during oocyte maturation facilitates GVBD by allowing Fyn up-regulation and the resulting stabilization of the interaction between actin and A-type lamins.//////////////////
FSH-induces multiple signaling cascades: evidence that activation of SRC, RAS and the EGF receptor are critical for granulosa cell differentiation. Wayne CM et al. Follicle stimulating hormone (FSH) regulates ovarian granulosa cell differentiation not only by activating adenylyl cyclase (AC) and protein kinase A (PKA) but also by other complex mechanisms. Using primary rat granulosa cell cultures, we provide novel evidence that FSH rapidly activates two small GTP binding proteins RAP1 and RAS. FSH activation of RAP1 requires cAMP-mediated activation of EPAC (exchange factor activated by cAMP)/RAPGEF3 whereas FSH activation of RAS and downstream signaling cascades involves multiple factors. Specifically, FSH activation of RAS required SRC family tyrosine kinase (SFK) and EGF receptor tyrosine kinase activities but not PKA. FSH induced phosphorylation of ERK1/2 was blocked by dominant-negative RAS as well as by inhibitors of EGF receptor tyrosine kinase, metalloproteinases involved in growth factor shedding and SRC family tyrosine kinases (SFKs). In contrast, FSH-induced phosphorylation of protein kinase B (PKB/AKT) and the Forkhead transcription factor, FOXO1a occurred by SFK dependent but RAS independent mechanisms. The SFKs, c-SRC and FYN and the SRC-related tyrosine kinase ABL were present and phosphorylated rapidly in response to FSH. Lastly, the EGF-like factor amphiregulin (AREG) activated RAS and ERK1/2 phosphorylation in granulosa cells by mechanisms that were selectively blocked an EGF receptor antagonist but not by an SFK antagonist. However, AREG mediated phosphorylation of PKB and FOXO1a required both EGF receptor and SFK activation. Moreover, we show that FSH induces AREG and that activation of the EGF receptor impacts granulosa cell differentiation and the expression of genes characteristic of the luteal cell phenotype. Thus, FSH orchestrates the coordinate activation of three diverse membrane-associated signaling cascades (AC, RAS and SFKs) that converge downstream to activate specific kinases (PKA, ERK1/2 and PKB/FOXO1a) that control granulosa cell function and differentiation.
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