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Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis.

Saare M, Lulla S, Tõnissoo T, Meier R, Kask K, Ruisu K, Karis A, Salumets A, Pooga M - PLoS ONE (2015)

Bottom Line: Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis.Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels.Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010, Tartu, Estonia; Competence Centre on Health Technologies, Tiigi 61b, 50410, Tartu, Estonia.

ABSTRACT
Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8), a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gαi1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.

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Related in: MedlinePlus

RIC8 protein expression pattern compared to NuMa or Gαi1/2 proteins during mouse oocyte maturation.(A-F) Mouse oocytes at meiosis I were double-labeled with RIC8 antibody (green) and NuMA or Gαi1/2 antibodies (red). DNA was stained with DAPI (blue). The overlapping regions of RIC8 and NuMA or Gαi1/2 (yellow to orange) at meiotic spindle indicated with white arrows. White arrowheads indicated cell cortex regions, where RIC8 and Gαi1/2 co-localize. Abbreviations: Ana/Tel I; anaphase/telophase of meiosis I; ms, meiotic spindle; pb, polar body. Scale bar: 10 μm.
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pone.0129131.g005: RIC8 protein expression pattern compared to NuMa or Gαi1/2 proteins during mouse oocyte maturation.(A-F) Mouse oocytes at meiosis I were double-labeled with RIC8 antibody (green) and NuMA or Gαi1/2 antibodies (red). DNA was stained with DAPI (blue). The overlapping regions of RIC8 and NuMA or Gαi1/2 (yellow to orange) at meiotic spindle indicated with white arrows. White arrowheads indicated cell cortex regions, where RIC8 and Gαi1/2 co-localize. Abbreviations: Ana/Tel I; anaphase/telophase of meiosis I; ms, meiotic spindle; pb, polar body. Scale bar: 10 μm.

Mentions: Before the germinal vesicle breakdown, dynamic changes take place in the chromatin configuration of germinal vesicle, which are associated with transcriptional activity. The localization of RIC8 was assessed at different maturation stages of nucleus, which were assigned based on the chromatin configuration. First, we discovered that when the chromatin started to condense and it was identified as a partial rim (PR) around the nucleolus, RIC8 remained diffusely localized across the germinal vesicle (Figs 2A, 2a and 3A), which was also typical for the secondary follicle stage in the ovary (Fig 1D). Later, at the full rim [28] stage, when chromatin had condensed densely around nucleolus, RIC8 accumulated in spots distributed around condensed chromatin in the germinal vesicle, with some foci attached to the nucleolus (Figs 2B, 2b, 3G and 3J). Further, when the nuclear lamina as well as the nucleolus started to disappear, the spots of RIC8 remained in the close vicinity of chromosomes (Figs 2C, 2c, 3D and 3d). With the organization of chromosomes to the metaphase plate, RIC8 distributed along the metaphase spindle (Figs 2E and 4A) and co-localized with tubulin (Fig 2E and 2F). In anaphase I, during the chromosome separation, RIC8 remained in the vicinity of chromosomes and co-localized with the meiotic spindle (Figs 2G, 2H and 4D). After completion of meiosis I, the secondary oocytes enter directly meiosis II, at which point they are arrested for the second time, as the chromosomes move to the metaphase II plate. During the second arrest, RIC8 was distributed to the metaphase spindle and diffusely in the cytoplasm (Fig 4G). After the ovulation, fertilization triggers the resumption and completion of meiosis II and metaphase is rapidly finished. During the final steps of meiosis II RIC8 was located in the spindle (Fig 2I–2L). In addition to the above-mentioned, a part of RIC8 protein was also detectable uniformly in the cytoplasm of oocyte at every examined stage, and also in some extent at the cell cortex (Fig 5A and 5D).


Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis.

Saare M, Lulla S, Tõnissoo T, Meier R, Kask K, Ruisu K, Karis A, Salumets A, Pooga M - PLoS ONE (2015)

RIC8 protein expression pattern compared to NuMa or Gαi1/2 proteins during mouse oocyte maturation.(A-F) Mouse oocytes at meiosis I were double-labeled with RIC8 antibody (green) and NuMA or Gαi1/2 antibodies (red). DNA was stained with DAPI (blue). The overlapping regions of RIC8 and NuMA or Gαi1/2 (yellow to orange) at meiotic spindle indicated with white arrows. White arrowheads indicated cell cortex regions, where RIC8 and Gαi1/2 co-localize. Abbreviations: Ana/Tel I; anaphase/telophase of meiosis I; ms, meiotic spindle; pb, polar body. Scale bar: 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4465189&req=5

pone.0129131.g005: RIC8 protein expression pattern compared to NuMa or Gαi1/2 proteins during mouse oocyte maturation.(A-F) Mouse oocytes at meiosis I were double-labeled with RIC8 antibody (green) and NuMA or Gαi1/2 antibodies (red). DNA was stained with DAPI (blue). The overlapping regions of RIC8 and NuMA or Gαi1/2 (yellow to orange) at meiotic spindle indicated with white arrows. White arrowheads indicated cell cortex regions, where RIC8 and Gαi1/2 co-localize. Abbreviations: Ana/Tel I; anaphase/telophase of meiosis I; ms, meiotic spindle; pb, polar body. Scale bar: 10 μm.
Mentions: Before the germinal vesicle breakdown, dynamic changes take place in the chromatin configuration of germinal vesicle, which are associated with transcriptional activity. The localization of RIC8 was assessed at different maturation stages of nucleus, which were assigned based on the chromatin configuration. First, we discovered that when the chromatin started to condense and it was identified as a partial rim (PR) around the nucleolus, RIC8 remained diffusely localized across the germinal vesicle (Figs 2A, 2a and 3A), which was also typical for the secondary follicle stage in the ovary (Fig 1D). Later, at the full rim [28] stage, when chromatin had condensed densely around nucleolus, RIC8 accumulated in spots distributed around condensed chromatin in the germinal vesicle, with some foci attached to the nucleolus (Figs 2B, 2b, 3G and 3J). Further, when the nuclear lamina as well as the nucleolus started to disappear, the spots of RIC8 remained in the close vicinity of chromosomes (Figs 2C, 2c, 3D and 3d). With the organization of chromosomes to the metaphase plate, RIC8 distributed along the metaphase spindle (Figs 2E and 4A) and co-localized with tubulin (Fig 2E and 2F). In anaphase I, during the chromosome separation, RIC8 remained in the vicinity of chromosomes and co-localized with the meiotic spindle (Figs 2G, 2H and 4D). After completion of meiosis I, the secondary oocytes enter directly meiosis II, at which point they are arrested for the second time, as the chromosomes move to the metaphase II plate. During the second arrest, RIC8 was distributed to the metaphase spindle and diffusely in the cytoplasm (Fig 4G). After the ovulation, fertilization triggers the resumption and completion of meiosis II and metaphase is rapidly finished. During the final steps of meiosis II RIC8 was located in the spindle (Fig 2I–2L). In addition to the above-mentioned, a part of RIC8 protein was also detectable uniformly in the cytoplasm of oocyte at every examined stage, and also in some extent at the cell cortex (Fig 5A and 5D).

Bottom Line: Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis.Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels.Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010, Tartu, Estonia; Competence Centre on Health Technologies, Tiigi 61b, 50410, Tartu, Estonia.

ABSTRACT
Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8), a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gαi1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.

No MeSH data available.


Related in: MedlinePlus