Dynamics of extracellular vesicle uptake by mural granulosa cells in mice.

Extracellular vesicles (EVs) play an important role in the development and function of mammalian ovarian follicles. However, the mechanisms by which they are taken up by the follicular granulosa cells remain unclear. In addition, while oocytes play a pivotal role in follicular development, the possible interactions between oocyte-derived paracrine factors (ODPFs) and EV signals are unknown. Therefore, this study aimed to elucidate the mechanism of EV uptake and the effects of ODPFs on EV uptake by follicular somatic mural granulosa cells in mice. Fluorescence-labeled transferrin (TRF) and cholera toxin B (CTB), substrates for clathrin- and caveolae-mediated endocytosis, respectively, were taken up by mural granulosa cells in vitro. Their uptake was inhibited by Pitstop 2 and genistein, inhibitors of clathrin and caveolae pathways, respectively. Mural granulosa cells took up EVs, and this uptake was suppressed by Pitstop 2 and genistein. Moreover, ODPFs promoted the uptake of EVs and CTB, but not TRF, by mural granulosa cells. These results suggest that mural granulosa cells take up EVs through both clathrin- and caveolae-mediated endocytosis and that oocytes may promote caveolae-mediated endocytosis to facilitate the uptake of EVs.

Accumulation of senescent cells in the stroma of aged mouse ovary.

Senescent cells play a detrimental role in age-associated pathogenesis by producing factors involved in senescence-associated secretory phenotype (SASP). The present study was conducted to examine the possibility that senescent cells are present in aged ovaries and, if so, to determine the tissue region where senescent cells accumulate using a mouse model. Female mice at 2-4 and 8-10 months were used as reproductively young and aged models, respectively; the latter included mice with and without reproductive experience. Cells positive for senescence-associated ß-galactosidase (SA-ß-Gal) staining, one of the markers of cellular senescence, were detected in the stromal region of aged, but not young, ovaries regardless of reproductive experience. Likewise, the localization of cells expressing CDKN2A (cyclin dependent kinase inhibitor 2A), another senescence marker, in the stromal region of aged ovaries was detected with immunohistochemistry. CDKN2A expression detected by western blotting was significantly higher in the ovaries of aged mice with reproductive experience than in those without the experience. Moreover, cells positive for both γH2AX (a senescence marker) and fluorescent SA-ß-Gal staining were present in those isolated from aged ovaries. In addition, the transcript levels of several SASP factors were significantly increased in aged ovaries. These results suggest that senescent cells accumulate in the ovarian stroma and may affect ovarian function in aged mice. Additionally, reproductive experience may promote accumulation.

The effect of ACVR1B/TGFBR1/ACVR1C signaling inhibition on oocyte and granulosa cell development during in vitro growth culture.

The signals of the transforming growth factor ß (TGF-ß) superfamily play a critical role in follicular development in mammals. ACVR1B/TGFBR1/ACVR1C receptors mediate the signaling of several TGF-ß superfamily ligands in granulosa cells. Although the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in follicular development has been confirmed using mutant mouse models, the detailed roles of the signaling in granulosa cell and oocyte development have not been clearly defined. In the present study, we examined the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in granulosa cells using an in vitro growth culture of oocyte-granulosa cell complexes (OGCs) and SB431542, a potent inhibitor of the receptor signaling. Although cumulus-oocyte complexes isolated from the control OGCs were able to undergo cumulus expansion, those isolated from OGCs grown with the inhibitor were not competent, even in the presence of in vivo-grown oocytes. The diameter of the oocytes in the SB431542-treated OGCs was comparable with that of the control; however, these oocytes were not competent for complete meiotic maturation or preimplantation development. Therefore, ACVR1B/TGFBR1/ACVR1C receptor signaling is not required for oocytes to increase their volume but is essential for the normal development of cumulus cells and oocyte developmental competence.

Paracrine regulation of granulosa cell development in the antral follicles in mammals.

Background: Development of ovarian follicles is regulated by a complex interaction of intra- and extra-follicular signals. Oocyte-derived paracrine factors (ODPFs) play a central role in this process in cooperation with other signals. Methods: This review provides an overview of the recent advances in our understanding of the paracrine regulation of antral follicle development in mammals. It specifically focuses on the regulation of granulosa cell development by ODPFs, along with other intrafollicular signals. Main Findings: Bi-directional communication between oocytes and surrounding cumulus cells is a fundamental mechanism that determines cumulus cell differentiation. Along with estrogen, ODPFs promote the expression of forkhead box L2, a critical transcription factor required for mural granulosa cells. Follicle-stimulating hormone (FSH) facilitates these processes by stimulating estrogen production in mural granulosa cells. Conclusion: Cooperative interactions among ODPFs, FSH, and estrogen are critical in determining the fate of cumulus and mural granulosa cells, as well as the development of oocytes.

Cooperative effects of oocytes and estrogen on the forkhead box L2 expression in mural granulosa cells in mice.

Forkhead box L2 (FOXL2) plays a critical role in the development and function of mammalian ovaries. In fact, the causative effects of FOXL2 misregulations have been identified in many ovarian diseases, such as primary ovarian insufficiency and granulosa cell tumor; however, the mechanism by which FOXL2 expression is regulated is not well studied. Here, we showed that FOXL2 expression in ovarian mural granulosa cells (MGCs) requires stimulation by both oocyte-derived signals and estrogen in mice. In the absence of oocytes or estrogen, expression of FOXL2 and its transcriptional targets, Cyp19a1 and Fst mRNA, in MGCs were significantly decreased. Moreover, expression levels of Sox9 mRNA, but not SOX9 protein, were significantly increased in the FOXL2-reduced MGCs. FOXL2 expression in MGCs was maintained with either oocytes or recombinant proteins of oocyte-derived paracrine factors, BMP15 and GDF9, together with estrogen, and this oocyte effect was abrogated with an ALK5 inhibitor, SB431542. In addition, the FOXL2 level was significantly decreased in MGCs isolated from Bmp15-/- /Gdf9+/- mice. Therefore, oocyte, probably with estrogen, plays a critical role in the regulation of FOXL2 expression in mural granulosa cells in mice.

Effect of fibroblast growth factor signaling on cumulus expansion in mice in vitro.

The expansion of cumulus cells associated with oocytes is an essential phenomenon in normal mammalian ovulation. Indeed, attenuated expression of cumulus expansion-related genes, including Has2, Ptgs2, Ptx3, and Tnfaip6, results in ovulation failure, leading to female subfertility or infertility. Moreover, emerging evidence suggests that proteins of the fibroblast growth factor (FGF) family, produced within ovarian follicles, regulate the development and function of cumulus cells; however, the effects of FGF signaling on cumulus expansion have not been investigated extensively. Herein, we investigate the effects of FGF signaling, particularly those of FGF8 secreted by oocytes, on epidermal growth factor-induced cumulus expansion in mice. The phosphorylation level of MAPK3/1, an intracellular mediator of FGF signaling, was significantly decreased in cumulus-oocyte complexes (COCs) following treatment with NVP-BGJ398, an FGF receptor inhibitor. Moreover, even though NVP-BGJ398 treatment did not affect cumulus cell expansion, it significantly upregulated the expression of Ptgs2 and Ptx3. In contrast, treatment with recombinant FGF8 did not affect the degree of cumulus expansion or the expression of expansion-related genes in COCs or oocytectomized cumulus cell complexes. Collectively, these results suggest that FGFs, other than FGF8, exert suppressive effects on the cumulus expansion process in mice.

Expression and regulation of estrogen receptor 2 and its coregulators in mouse granulosa cells.

The cooperative effects of estrogen and oocyte-derived paracrine factors (ODPFs) play critical roles in the normal development of ovarian follicles; however, the mechanism underlying this cooperation has not been well studied. The present study aimed to determine whether ODPFs affect estrogen signaling by regulating the expression of estrogen receptor (ESR) and its coregulators in mouse granulosa cells. Some transcripts encoding ESR coregulators were differentially expressed between cumulus and mural granulosa cells (MGCs). The transcript levels of ESR coregulators, including nuclear receptor corepressor 1 and activator 2, in cumulus cells were significantly suppressed by ODPFs; however, they increased when cumulus cell-oocyte complexes were treated with the transforming growth factor beta receptor I inhibitor, SB431542. Moreover, MGCs exhibited significantly higher ESR2 protein and transcript levels than those in cumulus cells. ODPFs promoted Esr2 expression in cumulus cells but had no effect on that in MGCs. Overall, regulation of the expression of ESR2 and its coregulators in cumulus cells by oocytes seems to be one of the mechanisms underlying estrogen-oocyte cooperation in well-developed antral follicles in mice.

Effects of oocyte-derived paracrine factors on release of extracellular vesicles by murine mural granulosa cells in vitro.

Both oocytes and extracellular vesicles (EV) have emerged as critical regulators of mammalian follicular development; however, the possible interaction between the oocyte-derived paracrine factor (ODPF) and EV signals has never been examined. Therefore, to explore the possibility of an interaction between oocyte and EV signals, the effects of ODPFs on the biogenesis of EVs as well as the expression levels of transcripts related to EV biogenesis in mural granulosa cells (MGCs) were examined using mice. The results showed that, while oocyte coculture has some effects on the expression levels of transcripts related to EV biogenesis, the number of EV particles present in the conditioned medium were not significantly different between ODPF-treated and non-treated MGCs. Therefore, oocytes have no effects on the EV biogenesis by MGCs, at least with respect to the numbers of EV particles.

Oocytes suppress FOXL2 expression in cumulus cells in mice†.

Cumulus cells and mural granulosa cells (MGCs) play distinct roles during follicular development, and normal development of these cell lineages is critical for the female fertility. Transcriptomic diversification between the two cell lineages is obviously a critical mechanism for their functional diversification; however, the transcriptional regulators responsible for this event have not been fully defined. In this study, we sought to identify key transcriptional regulators responsible for the differential gene expression between the two cell lineages. In silico analysis of transcriptomic comparison between cumulus cells and MGCs identified several candidate regulators responsible for the diversification of the two cell lineages. Among them, we herein focused on forkhead box L2 (FOXL2) and showed that expressions of FOXL2 as well as its target transcripts were differentially regulated between cumulus cells and MGCs. The lower expression of FOXL2 in cumulus cells seemed to be due to the suppression by oocyte-derived paracrine signals. These results suggest that FOXL2 is one of the critical transcription factors that determine cumulus cell and MGC lineages under the control of oocytes.

High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes.

CRISPR/Cas9 has been widely used for the efficient generation of genetically modified animals; however, this system could have unexpected off-target effects. In the present study, we confirmed the validity of a high-fidelity Cas9 variant, HypaCas9, for accurate genome editing in mouse zygotes. HypaCas9 efficiently modified the target locus while minimizing off-target effects even in a single-nucleotide mismatched sequence. Furthermore, by applying HypaCas9 to the discrimination of SNP in hybrid strain-derived zygotes, we accomplished allele-specific gene modifications and successfully generated mice with a monoallelic mutation in an essential gene. These results suggest that the improved accuracy of HypaCas9 facilitates the generation of genetically modified animals.

Generation of genetically modified mice using SpCas9-NG engineered nuclease.

Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a protospacer-adjacent motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5'-NGG sequence, SpCas9-NG recognized the 5'-NGA, 5'-NGC and 5'-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.

Expression and function of exportin 6 in full-grown and growing porcine oocytes.

Exportin 6, which functions specifically in the nuclear export of actin family proteins, has been reported to be absent in immature Xenopus oocytes, which have a huge nucleus containing a large amount of actin. In mammalian oocytes, however, the presence and the function of exportin 6 remain uninvestigated. In this study, we assessed the expression and effects of exportin 6 on meiotic resumption in porcine oocytes after cloning porcine exportin 6 cDNA and carrying out overexpression and expression inhibition by mRNA and antisense RNA injection, respectively. We found for the first time that exportin 6 was expressed in mammalian full-grown germinal-vesicle-stage oocytes and was involved in the nuclear export of actin. In contrast, exportin 6 was absent from the growing oocytes, which are meiotically incompetent and maintain the germinal-vesicle structure in the long term; the regulatory mechanism appeared to be active degradation. We examined the effects of exportin 6 on meiotic resumption of porcine oocytes and noted that its expression did not affect the onset time but increased the rate of germinal vesicle breakdown at 24 h via regulation of the nuclear actin level, which directly influences the physical strength of the germinal-vesicle membrane. Our results suggest that exportin 6 affects the nuclear transport of actin and meiotic resumption in mammalian oocytes.

Characterization of mRNA profiles of the exosome-like vesicles in porcine follicular fluid.

Extracellular vesicles such as exosomes contain several types of transcripts, including mRNAs and micro RNAs (miRNAs), and have emerged as important mediators of cell-to-cell communication. Exosome-like vesicles were identified in the ovarian follicles of several mammalian species. Although the miRNA contents have been extensively characterized, the detailed investigation of their mRNA profiles is lacking. Here, we characterize the mRNA profiles of exosome-like vesicles in ovarian follicles in a pig model. The mRNA contents of the exosome-like vesicles isolated from porcine follicular fluid were analyzed and compared with those from mural granulosa cells (MGCs) using the Illumina HiSeq platform. Bioinformatics studies suggested that the exosomal mRNAs are enriched in those encoding proteins involved in metabolic, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) -protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) pathways. While the mRNA profile of the exosome-like vesicles resembled that of MGCs, the vesicles contained mRNAs barely detectable in MGCs. Thus, while the majority of the vesicles are likely to be secreted from MGCs, some may originate from other cell types, including theca cells and oocytes, as well as the cells of non-ovarian organs/tissues. Therefore, the mRNA profiles unveiled several novel characteristics of the exosome-like vesicles in ovarian follicles.

Contributions of UBE2C and UBE2S to meiotic progression of porcine oocytes.

Vertebrate oocytes arrested at the first meiotic prophase must proceed to the second meiotic metaphase (MII) before fertilization. This meiotic process requires the precise control of protein degradation. Part of the protein degradation in oocytes is controlled by members of the ubiquitin-conjugating enzyme family, UBE2C and UBE2S, which are known to participate in mono-ubiquitination and poly-ubiquitination, respectively. Although UBE2 enzymes have been well studied in mitosis, their contribution to mammalian oocyte meiosis is relatively unknown and has been studied only in mice. Here, we investigated the contribution of UBE2C and UBE2S to porcine oocyte maturation using an RNA injection method. Overexpression of UBE2S prevented MII arrest of oocytes and led to the formation of a pronucleus (PN) at 48 h of culture. This effect was also observed for prolonged cultures of UBE2C-overexpressing oocytes, suggesting the effectiveness of poly-ubiquitination in the rapid escape from M-phase in porcine oocytes. Although the inhibition of either UBE2C or UBE2S by antisense RNA (asRNA) injection had no effect on oocyte maturation, asRNA-injected oocytes showed inhibited PN formation after parthenogenetic activation. These results indicated that ubiquitination of certain factors by UBE2S and UBE2C plays a role in the escape from MII arrest in porcine oocytes. Further investigations to identify the factors and how mono- and/or poly-ubiquitination contributes to protein degradation could provide a better understanding of UBE2 roles in oocyte maturation.

Effects of exportin 1 on nuclear transport and meiotic resumption in porcine full-grown and growing oocytes.

Exportin 1 (XPO1) is a nuclear transport receptor involved in the nuclear export of majority proteins in somatic cells. In mammalian oocytes, however, only the presence of XPO1 has been reported at mRNA and protein levels, and the definitive functions of XPO1 and its effects on the meiotic maturation of oocytes have never been directly examined. In the present study, the expression state and the nuclear-export function of porcine XPO1 were analyzed in porcine oocytes. In addition, we investigated the effects of the overexpression and inhibition of XPO1 on meiotic regulation in full-grown and growing oocytes by mRNA injection and inhibitor treatment. Endogenous XPO1 was stably expressed in porcine oocytes during the germinal vesicle (GV) stage, and the expression of exogenous XPO1 significantly decreased the nuclear localization of XPO1 cargos, snurportin 1, and WEE1B. Inhibition of XPO1 by a specific inhibitor, leptomycin B, delayed the GV breakdown (GVBD), whereas the overexpression of XPO1 by mRNA injection accelerated the GVBD. XPO1 overexpression overcame the meiotic arrest induced by WEE1B expression in full-grown oocytes. Surprisingly, the GVBD of porcine growing oocytes, which could not resume meiosis by the maturation culture in vitro, was induced by the expression of exogenous XPO1. These results showed the presence of XPO1 and its function as a nuclear export receptor in mammalian oocytes, including growing oocytes, and they suggest that the regulation of nuclear transport has a large influence on the GV maintenance and meiotic resumption of oocytes.

Efficient Generation of Genome-Modified Mice Using Campylobacter jejuni-Derived CRISPR/Cas.

Mammalian zygote-mediated genome-engineering by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas is currently used for the generation of genome-modified animals. Here, we report that a Campylobacter jejuni-derived orthologous CRISPR/Cas system recognizes a 5'-NNNVRYAC sequence as a protospacer-adjacent motif in mouse zygotes, and is applicable for efficient generation of knockout mice. Moreover, this novel CRISPR/Cas can be used for zygote-mediated knock-in at a unique locus, suggesting that this system could help to expand the feasibility of the zygote-mediated generation of genome-modified animals.

Effects of exosome-like vesicles on cumulus expansion in pigs in vitro.

Cell-secreted vesicles, such as exosomes, have recently been recognized as mediators of cell communication. A recent study in cattle showed the involvement of exosome-like vesicles in the control of cumulus expansion, a prerequisite process for normal ovulation; however, whether this is the case in other mammalian species is not known. Therefore, this study aimed to examine the presence of exosome-like vesicles in ovarian follicles and their effects on cumulus expansion in vitro in pigs. The presence of exosome-like vesicles in porcine follicular fluid (pFF) was confirmed by transmission electron microscopic observation, the detection of marker proteins, and RNA profiles specific to exosomes. Fluorescently labeled exosome-like vesicles isolated from pFF were incorporated into both cumulus and mural granulosa cells in vitro. Exosome-like vesicles were not capable of inducing cumulus expansion to a degree comparable to that induced by follicle-stimulating hormone (FSH). Moreover, exosome-like vesicles had no significant effects on the expression levels of transcripts required for the normal expansion process (HAS2, TNFAIP6, and PTGS2). Interestingly, FSH-induced expression of HAS2 and TNFAIP6 mRNA, but not of PTGS2 mRNA, was significantly increased by the presence of exosome-like vesicles; however, the degree of FSH-induced expansion was not affected. In addition, porcine exosome-like vesicles had no significant effects on the expansion of mouse cumulus-oocyte complexes. Collectively, the present results suggest that exosome-like vesicles are present in pFF, but they are not efficient in inducing cumulus expansion in pigs.

Efficient mutagenesis by CRISPR/Cas system during meiotic maturation of porcine oocytes.

Genome editing using the CRISPR/Cas system can induce mutations with high efficiency, and allows easier production of genome-modified animals than that offered by the conventional method where embryonic stem cells are used. However, studies using CRISPR/Cas systems have been mostly limited to proliferating somatic cells and pronuclear-stage fertilized eggs. In contrast, the efficiency of a CRISPR/Cas system in immature and maturing oocytes progressing through meiosis has not yet been assessed. In the present study, we evaluated the genome-modification efficiency of the CRISPR/Cas system during meiotic maturation of porcine oocytes. Additionally, the localization of the Cas9 protein in immature oocytes was analyzed in relation to nuclear transport and mutation induction. The results showed that CRISPR/Cas induced mutation with high efficiency even in maturing oocytes with condensed chromosomes, whereas mutations were not induced in GV-stage oocytes. The localization analysis of enhanced green fluorescent protein (EGFP)-tagged Cas9 (Cas9-EGFP) revealed that the nuclei contained lesser Cas9 than the cytoplasm in immature oocytes. Treatment with leptomycin B, a nuclear export inhibitor, increased the amount of nuclear Cas9 and enabled mutation induction in GV oocytes. Our results suggest that CRISPR/Cas systems can be applied to oocytes during meiotic maturation and be implemented in novel applications targeting female genomes.

Analyses of EMI functions on meiotic maturation of porcine oocytes.

Cyclin B (CCNB) accumulation is essential for regulating maturation/M-phase promoting factor activity during vertebrate oocyte maturation. Anaphase-promoting-complex/cyclosome (APC/C) degrades CCNB, allowing the cell cycle to progress; this complex is inhibited by Early mitotic inhibitors 1 and 2 (EMI1 and EMI2). The involvement of both EMI proteins in meiotic maturation has been reported in Xenopus and mouse oocytes, although a recent study described a marked difference in their respective function during meiotic resumption. Mouse is currently the only mammal in which the contribution of EMI to the oocyte maturation has been analyzed, so we used RNA injection methods to overexpress and knock down EMI1 and EMI2 to investigate their roles during porcine oocyte maturation. Up-regulation of either porcine EMI promoted precocious germinal vesicle breakdown (GVBD) with early CCNB1 accumulation in oocytes-which is consistent with their activities in mouse but not Xenopus oocytes. Knockdown of EMI1, but not EMI2, delayed GVBD and meiotic progression of oocytes from GVBD to meiotic metaphase I (MI). In contrast, knockdown of EMI2, but not EMI1, released oocytes from meiotic metaphase II (MII) arrest to produce a pronucleus. When injected oocytes were parthenogenetically activated, the up-regulation of EMI2, but not EMI1, prevented pronucleus formation. These results point to the similarities and differences of porcine EMI function with those of mouse versus Xenopus EMI, and generally contribute to our understanding of EMI function during mammalian oocyte maturation. Mol. Reprod. Dev. 83: 983-992, 2016 © 2016 Wiley Periodicals, Inc.

Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus-oocyte complexes.

Communication between oocytes and their companion somatic cells promotes the healthy development of ovarian follicles, which is crucial for producing oocytes that can be fertilized and are competent to support embryogenesis. However, how oocyte-derived signaling regulates these essential processes remains largely undefined. Here, we demonstrate that oocyte-derived paracrine factors, particularly GDF9 and GDF9-BMP15 heterodimer, promote the development and survival of cumulus-cell-oocyte complexes (COCs), partly by suppressing the expression of Ddit4l, a negative regulator of MTOR, and enabling the activation of MTOR signaling in cumulus cells. Cumulus cells expressed less Ddit4l mRNA and protein than mural granulosa cells, which is in striking contrast to the expression of phosphorylated RPS6 (a major downstream effector of MTOR). Knockdown of Ddit4l activated MTOR signaling in cumulus cells, whereas inhibition of MTOR in COCs compromised oocyte developmental competence and cumulus cell survival, with the latter likely to be attributable to specific changes in a subset of transcripts in the transcriptome of COCs. Therefore, oocyte suppression of Ddit4l expression allows for MTOR activation in cumulus cells, and this oocyte-dependent activation of MTOR signaling in cumulus cells controls the development and survival of COCs.