PDLIM7 Synergizes With PDLIM2 and p62/Sqstm1 to Inhibit Inflammatory Signaling by Promoting Degradation of the p65 Subunit of NF-κB.

Activation of NF-κB transcription factors is critical for innate immune cells to induce inflammation and fight against microbial pathogens. On the other hand, the excessive and prolonged activation of NF-κB causes massive inflammatory damage to the host, suggesting that regulatory mechanisms to promptly terminate NF-κB activation are important to prevent immunopathology. We have previously reported that PDLIM2, a PDZ-LIM domain-containing protein, is a nuclear ubiquitin E3 ligase that targets the p65 subunit of NF-κB for degradation, thereby suppressing NF-κB activation. Here we show that PDLIM7, another member of LIM protein family, is also a ubiquitin E3 ligase that inhibits NF-κB-mediated inflammatory responses. PDLIM7 directly polyubiquitinates p65 and promotes its proteasomal degradation. Moreover, PDLIM7 heterodimerizes with PDLIM2 to promote synergistic PDLIM2-mediated degradation of p65. Mechanistically, PDLIM7 promotes K63-linked ubiquitination of PDLIM2 and then the proteasome/autophagosome cargo protein p62/Sqstm1 binds to both polyubiquitinated PDLIM2 and the proteasome, thereby facilitating the delivery of the NF-κB-PDLIM2 complex to the proteasome and subsequent p65 degradation. Consistently, double knockdown of PDLIM7 and either PDLIM2 or p62/Sqstm1 results in augmented proinflammatory cytokine production compared to control cells or single knockdown cells. These data delineate a new role for PDLIM7 and p62/Sqstm1 in the regulation of NF-κB signaling by bridging a ubiquitin E3 ligase and the proteasome.

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.

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.

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.

Glucocorticoid metabolism in the bovine cumulus-oocyte complex matured in vitro.

Glucocorticoid action in target organs is regulated by relative activities of 11ß-HSD type 1 (HSD11B1) that mainly converts cortisone to active cortisol and type 2 (HSD11B2) that inactivates cortisol to cortisone. HSD11Bs have been shown to be expressed in the ovary of various species. However, little is known about the expression and activity of HSD11Bs in the bovine cumulus-oocyte complex (COC). In the present study, we investigated the expression and activities of HSD11Bs in in vitro-matured (IVM) bovine COCs. Bovine COCs were matured in M199 supplemented with or without FSH and FCS. The expression of HSD11B1 and HSD11B2 was measured by using quantitative RT-PCR in denuded oocytes (DO) and cumulus cells (CC). Reductive and oxidative activities of HSD11Bs were determined by radiometric conversion assay using labeled cortisol, cortisone or dexamethasone in intact COCs, DO or CC in the presence or absence of 11-keto-progesterone (11kP), a selective inhibitor of HSD11B2. The presence of HSD11Bs in the oocyte was examined by immunofluorescence microscopy. Oocytes exclusively expressed HSD11B2 and its expression and activity were largely unchanged during IVM. CC, on the other hand, exclusively expressed HSD11B1 and its expression and activity were upregulated as IVM progressed. As a result, the net glucocorticoid metabolism shifted from inactivation to activation towards the end of IVM. These results indicate that the bovine COC is capable of modulating local glucocorticoid concentration and, by doing so, may create an environment that is favorable to ovulating oocyte for maturation, fertilization and subsequent development.

Effects of porcine oocytes on the expression levels of transcripts encoding glycolytic enzymes in granulosa cells.

Oocytes play critical roles in regulating the expression of transcripts encoding the glycolytic enzymes phosphofructokinase, platelet (PFKP) and lactate dehydrogenase A (LDHA) in granulosa cells in mice, but whether this is the case in pigs or other mammals has not been adequately investigated. Therefore, the aim of this study was to determine whether porcine oocytes regulate the expression levels of these transcripts in granulosa cells in vitro. Porcine cumulus cells expressed higher levels of PFKP and LDHA transcripts than mural granulosa cells (MGCs). However, co-culturing with oocytes had no significant effect on the isolated cumulus cells. While murine oocytes promoted the expression of both Pfkp and Ldha transcripts by murine MGCs, porcine oocytes promoted the expression of only Pfkp, but not Ldha transcripts by murine MGCs. Neither murine nor porcine oocytes affected PFKP and LDHA expression by porcine MGCs. Moreover, in the presence of porcine follicular fluid, porcine oocytes maintained the expression of PFKP, but not LDHA by porcine cumulus cells. Therefore, porcine oocytes are capable of regulating the expression of PFKP but not LDHA in granulosa cells in coordination with unknown factor(s) present in the follicular fluid.

Finding of a highly efficient ZFN pair for Aqpep gene functioning in murine zygotes.

The generation efficiencies of mutation-induced mice when using engineered zinc-finger nucleases (ZFNs) have been generally 10 to 20% of obtained pups in previous studies. The discovery of high-affinity DNA-binding modules can contribute to the generation of various kinds of novel artificial chromatin-targeting tools, such as zinc-finger acetyltransferases, zinc-finger histone kinases and so on, as well as improvement of reported zinc-finger recombinases and zinc-finger methyltransferases. Here, we report a novel ZFN pair that has a highly efficient mutation-induction ability in murine zygotes. The ZFN pair induced mutations in all obtained mice in the target locus, exon 17 of aminopeptidase Q gene, and almost all of the pups had biallelic mutations. This high efficiency was also shown in the plasmid DNA transfected in a cultured human cell line. The induced mutations were inherited normally in the next generation. The zinc-finger modules of this ZFN pair are expected to contribute to the development of novel ZF-attached chromatin-targeting tools.

One-step generation of phenotype-expressing triple-knockout mice with heritable mutated alleles by the CRISPR/Cas9 system.

Triple-knockout mice generated by the one-step CRISPR/Cas9 system were examined for the effects of multiple gene modifications on each phenotype and individual gene function. Sixty embryos were transferred, and 9 pups were obtained; all 9 pups had mutations on 3 loci, and 7 pups showed mutations in all-alleles. F0 mice showed knockout phenotypes or no protein expression of target genes simultaneously, and these mutations were normally inherited in the next generation.

Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system.

The mammalian zygote-mediated CRISPR/Cas system can efficiently generate targeted genome-modified animals. However, this system is limited by the risk of off-target mutations. Here we show that offset-nicking by Cas9 nickase and paired gRNAs allows us to generate region deleted mice and targeted knock-in mice without off-target mutations.