Suk Namgoong

Effects of growth differentiation factor 9 and bone morphogenetic protein 15 on the in vitro maturation of porcine oocytes.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-β (TGF-β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ-GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos [a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.

Actin-capping proteins play essential roles in the asymmetric division of maturing mouse oocytes

ABSTRACT Actin polymerization is essential for various stages of mammalian oocyte maturation, including spindle migration, actin cap formation, polar body extrusion and cytokinesis. The heterodimeric actin-capping protein is an essential element of the actin cytoskeleton. It binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. However, the roles of capping protein in mammalian oocyte maturation are poorly understood. We investigated the roles of capping protein in mouse oocytes and found that it is essential for correct asymmetric spindle migration and polar body extrusion. Capping protein mainly localized in the cytoplasm during maturation. By knocking down or ectopically overexpressing this protein, we revealed that it is crucial for efficient spindle migration and maintenance of the cytoplasmic actin mesh density. Expression of the capping-protein-binding region of CARMIL (also known as LRRC16A) impaired spindle migration and polar body extrusion during oocyte maturation and decreased the density of the cytoplasmic actin mesh. Taken together, these findings show that capping protein is an essential component of the actin cytoskeleton machinery that plays crucial roles in oocyte maturation, presumably by controlling the cytoplasmic actin mesh density.

Superovulation induces defective methylation in line-1 retrotransposon elements in blastocyst

BackgroundSeries of epigenetic events happen during preimplantation development. Therefore assistant reproduction techniques (ART) have the potential to disrupt epigenetic regulation during embryo development. The purpose of this study was to investigate whether defects in methylation patterns in blastocyst due to superovulation originate from abnormal expression of Dnmts.MethodsLow- (6 IU) and high- (10 IU) dosage of PMSG was used to stimulate the female mice. The metaphase II(MII) oocytes, zygotes and blastocyst stage embryos were collected. Global methylation and methylation at H3K9 in zygote, and methylation at repeated sequence Line 1 and IAP in blastocysts were assayed. In addition, expression of Dnmts was examined in oocytes and zygotes.ResultsGlobal DNA methylation and methylation at H3K9 in zygotes derived from females after low- or high-dosage hormone treatment were unaltered compared to that in controls. Moreover, DNA methylation at IAP in blastocysts was also unaffected, regardless of hormone dosage. In contrast, methylation at Line1 decreased when high-dose hormone was administered. Unexpectedly, expression of Dnmt3a, Dnmt3b, Dnmt3L as well as maintenance Dnmt1o in oocytes and zygotes was not disrupted.ConclusionsThe results suggest that defects in embryonic methylation patterns do not originate from the disruption of Dnmt expression.

A specific inhibitor of CDK1, RO-3306, reversibly arrests meiosis during in vitro maturation of porcine oocytes

CDK1 plays pivotal role in meiotic progression of oocytes from G2 to metaphase II (MII) stage. In this study, we investigated the possibility of utilizing a selective inhibitor of CDK1, RO-3306, as a novel agent for the synchronization of oocyte maturation. Two groups of cumulus-oocyte complexes (COCs) were treated with 10 μM RO-3306. The first group was treated for 44 h, whereas the second group was transferred to drug-free medium after a 20 h treatment. MII-stage oocytes from each group were confirmed by cytoplasmic maturation and embryonic development assays. Treatment of immature porcine oocytes with RO-3306 for 20 h arrested them at the germinal vesicle (GV) stage. The GV-arrest effect of RO-3306 was reversible: when RO-3306-arrested COCs were subsequently cultured for 24h in the absence of RO-3306, 76.19 ± 2.68% of these oocytes reached the MII stage after 44 h of in vitro maturation, a rate similar to that of non-treated control oocytes (79.08 ± 3.23%). Furthermore, RO-3306-treated oocytes transferred to drug-free media did not differ significantly from controls (P>0.05) with respect to cleavage and blastocyst formation upon parthenogenetic activation. To explore the underlying molecular mechanisms, we examined the expression patterns of four representative maternal transcripts, CDK1, Cyclin B1, GDF9, and BMP15, by real-time polymerase chain reaction (PCR) and poly(A)-test PCR (PAT assay). RO-3306 treatment increased expression of CDK1 but had no effect on the expression of the other genes. These data suggest that RO-3306 efficiently blocks and synchronizes the meiotic progression of porcine oocytes at the GV stage without affecting their meiotic and cytoplasmic maturation.

The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho‐GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p‐cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocytes germinal‐vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p‐MLC) localized at the cortex, and its abundance decreased by the metaphase‐II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y‐27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p‐cofilin, and p‐MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML‐7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. Mol. Reprod. Dev. 82: 849–858, 2015.

Non-muscle tropomyosin (Tpm3) is crucial for asymmetric cell division and maintenance of cortical integrity in mouse oocytes

Tropomyosins are actin-binding cytoskeletal proteins that play a pivotal role in regulating the function of actin filaments in muscle and non-muscle cells; however, the roles of non-muscle tropomyosins in mouse oocytes are unknown. This study investigated the expression and functions of non-muscle tropomyosin (Tpm3) during meiotic maturation of mouse oocytes. Tpm3 mRNA was detected at all developmental stages in mouse oocytes. Tpm3 protein was localized at the cortex during the germinal vesicle and germinal vesicle breakdown stages. However, the overall fluorescence intensity of Tpm3 immunostaining was markedly decreased in metaphase II oocytes. Knockdown of Tpm3 impaired asymmetric division of oocytes and spindle migration, considerably reduced the amount of cortical actin, and caused membrane blebbing during cytokinesis. Expression of a constitutively active cofilin mutant and Tpm3 overexpression confirmed that Tpm3 protects cortical actin from depolymerization by cofilin. The data indicate that Tpm3 plays crucial roles in maintaining cortical actin integrity and asymmetric cell division during oocyte maturation, and that dynamic regulation of cortical actin by Tpm3 is critical to ensure proper polar body protrusion.

Small Molecule Inhibitor of Formin Homology 2 Domains (SMIFH2) Reveals the Roles of the Formin Family of Proteins in Spindle Assembly and Asymmetric Division in Mouse Oocytes

Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.

JMY Functions as Actin Nucleation-Promoting Factor and Mediator for p53-Mediated DNA Damage in Porcine Oocytes

Junction-mediating and regulatory protein(JMY) is a multifunctional protein with roles in the transcriptional co-activation of p53 and the regulation of actin nucleation promoting factors and, hence, cell migration; however, its role in the maturation of porcine oocytes is unclear. In the current study, we investigated functional roles of JMY in porcine oocytes. Porcine oocytes expressed JMY mRNA and protein, and the mRNA expression level decreased during oocyte maturation. Knockdown of JMY by RNA interference decreased the rate of polar body extrusion, validating its role in the asymmetric division of porcine oocytes. JMY knockdown also down-regulated the mRNA and protein levels of actin and Arp2/3. Furthermore, JMY accumulated in the nucleus in response to DNA damage, and JMY knockdown suppressed DNA damage-mediated p53 activation. In conclusion, our results show that JMY has important roles in oocyte maturation as a regulator of actin nucleation-promoting factors and an activator of p53 during DNA damage during DNA damages in porcine oocytes.

Structural basis for recognition of Emi2 by Polo-like kinase 1 and development of peptidomimetics blocking oocyte maturation and fertilization

In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr152 and Thr176) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.

CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against OCT4 decreased the percentages of OCT4-positive embryos to 37–50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP) vector targeting the OCT4 exon with Cas9 and sgRNA to create a knockin. We confirmed eGFP fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had eGFP sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig.