Metabolic Rewiring by Human Placenta-Derived Mesenchymal Stem Cell Therapy Promotes Rejuvenation in Aged Female Rats.

Aging is a degenerative process involving cell function deterioration, leading to altered metabolic pathways, increased metabolite diversity, and dysregulated metabolism. Previously, we reported that human placenta-derived mesenchymal stem cells (hPD-MSCs) have therapeutic effects on ovarian aging. This study aimed to identify hPD-MSC therapy-induced responses at the metabolite and protein levels and serum biomarker(s) of aging and/or rejuvenation. We observed weight loss after hPD-MSC therapy. Importantly, insulin-like growth factor-I (IGF-I), known prolongs healthy life spans, were markedly elevated in serum. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) analysis identified 176 metabolites, among which the levels of 3-hydroxybutyric acid, glycocholic acid, and taurine, which are associated with health and longevity, were enhanced after hPD-MSC stimulation. Furthermore, after hPD-MSC therapy, the levels of vitamin B6 and its metabolite pyridoxal 5'-phosphate were markedly increased in the serum and liver, respectively. Interestingly, hPD-MSC therapy promoted serotonin production due to increased vitamin B6 metabolism rates. Increased liver serotonin levels after multiple-injection therapy altered the expression of mRNAs and proteins associated with hepatocyte proliferation and mitochondrial biogenesis. Changes in metabolites in circulation after hPD-MSC therapy can be used to identify biomarker(s) of aging and/or rejuvenation. In addition, serotonin is a valuable therapeutic target for reversing aging-associated liver degeneration.

Oocyte Cytoplasmic Gas6 and Heparan Sulfate (HS) are Required to Establish the Open Chromatin State in Nuclei During Remodeling and Reprogramming.

BACKGROUND/AIMS: Previously, we found that silencing of growth arrest-specific gene 6 (Gas6) in oocytes impaired cytoplasmic maturation, resulting in failure of sperm chromatin decondensation (SCD) and pronuclear (PN) formation after fertilization. Thus, we conducted this study to determine the effect of Gas6 RNAi on downstream genes and to elucidate the working mechanism of Gas6 on oocyte cytoplasmic maturation and SCD. METHODS: Using RT-PCR, Western blot and immunofluorescence, the expression levels of various target genes and the localization of heparan sulfate (HS) were analyzed after Gas6 RNAi. The roles of Gas6 in HS biosynthesis, production of ATP and GSH, ROS generation and ΔΨm were also investigated. SCD and micrococcal nuclease (MNase) analyses were used to examine the effects of HS on the open chromatin state in sperm and somatic cell nuclei, respectively. RESULTS: Disruption of Gas6 expression led to the inhibition of HS biosynthesis through the reduction of several HS biosynthetic enzymes. The rescue experiment, HS treatment in vitro, significantly recovered SCD and PN formation, confirming that HS had the ability to induce sperm head remodeling during fertilization. Interestingly, excessive mitochondrial activation in Gas6-depleted MII oocytes caused ROS generation and glutathione (GSH) degradation via mitochondrial activation, such as elevated ΔΨm and ATP production. Indeed, HS-treated NIH3T3 cell nuclei showed an open chromatin state, as determined by diffuse DAPI staining and increased sensitivity to MNase. CONCLUSION: We propose that the addition of HS to sperm and/or oocyte maturation would improve the efficiency of in vitro fertilization and somatic cell nuclear transfer (SCNT) reprogramming.

RASD1 Knockdown Results in Failure of Oocyte Maturation.

BACKGROUND: Ras dexamethasone-induced protein (RASD1) is a member of Ras superfamily of small GTPases. RASD1 regulates various signaling pathways involved in iron homeostasis, growth hormone secretion, and circadian rhythm. However, RASD1 function in oocyte remains unknown. METHODS: Using immunohistochemistry, immunofluorescence, and quantitative real-time RT-PCR, RASD1 expression in mouse ovary and RASD1 role in oocyte maturation-related gene expression, spindle formation, and chromosome alignment were analyzed. RNAi microinjection and time-lapse video microscopy were used to examine the effect of Rasd1 knockdown on oocyte maturation. RESULTS: RASD1 was highly detected in oocytes transitioning from primordial to secondary follicles. Rasd1 was highly expressed in germinal vesicle (GV), during GV breakdown, and in metaphase I (MI) stage as oocytes mature, and its expression was significantly downregulated in MII stage. With knockdown of Rasd1, maturation in GV oocytes was arrested at MI stage, showing disrupted meiotic spindling and chromosomal misalignment. In addition, Obox4 and Arp2/3, engaged in MI-MII transition and cytokinesis, respectively, were misregulated in GV oocytes by Rasd1 knockdown. CONCLUSION: These findings suggest that RASD1 is a novel factor in MI-MII oocyte transition and may be involved in regulating the progression of cytokinesis and spindle formation, controlling related signaling pathways during oocyte maturation.

Obox4-silencing-activated STAT3 and MPF/MAPK signaling accelerate nuclear membrane breakdown in mouse oocytes.

Mouse oocytes begin to mature in vitro once liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox 4 (Obox4) is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I-II (MI-MII) transition. This study further examines the molecular mechanisms of OBOX4 in regulating GV nuclear membrane breakdown. Maturation-promoting factor (MPF) and MAPK are normally inactive in GV stage oocytes but were activated prematurely in arrested GV stage oocytes by 3-isobutyl-1-metyl-xanthine (IBMX) in vitro after Obox4 RNA interference (RNAi). Furthermore, signal transducer and activator of transcription 3 (STAT3) was significantly activated by Obox4 RNAi. We confirmed that this Obox4 RNAi-induced premature STAT3 and MPF/MAPK activation at the GV stage provoked subsequent GV breakdown (GVBD) despite the opposing force of high cAMP in the IBMX-supplemented medium to maintain intact GV. When cumulus-oocyte complexes were exposed to interferon α (IFNA), a STAT3 activator, oocytes matured and cumulus cells expanded to resume nuclear maturation in IBMX-supplemented medium, suggesting that STAT3 activation is sufficient for stimulating the continuation of meiosis. Using Stattic, a specific STAT3 inhibitor, we confirmed that GVBD involves STAT3 activation in Obox4-silenced oocytes. Based on these findings, we concluded that i) Obox4 is an important upstream regulator of MPF/MAPK and STAT3 signaling, and ii) Obox4 is a key regulator of the GV arrest mechanism in oocytes.

Transcription elongation factor Tcea3 regulates the pluripotent differentiation potential of mouse embryonic stem cells via the Lefty1-Nodal-Smad2 pathway.

Self-renewal and pluripotency are hallmark properties of pluripotent stem cells, including embryonic stem cells (ESCs) and iPS cells. Previous studies revealed the ESC-specific core transcription circuitry and showed that these core factors (e.g., Oct3/4, Sox2, and Nanog) regulate not only self-renewal but also pluripotent differentiation. However, it remains elusive how these two cell states are regulated and balanced during in vitro replication and differentiation. Here, we report that the transcription elongation factor Tcea3 is highly enriched in mouse ESCs (mESCs) and plays important roles in regulating the differentiation. Strikingly, altering Tcea3 expression in mESCs did not affect self-renewal under nondifferentiating condition; however, upon exposure to differentiating cues, its overexpression impaired in vitro differentiation capacity, and its knockdown biased differentiation toward mesodermal and endodermal fates. Furthermore, we identified Lefty1 as a downstream target of Tcea3 and showed that the Tcea3-Lefty1-Nodal-Smad2 pathway is an innate program critically regulating cell fate choices between self-replication and differentiation commitment. Together, we propose that Tcea3 critically regulates pluripotent differentiation of mESCs as a molecular rheostat of Nodal-Smad2/3 signaling.

Single nucleotide polymorphisms in fibroblast growth factor 23 gene, FGF23, are associated with prostate cancer risk.

OBJECTIVE: To determine whether sequence variants within the FGF23 gene are associated with the risk of developing prostate cancer in a Korean population. PATIENTS AND METHODS: Five common single nucleotide polymorphisms (SNPs) in the FGF23 gene were assessed in 272 patients with prostate cancer and 173 control subjects with benign prostatic hyperplasia. Single-locus analyses were conducted using conditional logistic regression. In addition, we performed a haplotype analysis for the five FGF23 SNPs tested. RESULTS: Three SNPs in the FGF23 gene (rs11063118, rs13312789 and rs7955866) were associated with an increased risk of prostate cancer in our study population. Odds ratios for homozygous variants vs wild-type variants ranged from 1.68 (95% confidence interval [CI]: 1.15-2.46) to 1.79 (95% CI: 1.16-2.75). CONCLUSION: This is the first study showing that genetic variations in FGF23 increase prostate cancer susceptibility.

The Antioxidant Auraptene Improves Aged Oocyte Quality and Embryo Development in Mice.

Decrease in quality of postovulatory aged oocytes occurs due to oxidative stress and leads to low fertilization and development competence. It is one of the main causes that exerting detrimental effect on the success rate in assisted reproductive technology (ART). Auraptene (AUR), a citrus coumarin, has been reported to possess an antioxidant effects in other tissues. In this study, we aimed to confirm the potential of AUR to delay the oocyte aging process by alleviating oxidative stress. Superovulated mouse oocytes in metaphase of second meiosis (MII) were exposed to 0, 1 or 10 µM AUR for 12 h of in vitro aging. AUR addition to the culture medium recovered abnormal spindle and chromosome morphology and mitigated mitochondrial distribution and mitochondrial membrane potential (ΔΨ) in aged oocytes. AUR-treated aged oocytes also showed suppressed oxidative stress, with lower reactive oxygen species (ROS) levels, higher glutathione (GSH) levels and increased expression of several genes involved in antioxidation. Furthermore, AUR significantly elevated the fertilization and embryo developmental rates. Oocytes aged with 1 µM AUR exhibited morphokinetics that were very similar to those of the control group. Altogether, these data allowed us to conclude that AUR improved the quality of aged oocytes and suggest AUR as an effective clinical supplement candidate to prevent postovulatory aging.

Expression pattern of endothelin system components and localization of smooth muscle cells in the human pre-ovulatory follicle.

BACKGROUND: Whether ovarian follicular rupture involves contractile activity or not has been debated for decades. Recently, study in the rodents has indicated that an endogenously produced potent vasoconstrictive peptide, endothelin-2 (EDN2), may induce follicular constriction immediately prior to ovulation. This study was aimed to systematically characterize the human ovarian endothelin system and localize smooth muscle cells to assess the possible involvement of contractile activity in human ovulation. METHODS: This is a prospective experimental study. Study subjects were 20 women aged 20-38 years who underwent IVF owing to tubal or male factors. Expression patterns of messenger RNAs (mRNAs) for EDN1, EDN2, EDN3, endothelin-converting enzyme-1 (ECE1 and ECE2), endothelin receptor A (ET(A)) and ET(B) in the granulosa cells (GCs) and cumulus cells and endothelin peptide concentration in the pre-ovulatory follicles were measured at 36 h after hCG injection. In addition, localization of ovarian smooth muscle cells and endothelin receptor expression were determined in normal (non-IVF patient) ovaries. RESULTS: Pre-ovulatory follicles express mRNA for EDN1 and EDN2, ECE1, ECE2, ET(A) and ET(B), but not EDN3, contain highly concentrated endothelin peptides (105.9 pg/ml) and are surrounded by theca externa that are made mostly of multicell layer non-vascular smooth muscle cells. ET(A) expression is localized in the smooth muscle cells of theca externa, theca interna and GC, whereas ET(B) expression is confined to theca interna. CONCLUSIONS: Pre-ovulatory follicles contain highly concentrated endothelins and are surrounded by non-vascular smooth muscle cells that express endothelin receptor, suggesting involvement of endothelin-induced contractile action in ovulation in the human ovary.

Obox4 critically regulates cAMP-dependent meiotic arrest and MI-MII transition in oocytes.

Extra follicular oocytes spontaneously resume meiosis in vitro, but the intact germinal vesicle (GV) is retained if the oocytes are cultured in medium containing phosphodiesterase (PDE) inhibitors or cAMP analogues. On the basis of our finding that Obox4 is prominently expressed in oocytes, the present study was conducted to determine the functional role of the homeodomain-containing factor Obox4 during in vitro oocyte maturation. After microinjection of Obox4 dsRNA into the cytoplasm of GV oocytes cultured in M16 medium, oocytes were arrested at metaphase I (MI, 77.7%) and metaphase II (MII, 22.3%). Surprisingly, however, 89% of Obox4 RNAi-treated oocytes resumed meiosis and developed to MI and MII when cultured in medium containing 0.2 mM 3-isobutyl-1-methyl-xanthine (IBMX), in which untreated oocytes maintain intact GVs. Spindles were aberrant, and chromosomes were severely aggregated with decreased MPF and MAP kinase activities in arrested MI oocytes after exposure to Obox4 RNAi. Oocytes overexpressing Obox4 retained intact GVs when cultured in M16 medium. Taken together, for the first time to our knowledge, these findings indicate that Obox4 plays a key role in the cAMP-dependent signaling cascades that maintain GV arrest. Oocytes not expressing Obox4 failed to maintain intact GVs in IBMX-supplemented medium, while GVs remained intact when oocytes were kept in plain medium and overexpressing Obox4, suggesting that Obox4 plays a critical role in cAMP-dependent cascade for maintaining intact GVs.

GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function.

Previously, we reported that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation by suppressing mitophagy and inducing mitochondrial dysfunction, resulting in fertilization failure. Here, we show that oocyte aging is accompanied by an increase in meiotic defects associated with chromosome misalignment and abnormal spindle organization. Intriguingly, decreased Gas6 mRNA and protein expression were observed in aged oocytes from older females. We further explored the effect of GAS6 on the quality and fertility of aged mouse oocytes using a GAS6 rescue analysis. After treatment with the GAS6 protein, aged oocytes matured normally to the meiosis II (MII) stage. Additionally, maternal age-related meiotic defects were reduced by GAS6 protein microinjection. Restoring GAS6 ameliorated the mitochondrial dysfunction induced by maternal aging. Ultimately, GAS6-rescued MII oocytes exhibited increased ATP levels, reduced ROS levels and elevated glutathione (GSH) levels, collectively indicating improved mitochondrial function in aged oocytes. Thus, the age-associated decrease in oocyte quality was prevented by restoring GAS6. Importantly, GAS6 protein microinjection in aged oocytes also rescued fertility. We conclude that GAS6 improves mitochondrial function to achieve sufficient cytoplasmic maturation and attenuates maternal age-related meiotic errors, thereby efficiently safeguarding oocyte quality and fertility.

Bcl2l10 mediates the proliferation, invasion and migration of ovarian cancer cells.

Bcl2l10, also known as Diva, Bcl­b and Boo, is a member of the Bcl2 family of proteins, which are involved in signaling pathways that regulate cell apoptosis and autophagy. Previously, it was demonstrated that Bcl2l10 plays a crucial role in the completion of oocyte meiosis and is a key regulator of Aurora kinase A (Aurka) expression and activity in oocytes. Aurka is overexpressed in several types of solid tumors and has been considered a target of cancer therapy. Based on these previous results, in the present study, the authors aimed to investigate the regulatory role of Bcl2l10 in A2780 and SKOV3 human ovarian cancer cells. The protein expression of Bcl2l10 was examined in human cancer tissues and cell lines, including the ovaries, using a tissue microarray and various human ovarian cancer cell lines. It was found that Bcl2l10 regulated the protein stability and activities of Aurka in ovarian cancer cells. Although apoptosis was not affected, the cell cycle was arrested at the G0/G1 phase by Bcl2l10 knockdown. Of note, cell viability and motility were markedly increased by Bcl2l10 knockdown. On the whole, the findings of this study suggest that Bcl2l10 functions as tumor suppressor gene in ovarian cancer.

Human placenta-derived mesenchymal stem cells stimulate ovarian function via miR-145 and bone morphogenetic protein signaling in aged rats.

BACKGROUND: Aging has detrimental effects on the ovary, such as a progressive reduction in fertility and decreased hormone production, that greatly reduce the quality of life of women. Thus, the current study was undertaken to investigate whether human placenta-derived mesenchymal stem cell (hPD-MSC) treatment can restore the decreases in folliculogenesis and ovarian function that occur with aging. METHODS: Acclimatized 52-week-old female SD rats were randomly divided into four groups: single hPD-MSC (5 × 105) therapy, multiple (three times, 10-day intervals) hPD-MSC therapy, control (PBS), and non-treated groups. hPD-MSC therapy was conducted by tail vein injection into aged rats. The rats were sacrificed 1, 2, 3, and 5 weeks after the last injection. hPD-MSC tracking and follicle numbers were histologically confirmed. The serum levels of sex hormones and circulating miRNAs were detected by ELISA and qRT-PCR, respectively. TGF-ß superfamily proteins and SMAD proteins in the ovary were detected by Western blot analysis. RESULTS: We observed that multiple transplantations of hPD-MSCs more effectively promoted primordial follicle activation and ovarian hormone (E2 and AMH) production than a single injection. After hPD-MSC therapy, the levels of miR-21-5p, miR-132-3p, and miR-212-3p, miRNAs associated with the ovarian reserve, were increased in the serum. Moreover, miRNAs (miR-16-5p, miR-34a-5p, and miR-191-5p) with known adverse effects on folliculogenesis were markedly suppressed. Importantly, the level of miR-145-5p was reduced after single- or multiple-injection hPD-MSC therapy, and we confirmed that miR-145-5p targets Bmpr2 but not Tgfbr2. Interestingly, downregulation of miR-145-5p led to an increase in BMPR2, and activation of SMAD signaling concurrently increased primordial follicle development and the number of primary and antral follicles. CONCLUSIONS: Our study verified that multiple intravenous injections of hPD-MSCs led to improved ovarian function via miR-145-5p and BMP-SMAD signaling and proposed the future therapeutic potential of hPD-MSCs to promote ovarian function in women at advanced age to improve their quality of life during climacterium.

Role of Bcl2-like 10 (Bcl2l10) in Regulating Mouse Oocyte Maturation.

Previously, we have shown that Bcl2l10 is highly expressed in metaphase II (MII)-stage oocytes. The objective of this study was to characterize Bcl2l10 expression in ovaries and to examine the function of Bcl2l10 in oocyte maturation using RNA interference. Bcl2l10 transcript expression was ovary and oocyte specific. Bcl2l10 was highly expressed in oocytes and pronuclear-stage embryos; however, its expression decreased at the two-cell stage and dramatically disappeared thereafter. Microinjection of Bcl2l10 double-stranded RNA into the cytoplasm of germinal vesicle oocytes resulted in a marked decrease in Bcl2l10 mRNA and protein and metaphase I (MI) arrest (78.9%). Most MI-arrested oocytes exhibited abnormalities in their spindles and chromosome configurations. Bcl2l10 RNA interference had an obvious effect on the activity of maturation-promoting factor but not on that of mitogen-activated protein kinase. We concluded that the role of Bcl2l10 is strongly associated with oocyte maturation, especially at the MI-MII transition.

Gas6 is a reciprocal regulator of mitophagy during mammalian oocyte maturation.

Previously, we found that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation through mitochondrial overactivation with concurrent failure of pronuclear formation after fertilization. In this study, we report that Gas6 regulates mitophagy and safeguards mitochondrial activity by regulating mitophagy-related genes essential to the complete competency of oocytes. Based on RNA-Seq and RT-PCR analysis, in Gas6-silenced MII oocytes, expressions of mitophagy-related genes were decreased in Gas6-silenced MII oocytes, while mitochondrial proteins and Ptpn11, the downstream target of Gas6, was increased. Interestingly, GAS6 depletion induced remarkable MTOR activation. Gas6-depleted MII oocytes exhibited mitochondrial accumulation and aggregation caused by mitophagy inhibition. Gas6-depleted MII oocytes had a markedly lower mtDNA copy number. Rapamycin treatment rescued mitophagy, blocked the increase in MTOR and phosphorylated-MTOR, and increased the mitophagy-related gene expression in Gas6-depleted MII oocytes. After treatment with Mdivi-1, a mitochondrial division/mitophagy inhibitor, all oocytes matured and these MII oocytes showed mitochondrial accumulation but reduced Gas6 expression and failure of fertilization, showing phenomena very similar to the direct targeting of Gas6 by RNAi. Taken together, we conclude that the Gas6 signaling plays a crucial role in control of oocytes cytoplasmic maturation by modulating the dynamics and activity of oocyte mitochondria.

Gene Expression Analysis in Mammalian Oocytes and Embryos by Quantitative Real-Time RT-PCR.

This chapter describes methods for preparing oocytes and embryos to analyze their gene expression at specific developmental stages. We illustrate how to collect germinal vesicles (GVs) and mature metaphase II (MII) stage oocytes, as well as how to collect embryos at specific developmental stages from the pronucleus (PN) to the blastocyst stage from female mice. We also describe how to prepare mRNAs from these precious cells to analyze the expression of the target genes. The materials and methods in this chapter are used mainly for mouse oocytes and embryos, but with subtle modifications, they may be applicable for most mammalian species.

Establishment of ovarian reconstruction system in culture for functional genomic analysis.

In a previous study, we reported a list of genes expressed differentially in primordial and primary follicles [Park et al., Fertil. Steril., 83, 410-418 (2005)]. An innovative experimental system is required to evaluate the functions of these genes in folliculogenesis, particularly primordial-primary follicle transition. In this study, ovarian tissues were dissociated, and isolated cells were transfected using small interfering RNAs (siRNAs) for disrupting a specific gene, followed by ovarian reconstruction via calcium alginate encapsulation. The effects of RNA interference (RNAi) on follicular development were evaluated by a histological observation of the reconstructed ovarian tissue. Interestingly, follicular formation and development showed differences between control and experimental groups. Thus, even though this system includes some problems that need to be solved, ovarian reconstruction following the modification of gene expression of individual ovarian component cells could lead the way to methods of studying molecular mechanisms of primordial-primary follicle transition.

Bcl2l10, a new Tpx2 binding partner, is a master regulator of Aurora kinase A in mouse oocytes.

Previously, we demonstrated that Bcl-2-like 10 (Bcl2l10) is associated with meiotic spindle assembly and that the gene that is most strongly down-regulated by Bcl2l10 RNAi is targeting protein for Xklp2 (Tpx2). Tpx2 is a well-known cofactor that controls the activity and localization of Aurora kinase A (Aurka) during mitotic spindle assembly. Therefore, this study was conducted (1) to identify the associations among Bcl2l10, Tpx2, and Aurka and (2) to understand how Bcl2l10 regulates meiotic spindle assembly in mouse oocytes. Bcl2l10, Tpx2, and Aurka co-localized on the meiotic spindles, and Bcl2l10 was present in the same complex with Tpx2. Tpx2 and Aurka expression decreased whereas phospho-Aurka increased in Bcl2l10 RNAi-treated oocytes. Counterbalancing changes in the levels of these 2 activators, Tpx2 and phospho-Aurka, resulted in decreased Aurka catalytic activity after Bcl2l10 RNAi treatment. Bcl2l10 RNAi decreased the expression of microtubule organizing center (MTOC)-related proteins, disturbed MTOC formation and disrupted meiotic spindle assembly. Our data demonstrate that Bcl2l10 is a binding partner of Tpx2 and a new regulator of the complex controlling the organization of microtubules and MTOC biogenesis in meiotic spindle assembly. The discovery of Bcl2l10 as a new effector of Aurka suggests that Bcl2l10 may have diverse functions in mitotic cells.

The role of Rad51 in safeguarding mitochondrial activity during the meiotic cell cycle in mammalian oocytes.

Rad51 is a conserved eukaryotic protein that mediates the homologous recombination repair of DNA double-strand breaks that occur during mitosis and meiosis. In addition, Rad51 promotes mitochondrial DNA synthesis when replication stress is increased. Rad51 also regulates cell cycle progression by preserving the G2/M transition in embryonic stem cells. In this study, we report a novel function of Rad51 in regulating mitochondrial activity during in vitro maturation of mouse oocytes. Suppression of Rad51 by injection of Rad51 dsRNA into germinal vesicle-stage oocytes resulted in arrest of meiosis in metaphase I. Rad51-depleted oocytes showed chromosome misalignment and failures in spindle aggregation, affecting the completion of cytokinesis. We found that Rad51 depletion was accompanied by decreased ATP production and mitochondrial membrane potential and increased DNA degradation. We further demonstrated that the mitochondrial defect activated autophagy in Rad51-depleted oocytes. Taken together, we concluded that Rad51 functions to safeguard mitochondrial integrity during the meiotic maturation of oocytes.

The miR-125 family is an important regulator of the expression and maintenance of maternal effect genes during preimplantational embryo development.

Previously, we reported that Sebox is a new maternal effect gene (MEG) that is required for early embryo development beyond the two-cell (2C) stage because this gene orchestrates the expression of important genes for zygotic genome activation (ZGA). However, regulators of Sebox expression remain unknown. Therefore, the objectives of the present study were to use bioinformatics tools to identify such regulatory microRNAs (miRNAs) and to determine the effects of the identified miRNAs on Sebox expression. Using computational algorithms, we identified a motif within the 3'UTR of Sebox mRNA that is specific to the seed region of the miR-125 family, which includes miR-125a-5p, miR-125b-5p and miR-351-5p. During our search for miRNAs, we found that the Lin28a 3'UTR also contains the same binding motif for the seed region of the miR-125 family. In addition, we confirmed that Lin28a also plays a role as a MEG and affects ZGA at the 2C stage, without affecting oocyte maturation or fertilization. Thus, we provide the first report indicating that the miR-125 family plays a crucial role in regulating MEGs related to the 2C block and in regulating ZGA through methods such as affecting Sebox and Lin28a in oocytes and embryos.

Expression of Beta-Defensin 131 Promotes an Innate Immune Response in Human Prostate Epithelial Cells.

Previously, using the Illumina HumanHT-12 microarray we found that ß-defensin 131 (DEFB131), an antimicrobial peptide, is upregulated in the human prostate epithelial cell line RWPE-1 upon stimulation with lipoteichoic acid (LTA; a gram-positive bacterial component), than that in the untreated RWPE-1 cells. In the current study, we aimed to investigate the role of DEFB131 in RWPE-1 cells during bacterial infection. We examined the intracellular signaling pathways and nuclear responses in RWPE-1 cells that contribute to DEFB131 gene induction upon stimulation with LTA. Chromatin immunoprecipitation was performed to determine whether NF-κB directly binds to the DEFB131 promoter after LTA stimulation in RWPE-1 cells. We found that DEFB131 expression was induced by LTA stimulation through TLR2 and p38MAPK/NF-κB activation, which was evident in the phosphorylation of both p38MAPK and IκBα. We also found that SB203580 and Bay11-7082, inhibitors of p38MAPK and NF-κB, respectively, suppressed LTA-induced DEFB131 expression. The chromatin immunoprecipitation assay showed that NF-κB directly binds to the DEFB131 promoter, suggesting that NF-κB is a direct regulator, and is necessary for LTA-induced DEFB131 expression in RWPE-1 cells. Interestingly, with DEFB131 overexpression in RWPE-1 cells, the accumulation of mRNA and protein secretion of cytokines (IL-1α, IL-1ß, IL-6, and IL-12α) and chemokines (CCL20, CCL22, and CXCL8) were significantly enhanced. In addition, DEFB131-transfected RWPE-1 cells markedly induced chemotactic activity in THP-1 monocytes. We concluded that DEFB131 induces cytokine and chemokine upregulation through the TLR2/NF-κB signaling pathway in RWPE-1 cells during bacterial infection and promotes an innate immune response.