Down-Regulation of Neogenin Decreases Proliferation and Differentiation of Spermatogonia during the Early Phase of Spermatogenesis.

Non-obstructive azoospermia is a major clinical issue associated with male infertility that remains to be addressed. Although neogenin is reportedly abundantly expressed in the testis, its role in mammalian spermatogenesis is unknown. We systematically investigated the role of neogenin during spermatogenesis by performing loss-of-function studies. Testis-specific neogenin conditional knock-out (cKO) mice were generated using CRISPR/Cas9 and neogenin-targeting guide RNAs. We analyzed the expression profiles of germ cell factors by RT-PCR and Western blotting. Neogenin localized mainly to spermatogonia in seminiferous tubules of mouse testes. RT-PCR and Western blot analyses further demonstrated that neogenin expression varied during spermatogenesis and was dramatically increased at postnatal day 12-25 during the pubertal stage. In neogenin-cKO mouse testes, the ratio of primary and secondary spermatocytes was significantly decreased compared with the control, while the number of apoptotic testicular cells was significantly increased. Taken together, these results suggest that neogenin plays a pivotal role in the maintenance and proliferation of spermatogonia during the early stage of spermatogenesis in mice.

Mitochondrial genome mutations and neuronal dysfunction of induced pluripotent stem cells derived from patients with Alzheimer's disease.

OBJECTIVES: Patient-derived induced pluripotent stem cells (iPSCs) are materials that can be used for autologous stem cell therapy. We screened mtDNA mutations in iPSCs and iPSC-derived neuronal cells from patients with Alzheimer's disease (AD). Also, we investigated whether the mutations could affect mitochondrial function and deposition of ß-amyloid (Aß) in differentiated neuronal cells. MATERIALS AND METHODS: mtDNA mutations were measured and compared among iPSCs and iPSC-derived neuronal cells. The selected iPSCs carrying mtDNA mutations were subcloned, and then their growth rate and neuronal differentiation pattern were analyzed. The differentiated cells were measured for mitochondrial respiration and membrane potential, as well as deposition of Aß. RESULTS: Most iPSCs from subjects with AD harbored ≥1 mtDNA mutations, and the number of mutations was significantly higher than that from umbilical cord blood. About 35% and 40% of mutations in iPSCs were shared with isogenic iPSCs and their differentiated neuronal precursor cells, respectively, with similar or different heteroplasmy. Furthermore, the mutations in clonal iPSCs were stable during extended culture and neuronal differentiation. Finally, mtDNA mutations could induce a growth advantage with higher viability and proliferation, lower mitochondrial respiration and membrane potential, as well as increased Aß deposition. CONCLUSION: This study demonstrates that mtDNA mutations in patients with AD could lead to mitochondrial dysfunction and accelerated Aß deposition. Therefore, early screening for mtDNA mutations in iPSC lines would be essential for developing autologous cell therapy or drug screening for patients with AD.

Artificial Oocyte: Development and Potential Application.

Millions of people around the world suffer from infertility, with the number of infertile couples and individuals increasing every year. Assisted reproductive technologies (ART) have been widely developed in recent years; however, some patients are unable to benefit from these technologies due to their lack of functional germ cells. Therefore, the development of alternative methods seems necessary. One of these methods is to create artificial oocytes. Oocytes can be generated in vitro from the ovary, fetal gonad, germline stem cells (GSCs), ovarian stem cells, or pluripotent stem cells (PSCs). This approach has raised new hopes in both basic research and medical applications. In this article, we looked at the principle of oocyte development, the landmark studies that enhanced our understanding of the cellular and molecular mechanisms that govern oogenesis in vivo, as well as the mechanisms underlying in vitro generation of functional oocytes from different sources of mouse and human stem cells. In addition, we introduced next-generation ART using somatic cells with artificial oocytes. Finally, we provided an overview of the reproductive application of in vitro oogenesis and its use in human fertility.

Viscous Cervical Environment-on-a-Chip for Selecting High-Quality Sperm from Human Semen.

When ejaculated sperm travels through the vagina to the uterus, mucus secreted by the cervical canal generally filters out sperm having low motility and poor morphology. To investigate this selection principle in vivo, we developed a microfluidic sperm-sorting chip with a viscous medium (polyvinylpyrrolidone: PVP) to imitate the biophysical environment mimic system of the human cervical canal. The material property of the PVP solution was tuned to the range of viscosities of cervical mucus using micro-viscometry. The selection of high-quality human sperm was experimentally evaluated in vitro and theoretically analyzed by the convection-diffusion mechanism. The convection flow is shown to be dominant at low viscosity of the medium used in the sperm-sorting chip when seeded with raw semen; hence, the raw semen containing sperm and debris convectively flow together with suppressed relative dispersions. Also, it was observed that the sperm selected via the chip not only had high motilities but also normal morphologies and high DNA integrity. Therefore, the biomimetic sperm-sorting chip with PVP medium is expected to improve male fertility by enabling the selection of high-quality sperm as well as uncovering pathways and regulatory mechanisms involved in sperm transport through the female reproductive tract for egg fertilization.

Upregulation of Low-Density Lipoprotein Receptor of the Steroidogenesis Pathway in the Cumulus Cells Is Associated with the Maturation of Oocytes and Achievement of Pregnancy.

The maturation of the oocyte is influenced by cumulus cells (CCs) and associated with pregnancy rate, whereas the influencing factors have not been completely elucidated in the CCs. In this study, we identified new regulators of CCs for high-quality oocytes and successful pregnancies during assisted reproductive techniques. CCs were collected from cumulus-oocyte complexes (COCs) in young (≤33 years old) and old (≥40 years old) women undergoing intracytoplasmic sperm injection (ICSI) procedures. We screened for factors differentially expressed between young vs. old CCs and pregnancy vs. non-pregnancy using whole mRNA-seq-next-generation sequencing (NGS). We characterized the transcriptome of the CCs to identify factors critical for achieving pregnancy in IVF cycles. Women in the young and old pregnancy groups exhibited the up- and downregulation of multiple genes compared with the non-pregnancy groups, revealing the differential regulation of several specific genes involved in ovarian steroidogenesis in CCs. It was shown that the low-density lipoprotein (LDL) receptor to the steroidogenesis pathway was upregulated in CCs with higher maturity rates of oocytes in the pregnancy group. In conclusion, a higher pregnancy rate is related to the signaling pathway of steroidogenesis by the LDL receptor in infertile women undergoing IVF procedures.

Genetic Polymorphisms in miR-604A>G, miR-938G>A, miR-1302-3C>T and the Risk of Idiopathic Recurrent Pregnancy Loss.

The purpose of this study was to investigate whether polymorphisms in five microRNAs (miRNAs), miR-604A>G, miR-608C>G, 631I/D, miR-938G>A, and miR-1302-3C>T, are associated with the risk of idiopathic recurrent pregnancy loss (RPL). Blood samples were collected from 388 patients with idiopathic RPL (at least two consecutive spontaneous abortions) and 227 control participants. We found the miR-604 AG and AG + GG genotypes of miR-604, the miR-938 GA and GA + AA genotypes of miR-938, and the miR-1302-3CT and CT + TT genotypes of miR-1302-3 are less frequent than the wild-type (WT) genotypes, miR-604AA, miR-938GG, and miR-1302-3CC, respectively, in RPL patients. Using allele-combination multifactor dimensionality reduction (MDR) analysis, we found that eight haplotypes conferred by the miR-604/miR-608/miR-631/miR-938/miR-1302-3 allele combination, A-C-I-G-T, A-C-I-A-C, G-C-I-G-C, G-C-I-G-T, G-G-I-G-C, G-G-I-G-T, G-G-I-A-C, G-G-D-G-C, three from the miR-604/miR-631/miR-938/miR-1302-3 allele combination, A-I-G-T, G-I-G-C, G-I-A-T, one from the miR-604/miR-631/miR-1302-3 allele combination, G-I-C, and two from the miR-604/miR-1302-3 allele combination, G-C and G-T, were less frequent in RPL patients, suggesting protective effects (all p < 0.05). We also identified the miR-604A>G and miR-938G>A polymorphisms within the seed sequence of the mature miRNAs and aligned the seed sequences with the 3'UTR of putative target genes, methylenetetrahydrofolate reductase (MTHFR) and gonadotropin-releasing hormone receptor (GnRHR), respectively. We further found that the binding affinities between miR-604/miR-938 and the 3'UTR of their respective target genes (MTHFR, GnRHR) were significantly different for the common (miR-604A, miR-938G) and variant alleles (miR-604G, miR-938A). These results reveal a significant association between the miR-604A>G and miR-938G>A polymorphisms and idiopathic RPL and suggest that miRNAs can affect RPL in Korean women.

MIT-001 Restores Human Placenta-Derived Mesenchymal Stem Cells by Enhancing Mitochondrial Quiescence and Cytoskeletal Organization.

Inflammation is a major cause of several chronic diseases and is reported to be recovered by the immuno-modulation of mesenchymal stem cells (MSCs). While most studies have focussed on the anti-inflammatory roles of MSCs in stem cell therapy, the impaired features of MSCs, such as the loss of homeostasis by systemic aging or pathologic conditions, remain incompletely understood. In this study, we investigated whether the altered phenotypes of human placenta-derived MSCs (hPD-MSCs) exposed to inflammatory cytokines, including TNF-α and IFN-γ, could be protected by MIT-001, a small anti-inflammatory and anti-necrotic molecule. MIT-001 promoted the spindle-like shape and cytoskeletal organization extending across the long cell axis, whereas hPD-MSCs exposed to TNF-α/IFN-γ exhibited increased morphological heterogeneity with an abnormal cell shape and cytoskeletal disorganization. Importantly, MIT-001 improved mitochondrial distribution across the cytoplasm. MIT-001 significantly reduced basal respiration, ATP production, and cellular ROS levels and augmented the spare respiratory capacity compared to TNF-α/IFN-γ-exposed hPD-MSCs, indicating enhanced mitochondrial quiescence and homeostasis. In conclusion, while TNF-α/IFN-γ-exposed MSCs lost homeostasis and mitochondrial quiescence by becoming over-activated in response to inflammatory cytokines, MIT-001 was able to rescue mitochondrial features and cellular phenotypes. Therefore, MIT-001 has therapeutic potential for clinical applications to treat mitochondrion-related inflammatory diseases.

CXCL12 enhances pregnancy outcome via improvement of endometrial receptivity in mice.

Successful pregnancy inevitably depends on the implantation of a competent embryo into a receptive endometrium. Although many substances have been suggested to improve the rate of embryo implantation targeting enhancement of endometrial receptivity, currently there rarely are effective evidence-based treatments to prevent or cure this condition. Here we strongly suggest minimally-invasive intra-uterine administration of embryo-secreted chemokine CXCL12 as an effective therapeutic intervention. Chemokine CXCL12 derived from pre- and peri-implanting embryos significantly enhances the rates of embryo attachment and promoted endothelial vessel formation and sprouting in vitro. Consistently, intra-uterine CXCL12 administration in C57BL/6 mice improved endometrial receptivity showing increased integrin ß3 and its ligand osteopontin, and induced endometrial angiogenesis displaying increased numbers of vessel formation near the lining of endometrial epithelial layer with higher CD31 and CD34 expression. Furthermore, intra-uterine CXCL12 application dramatically promoted the rates of embryo implantation with no morphologically retarded embryos. Thus, our present study provides a novel evidence that improved uterine endometrial receptivity and enhanced angiogenesis induced by embryo-derived chemokine CXCL12 may aid to develop a minimally-invasive therapeutic strategy for clinical treatment or supplement for the patients with repeated implantation failure with less risk.

Role of RGMc as a Neogenin Ligand in Follicular Development in the Ovary.

There is currently no cure for infertility in women with a poor ovarian response (POR). Neogenin is reported to be abundantly expressed in the ovary; however, its role in mammalian follicular development is unclear and its ligand and signaling pathway remain uncertain. We systematically investigated the role of neogenin and the ligand repulsive guidance molecule c (RGMc) during follicular development. We treated hyperstimulated mouse ovaries with RGMc and analyzed follicular development. Furthermore, we investigated clusters of up/downregulated genes in RGMc-treated ovaries using whole-transcriptome next-generation sequencing (NGS). In addition, we investigated whether expression of up/downregulated factors identified by NGS was also altered in cumulus cells (CCs) of patients with a POR. The number of oocytes was 40% higher in RGMc-treated ovaries than in control ovaries. NGS data indicated that prostaglandin D2 (PGD2) was involved in the RGMc signaling pathway during follicular development. RGMc treatment significantly elevated the PGD2 level in culture medium of CCs obtained from patients with a POR. Our results demonstrate that RGMc as neogenin ligand promotes follicular development in ovaries via the PGD2 signaling pathway. Therefore, it may be possible to use RGMc for ovarian stimulation in patients with a POR.

The mitochondrial-derived peptide MOTS-c promotes homeostasis in aged human placenta-derived mesenchymal stem cells in vitro.

Mesenchymal stem cells (MSCs) are multipotent cells with critical roles in homeostasis and regeneration. MSCs undergo aging in response to various stresses, and this causes many diseases including degenerative disorders. Thus, regulation of aging factors is crucial for healthy aging. Mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) was recently reported to regulate metabolic homeostasis. Here, we investigated the restorative effects of MOTS-c on aged human placenta-derived MSCs (hPD-MSCs). MOTS-c promoted the morphology of old hPD-MSCs. MOTS-c significantly activated AMP-activated protein kinase, which is the main target pathway of MOTS-c, and inhibited its antagonistic effector mTORC1. MOTS-c considerably enhanced mitochondrial homeostasis by decreasing oxygen consumption and reactive oxygen species production. The mitochondrial state of MOTS-c-treated old hPD-MSCs was more similar to that of young hPD-MSCs than the mitochondrial state of non-treated old hPD-MSCs. MOTS-c also decreased lipid synthesis. In conclusion, we demonstrated that MOTS-c promotes homeostasis in aged hPD-MSCs.

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.

Eupatilin treatment inhibits transforming growth factor beta-induced endometrial fibrosis in vitro.

OBJECTIVE: Endometrial fibrosis, the primary pathological feature of intrauterine adhesion, may lead to disruption of endometrial tissue structure, menstrual abnormalities, infertility, and recurrent pregnancy loss. At present, no ideal therapeutic strategy exists for this fibrotic disease. Eupatilin, a major pharmacologically active flavone from Artemisia, has been previously reported to act as a potent inducer of dedifferentiation of fibrotic tissue in the liver and lung. However, the effects of eupatilin on endometrial fibrosis have not yet been investigated. In this study, we present the first report on the impact of eupatilin treatment on transforming growth factor beta (TGF-ß)-induced endometrial fibrosis. METHODS: The efficacy of eupatilin on TGF-ß-induced endometrial fibrosis was assessed by examining changes in morphology and the expression levels of fibrosis markers using immunofluorescence staining and quantitative real-time reverse-transcription polymerase chain reaction. RESULTS: Eupatilin treatment significantly reduced the fibrotic activity of TGF-ß-induced endometrial fibrosis in Ishikawa cells, which displayed more circular shapes and formed more colonies. Additionally, the effects of eupatilin on fibrotic markers including alpha-smooth muscle actin, hypoxia-inducible factor 1 alpha, collagen type I alpha 1 chain, and matrix metalloproteinase-2, were evaluated in TGF-ß-induced endometrial fibrosis. The expression of these markers was highly upregulated by TGF-ß pretreatment and recovered to the levels of control cells in response to eupatilin treatment. CONCLUSION: Our findings suggest that suppression of TGF-ß-induced signaling by eupatilin might be an effective therapeutic strategy for the treatment of endometrial fibrosis.

Endometrial profilin 1: a key player in embryo-endometrial crosstalk.

OBJECTIVE: Despite extensive research on implantation failure, little is known about the molecular mechanisms underlying the crosstalk between the embryo and the maternal endometrium, which is critical for successful pregnancy. Profilin 1 (PFN1), which is expressed both in the embryo and in the endometrial epithelium, acts as a potent regulator of actin polymerization and the cytoskeletal network. In this study, we identified the specific role of endometrial PFN1 during embryo implantation. METHODS: Morphological alterations depending on the status of PFN1 expression were assessed in PFN1-depleted or control cells grown on Matrigel-coated cover glass. Day-5 mouse embryos were cocultured with Ishikawa cells. Comparisons of the rates of F-actin formation and embryo attachment were performed by measuring the stability of the attached embryo onto PFN1-depleted or control cells. RESULTS: Depletion of PFN1 in endometrial epithelial cells induced a significant reduction in cell-cell adhesion displaying less formation of colonies and a more circular cell shape. Mouse embryos co-cultured with PFN1-depleted cells failed to form actin cytoskeletal networks, whereas more F-actin formation in the direction of surrounding PFN1-intact endometrial epithelial cells was detected. Furthermore, significantly lower embryo attachment stability was observed in PFN1-depleted cells than in control cells. This may have been due to reduced endometrial receptivity caused by impaired actin cytoskeletal networks associated with PFN1 deficiency. CONCLUSION: These observations definitively demonstrate an important role of PFN1 in mediating cell-cell adhesion during the initial stage of embryo implantation and suggest a potential therapeutic target or novel biomarker for patients suffering from implantation failure.

Motility enhancement of human spermatozoa using electrical stimulation in the nano-Ampere range with enzymatic biofuel cells.

Sperm motility is a crucial factor for normal fertilisation that is partly supported by mitochondrial activity. Enzymatic biofuel cells (EBFCs) generate electric currents by an electron grade from anodic to cathodic electrodes in a culture media. We demonstrate that electrical stimulation by EBFC at the nano-Ampere range enhances sperm motility that can potentially allow the development of a new therapeutic tool for male infertility, including poor motility. EBFC was set up with three different electrical currents (112 nA/cm2 and 250 nA/cm2) at two different times (1 h, 2 h). Each sample was evaluated for its motility by computer-assisted sperm analyses and sperm viability testing. In the expanded study, we used the optimal electrical current of the EBFC system to treat asthenozoospermia and sperm with 0% motility. Results showed that optimal electrical stimulation schemes with EBFCs enhanced sperm motility by 30-40% compared with controls. Activated spermatozoa led to tyrosine phosphorylation in the tail area of the sperm following the electrical stimulation in the nano-Ampere range. However, the electrically stimulated group did not exhibit increased acrosomal reaction rates compared with the control group. In cases related to asthenozoospermia, 40% of motility was recovered following the electrical stimulation at the nano-Ampere range. However, motility is not recovered in sperm with 0% motility. In conclusion, we found that sperm motility was enhanced by exposure to electrical currents in the nano-Ampere range induced by optimal EBFCs. Electrical stimulation enhanced the motility of the sperm though tyrosine phosphorylation in spermatozoa. Therefore, our results show that electrical currents in the nano-Ampere range can be potentially applied to male infertility therapy as enhancers of sperm motility in assisted reproductive technology.

Association Between Functional Activity of Mitochondria and Actin Cytoskeleton Instability in Oocytes from Advanced Age Mice.

Mitochondrial dysfunction is strongly associated with the oocyte quality and aging, wherein the aged oocytes are related to the actin cytoskeleton integrity; however, whether this integrity is associated with mitochondrial dysfunction in oocytes from aged mice remains unclear. In the present study, we investigated the relationship between mitochondrial dysfunction and actin cytoskeleton instability in oocytes from the aged mice. We performed comparable analysis of mitochondrial motility between young, 1.5 µM cytochalasin B (CB)-treated young oocytes, and aged oocytes by confocal live imaging. Moreover, we analyzed the relationships between mitochondrial motility and maturation ratios, including ATP production ratio of the young, CB-treated young, and aged oocytes. Actin cytoskeleton instability in the aged oocytes and CB-treated young oocytes led to a significant decrease in the mitochondrial motility and low ATP productive ratios compared to those in the young group. Our data suggest that the actin cytoskeleton instability is presumably the primary cause for the loss of mitochondrial function in the aged murine oocytes.

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.

The association of miR-25T>C, miR-32C>A, miR-125C>T, and miR-222G>T polymorphisms with a risk of primary ovarian insufficiency in Korean women.

OBJECTIVE: The purpose of this study was to investigate the association of microRNA polymorphisms (miR-25T>C, miR-32C>A, miR-125C>T, and miR-222G>T) with primary ovarian insufficiency (POI) in Korean women. METHODS: We conducted a case-control study of Korean women: 142 participants with POI and 266 controls with at least 1 live birth and no history of pregnancy loss. RESULTS: The haplotype-based multifactor dimensionality reduction analysis revealed that the T-C-T-G (miR-25/-32/-125/-222), T-A-C-G (miR-25/-32/-125/-222), C-T-G (miR-32/-125/-222), A-C-G (miR-32/-125/-222), T-G (miR-122/-222), C-T (miR-32/-125), and C-C (miR-25/-32) inferred haplotypes were significantly less frequent in POI (P < 0.05), which suggested potential protective effects. Participants with POI had significantly increased luteinizing hormone levels (P < 0.05), but hormonal levels, including luteinizing hormone, were not significantly different between POI women and control women with miR-32/-125/-222. CONCLUSIONS: After considering multiple comparisons, we concluded that miR-25T>C, miR-32C>A, miR-125C>T, and miR-222G>T had no relation with POI.

Activating P2X7 Receptors Increases Proliferation of Human Pancreatic Cancer Cells via ERK1/2 and JNK.

OBJECTIVES: The aim of this study was to investigate the effects of the activated P2X7 receptors on the proliferation and growth of human pancreatic cancer cells. METHODS: Proliferation was measured by incorporating bromodeoxyuridine into pancreatic cancer cells, MIA PaCa-2 and HPAC. Expression of P2 receptors and signal molecules was examined using quantitative reverse transcription/polymerase chain reaction and/or Western blot. Proliferative effects of the P2X7 receptors in vivo were examined using a xenotransplant model of pancreatic cancer cell lines. RESULTS: Incubating pancreatic cancer cells with adenosine triphosphate (ATP) and 2'(3')-O-(4-Benzoylbenzoyl)ATP resulted in a dose-dependent increase of cell proliferation. The P2 receptor antagonist, KN-62, and small interfering RNA against P2X7 receptors, significantly decreased the proliferative effects of ATP. The ATP-induced proliferation was mediated by protein kinase C, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK); specifically, ATP increased the phosphorylation of ERK1/2 and JNK. The expression of inducible nitric oxide synthase was decreased by P2X7 receptor activation. In a xenotransplant model, applying ATP significantly increased the growth of induced tumors. CONCLUSIONS: The P2X7 receptor activation by extracellular nucleotides increased proliferation and growth of human pancreatic cancer cells via ERK1/2 and JNK. This supports the pathophysiological role of P2X7 receptors in pancreatic disease and recovery.

Knockdown of PRKAR2B Results in the Failure of Oocyte Maturation.

BACKGROUND/AIMS: Cyclic adenosine monophosphate (cAMP)-dependent type 2 regulatory subunit beta (Prkar2b) is a regulatory isoform of cAMP-dependent protein kinase (PKA), which is the primary target for cAMP actions. In oocytes, PKA and the pentose phosphate pathway (PPP) have important roles during the germinal vesicle (GV) stage arrest of development. Although the roles of the PKA signal pathway have been studied in the development of oocyte, there has been no report on the function of PRKAR2B, a key regulator of PKA. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), immunohistochemistry, and immunofluorescence, we determined the relative expression of Prkar2b in various tissues, including ovarian follicles, during oocyte maturation. Prkar2b-interfering RNA (RNAi) microinjection was conducted to confirm the effect of Prkar2b knockdown, and immunofluorescence, qRT-PCR, and time-lapse video microscopy were used to analyze Prkar2b-deficient oocytes. RESULTS: Prkar2b is strongly expressed in the ovarian tissues, particularly in the growing follicle. During oocyte maturation, the highest expression of Prkar2b was during metaphase I (MI), with a significant decrease at metaphase II (MII). RNAi-mediated Prkar2b suppression resulted in MI-stage arrest during oocyte development, and these oocytes exhibited abnormal spindle formation and chromosome aggregation. Expression of other members of the PKA family (except for Prkaca) were decreased, and the majority of the PPP factors were also reduced in Prkar2b-deficient oocytes. CONCLUSION: These results suggest that Prkar2b is closely involved in the maturation of oocytes by controlling spindle formation and PPP-mediated metabolism.

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.