Growth Invitro Research Articles

Zoledronic acid sensitizes breast cancer cells to fulvestrant via ERK/HIF-1 pathway inhibition invivo.

Previous studies have reported that hypoxia-inducible factor (HIF)-1α confers endocrine resistance and that zoledronic acid (ZOL) decreases HIF‑1α expression in estrogen receptor‑positive breast cancer. The present study investigated the effect of the combination treatment with ZOL and fulvestrant and its possible mechanism for HIF‑1α inhibition invitro and invivo. First, cell proliferation, clonogenic ability and HIF‑1α expression by western blotting were determined in MCF‑7 breast cancer cells stably expressing HIF‑1α invitro. Next, a mouse xenograft model was established with the HIF‑1α‑overexpressing MCF‑7 breast cancer cells, and treated with PBS, fulvestrant, ZOL or fulvestrant plus ZOL. Tumor volumes were compared and animal [18F]‑fluoromisonidazole (FMISO) positron emission tomography‑computer tomography (PET‑CT) was used to detect the hypoxic status of the xenograft tumors. Protein expression levels of HIF‑1α in the xenograft tumors were detected by immunohistochemistry and western blotting. The results demonstrated that the HIF-1α-overexpressing xenograft tumors grew faster and larger compared with control tumors. The animal [18F]‑FMISO PET‑CT also confirmed these results. [18F]‑FMISO uptake was significantly higher in HIF‑1α‑overexpressing xenograft tumors compared with control tumors. In addition, the combination treatment with ZOL and fulvestrant acted synergistically in the mouse xenograft model invivo to significantly reduce tumor burden. Similarly, combination of ZOL and fulvestrant significantly reduced tumor cell growth invitro. ZOL alone did not inhibit the tumor growth of MCF‑7 cells stably expressing HIF‑1α. Furthermore, ZOL significantly inhibited extracellular signal‑regulated kinase (ERK) 1/2 phosphorylation, while phosphoinositide 3‑kinase/AKT signaling was not affected. In conclusion, the present study demonstrated that ZOL significantly increased the sensitivity of breast cancer cells to fulvestrant through inhibition of the ERK/HIF-1α pathway.

EFFECT OF PROGESTERONE ON SURVIVAL, IN VITRO GROWTH AND IN VITRO MATURATION OF FOLLICLE DERIVED FROM MOUSE POLYCYSTIC OVARY.

Polycystic ovarian syndrome is the most common endocrine disorder in women of reproductive age. The purpose of the present study is to investigate the role of progesterone on survival, in vitro growth and in vitro maturation of follicles and the maturation of oocytes derived from mouse polycystic ovary. To induce polycystic ovary (PCO) female NMRI mice 21 days old were injected daily with testosterone enanthate 1 mg/100g body weight dissolved in sesame oil for 4 weeks (PCO group), while non-PCO group were injected only with vehicle. Follicles were derived from both groups and cultured in MEM-α medium either with progesterone or without progesterone. Sizes of follicles were measured in days 1, 3, 6, 9 and 12. After 12 days follicles were transferred to mature medium, follicles from two groups were cultured for 24 and 48 h and the in vitro maturation of oocytes was assessed. In PCO groups with progesterone, survival and in vitro growth of follicles, significantly increased as compared with PCO groups without progesterone (p<0.05). The in vitro maturation rate in PCO group with progesterone was significantly higher than in those not treated by progesterone (p<0.05). This study demonstrated that progesterone can improve survival, in vitro growth and in vitro maturation of follicles derived from mouse polycystic ovary.

The F1 -ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit.

The F-ATPases (also called the F1 Fo -ATPases or ATP synthases) are multi-subunit membrane-bound molecular machines that produce ATP in bacteria and in eukaryotic mitochondria and chloroplasts. The structures and enzymic mechanisms of their F1 -catalytic domains are highly conserved in all species investigated hitherto. However, there is evidence that the F-ATPases from the group of protozoa known as Euglenozoa have novel features. Therefore, we have isolated pure and active F1 -ATPase from the euglenozoan parasite, Trypanosoma brucei, and characterized it. All of the usual eukaryotic subunits (α, β, γ, δ, and ε) were present in the enzyme, and, in addition, two unique features were detected. First, each of the three α-subunits in the F1 -domain has been cleaved by proteolysis invivo at two sites eight residues apart, producing two assembled fragments. Second, the T.brucei F1 -ATPase has an additional subunit, called p18, present in three copies per complex. Suppression of expression of p18 affected invitro growth of both the insect and infectious mammalian forms of T.brucei. It also reduced the levels of monomeric and multimeric F-ATPase complexes and diminished the invivo hydrolytic activity of the enzyme significantly. These observations imply that p18 plays a role in the assembly of the F1 domain. These unique features of the F1 -ATPase extend the list of special characteristics of the F-ATPase from T.brucei, and also, demonstrate that the architecture of the F1 -ATPase complex is not strictly conserved in eukaryotes.

129 Embryo Production from Fully Grown and Growing Stage Oocytes in Japanese Black Calves

Before fattening of Japanese Black female calves, the ovaries are sometimes removed and discarded. Production of embryos from the oocytes residing in such ovaries is beneficial for the rescue of genetic resources. The aim of this study was to establish an embryo production system using oocytes collected from the ovaries of calves just before fattening and to investigate the correlation between the developmental competence of oocytes and the onset of puberty. Ovaries were collected from Japanese Black calves (9.5 ± 0.1 months old, n = 30, 3 replicates) in a fattening farm and separated according to the presence or absence of corpus luteum as the indicator of puberty (CL+ and CL– groups, respectively). Immature fully grown oocytes (IM oocytes), ~120 μm in diameter, were aspirated from follicles of 2 to 6 mm in diameter (CL+; n = 132, CL–; n = 41) and cultured for 22 to 23 h for maturation (IVM). After in vitro fertilization (IVF) for 6 h (designated Day 0), the oocytes were cultured for 9 days (in vitro culture, IVC) (Matoba et al. 2014 J. Dairy Sci. 97, 743-753). Growing oocytes, ~100 μm in diameter, were also collected by dissecting the follicles smaller than 1 mm in diameter. The growing oocytes were cultured for 14 days on membrane inserts for in vitro growth (IVG) (Hirao et al. 2013 Biol. Reprod. 89, 1-11). Then, IVG oocytes (CL+; n = 29, CL–; n = 32) were subjected to IVM, IVF, and IVC. Presumptive zygotes were cultured individually in microwells in culture dishes. Mitochondrial DNA (mtDNA) copy numbers (COX1 gene) of oocytes were examined (Takeda et al. 2010 Mitochondrion 10, 137-142). A comparison was made between the oocytes derived from calf ovaries and those of oocytes collected from cow ovaries by transvaginal ovum pick-up or by aspiration of the ovaries obtained at a local slaughterhouse. In IM oocytes, the rate of embryos developed to the blastocyst stage on Day 7 to 9 was higher in the CL+ group than in the CL– group (38.9 ± 2.6 v. 10.0 ± 7.1%, respectively; P &lt; 0.05, t-test). However, IVG oocytes were compared, there was no significant difference in the blastocyst formation rate between the CL+ or CL– groups (34.7 ± 5.2 v. 19.8 ± 10.1%, respectively). The mtDNA copy numbers of matured oocytes were similar between IM and IVG oocytes irrespective of the maturity of the donor animals. In conclusion, we demonstrated the possibility of embryo production by IVM/IVF/IVC using fully grown and growing oocytes that are present in the ovaries of calves before fattening. Puberty positively affected the developmental competence of IM oocytes but the effect was not significant in IVG oocytes. Utilisation of both fully grown oocytes and growing oocytes may double the chance of rescuing genetic resources of high-breeding-value calves. This study was partly supported by grants from the Ito Foundation. We thank staff at Mie-Katoubokujou for allowing access to calves’ ovaries.

Oncogenic MYC Activates a Feedforward Regulatory Loop Promoting Essential Amino Acid Metabolism and Tumorigenesis

Most tumor cells exhibit obligatory demands foressential amino acids (EAAs), but the regulatorymechanisms whereby tumor cells take up EAAsand EAAs promote malignant transformation remainto be determined. Here, we show that oncogenic MYC, solute carrier family (SLC) 7 member 5 (SLC7A5), and SLC43A1 constitute a feedforward activation loop to promote EAA transport and tumorigenesis. MYC selectively activates Slc7a5 and Slc43a1 transcription through direct binding to specific E box elements within both genes, enabling effective EAA import. Elevated EAAs, in turn, stimulate Myc mRNA translation, in part through attenuation of the GCN2-eIF2α-ATF4 amino acid stress response pathway, leading to MYC-dependent transcriptional amplification. SLC7A5/SLC43A1 depletion inhibits MYC expression, metabolic reprogramming, and tumor cell growth invitro and invivo. These findings thus reveal a MYC-SLC7A5/SLC43A1 signaling circuit that underlies EAA metabolism, MYC deregulation, and tumorigenesis.

The Role of Extracellular Histones in Influenza Virus Pathogenesis

Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth invitro, invivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia.

MiR-320a modulates cell growth and chemosensitivity via regulating ADAM10 in gastric cancer.

MicroRNAs (miRNAs) may function as tumor suppressor or onco‑miRNAs and have critical roles in the pathogenesis of gastric cancer (GC). The exact function and mechanism of miRNA (miR)‑320a in GC remains to be elucidated. The present study performed gain‑ and loss‑of‑function analyses by transfecting cells with mimics or inhibitors and subsequently performing colony formation, proliferation and cisplatin‑sensitivity assays. Additionally, invivo xenograft models were also performed. Bioinformatics algorithms, luciferase reporter activity assay and western blotting were used to predict the potential target of miR‑320a. Additionally, the effect of knockdown or overexpression of ADAM metallopeptidase domain 10 (ADAM10) on cell growth and chemosensitivity was examined. The expression of miR‑320a and ADAM10 was also determined in primary tumors. The present study revealed that the expression of miR‑320a was reduced in GC cells and ectopic miR‑320a expression significantly inhibited cell growth invitro and invivo and enhanced the sensitivity of GC cells to cisplatin. ADAM10 was a direct target of miR‑320a in GC. Knockdown of ADAM10 attenuated the proliferative ability of GC cells, and increased the sensitivity of GC cells to cisplatin. The upregulated ADAM10 accelerated cell growth rate and reduced the cisplatin‑sensitivity of cells. Clinically, a significantly negative correlation was identified between the expression of miR‑320a and mRNA levels of ADAM10 in tumors. The findings of the present study suggested that miR‑320a may function as a tumor suppressor in GC progression and potential therapeutic strategies for GC may be based on the miR‑320a/ADAM10 axis.

Epigenetic Silencing of miRNA-34a in Human Cholangiocarcinoma via EZH2 and DNA Methylation: Impact on Regulation of Notch Pathway

Aberrant expression and regulation of miRNAs have been implicated in multiple stages of tumorigenic processes. The current study was designed to explore the biological function and epigenetic regulation of miR-34a in human cholangiocarcinoma (CCA). Our data show that the expression of miR-34a is decreased significantly in CCA cells compared with non-neoplastic biliary epithelial cells. Forced overexpression of miR-34a in CCA cells inhibited their proliferation and clonogenic capacity invitro, and suppressed tumor xenograft growth in severe combined immunodeficiency mice. We identified three key components of the Notch pathway, Notch1, Notch2, and Jagged 1, as direct targets of miR-34a. Our further studies show that down-regulation of miR-34a is caused by Enhancer of zeste homolog 2 (EZH2)-mediated H3 lysine 27 trimethylation as well as DNA methylation. Accordingly, treatment with the EZH2 inhibitor, selective S-adenosyl-methionine-competitive small-molecule (GSK126), or the DNA methylation inhibitor, 5-Aza-2'-deoxycytidine, partially restored miR-34a levels in human CCA cells. Immunohistochemical staining and Western blot analyses showed increased EZH2 expression in human CCA tissues and cell lines. We observed that GSK126 significantly reduced CCA cell growth invitro and intrahepatic metastasis invivo. Our findings provide novel evidence that miR-34a expression is silenced epigenetically by EZH2 and DNA methylation, which promotes CCA cell growth through activation of the Notch pathway. Consequently, these signaling cascades may represent potential therapeutic targets for effective treatment of human CCA.

In Vivo Expression of miR-32 Induces Proliferation in Prostate Epithelium

miRNAs are important regulators of gene expression and are often deregulated in cancer. We have previously shown that miR-32 is an androgen receptor-regulated miRNA overexpressed in castration-resistant prostate cancer and that miR-32 can improve prostate cancer cell growth invitro. To assess the effects of miR-32 invivo, we developed transgenic mice overexpressing miR-32 in the prostate. The study indicated that transgenic miR-32 expression increases replicative activity in the prostate epithelium. We further observed an aging-associated increase in the incidence of goblet cell metaplasia in the prostate epithelium. Furthermore, aged miR-32 transgenic mice exhibited metaplasia-associated prostatic intraepithelial neoplasia at a low frequency. When crossbred with mice lacking the other allele of tumor-suppressor Pten (miR-32xPten+/- mice), miR-32 expression increased both the incidence and the replicative activity of prostatic intraepithelial neoplasia lesions in the dorsal prostate. The miR-32xPten+/- mice also demonstrated increased goblet cell metaplasia compared with Pten+/- mice. By performing a microarray analysis of mouse prostate tissue to screen downstream targets and effectors of miR-32, we identified RAC2 as a potential, and clinically relevant, target of miR-32. We also demonstrate down-regulation of several interesting, potentially prostate cancer-relevant genes (Spink1, Spink5, and Casp1) by miR-32 in the prostate tissue. The results demonstrate that miR-32 increases proliferation and promotes metaplastic transformation in mouse prostate epithelium, which may promote neoplastic alterations in the prostate.

Reconstitution of mouse oogenesis in a dish from pluripotent stem cells.

This protocol is an extension to: Nat. Protoc. 8, 1513-1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take ∼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals.

Enhancer Reprogramming Promotes Pancreatic Cancer Metastasis

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies, owing in part to its propensity for metastasis. Here, we used an organoid culture system to investigate how transcription and the enhancer landscape become altered during discrete stages of disease progression in a PDA mouse model. This approach revealed that the metastatic transition is accompanied by massive and recurrent alterations in enhancer activity. We implicate the pioneer factor FOXA1 as a driver of enhancer activation in this system, a mechanism that renders PDA cells more invasive and less anchorage-dependent for growth invitro, as well as more metastatic invivo. In this context, FOXA1-dependent enhancer reprogramming activates a transcriptional program of embryonic foregut endoderm. Collectively, our study implicates enhancer reprogramming, FOXA1 upregulation, and a retrograde developmental transition in PDA metastasis.

LncRNA ODRUL Contributes to Osteosarcoma Progression through the miR-3182/MMP2 Axis

Recent findings have shown that lncRNA dysregulation is involved in many cancers, including osteosarcoma (OS). In a previous study, we reported a novel lncRNA, ODRUL, that could promote doxorubicin resistance in OS. We now report the function and underlying mechanism of ODRUL in regulating OS progression. We show that ODRUL is upregulated in OS tissues and cell lines and correlates with poor prognosis. ODRUL knockdown significantly inhibits OS cell proliferation, migration, invasion, and tumor growth invitro and invivo by decreasing matrix metalloproteinase (MMP) expression. A microarray screen combined with online database analysis showed that miR-3182 is upregulated and MMP2 is downregulated in sh-ODRUL-expressing MG63 cells and that miR-3182 harbors potential binding sites for ODRUL and the 3' UTR of MMP2 mRNA. In addition, miR-3182 expression and function are inversely correlated with ODRUL expression invitro and invivo. A luciferase reporter assay demonstrated that ODRUL could directly interact with miR-3182 and upregulate MMP2 expression via its competing endogenous RNA activity on miR-3182 at the posttranscriptional level. Taken together, our study has elucidated the role of oncogenic ODRUL in OS progression and may provide a new target in OS therapy.

Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth invitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury invivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein-4 increases oocyte diameter, but impairs subsequent developmental competence.

Bone morphogenetic protein-4 (BMP-4) inhibits luteinization of granulosa cells during invitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than invivo-grown oocytes. We herein investigated whether an extended IVG culture with BMP-4 improves oocyte growth and development to blastocysts after invitro fertilization. Oocyte-granulosa cell complexes (OGCs) were cultured for 14 or 16days with BMP-4 (10ng/mL), while a 12 day culture with BMP-4 served as the invitro control. OGC viability was maintained for the 16 day culture with BMP-4 (83.2%), but was significantly lower without BMP-4 (58.9%) than the control (83.0%). Prolong-cultured oocytes at 16days had statistically greater diameter (114.6μm) than the control (111.7μm). IVG oocytes with BMP-4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP-4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP-4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes.

Relationship between in vitro growth of bovine oocytes and steroidogenesis of granulosa cells cultured in medium supplemented with bone morphogenetic protein-4 and follicle stimulating hormone

Bone morphogenetic protein-4 (BMP-4) and FSH play important regulatory roles in follicular growth and steroidogenesis in vivo. The purpose of this study was to investigate the effects of BMP-4 and FSH on in vitro growth (IVG) and steroidogenesis of bovine oocyte-cumulus-granulosa complexes (OCGCs). We cultured OCGCs collected from early antral follicles (0.5–1 mm) in medium without BMP-4 and FSH for 4 days and investigated the appearance of OCGCs and their steroidogenesis. During the first 4 days of IVG, morphologically normal OCGCs produced more estradiol-17β (E2), but less progesterone (P4). Morphologically normal OCGCs were subjected to an additional culture in medium supplemented with BMP-4 (0, 10, and 50 ng/mL) and FSH (0 and 0.5 ng/mL) until day 12. We examined the viability and steroidogenesis of OCGCs after 8 and 12 days of culture. Oocyte growth, characteristics of granulosa cells, and the maturational competence of oocytes were also investigated. On day 8, the viability of OCGCs cultured without FSH was higher in the 10 ng/mL BMP-4 group than in the 50 ng/mL BMP-4 group (P < 0.05). No significant difference was observed in the viability of groups cultured with FSH, regardless of the addition of BMP-4, and FSH improved the viability of 50 ng/mL BMP-4 group similar to 10 ng/mL BMP-4 group. The total number of granulosa cells was larger in 10 ng/mL BMP-4 group cultured with FSH than in 50 ng/mL BMP-4 group cultured with FSH on day 8 (P < 0.05). E2 production decreased from days 8–12, and P4 production increased throughout IVG culture, regardless of the addition of BMP-4 and FSH (P < 0.05). No significant differences in E2 production were observed between groups from days 4–8, regardless of whether BMP-4 was added without FSH; however, E2 production in the group cultured with 50 ng/mL BMP-4 was suppressed by FSH. BMP-4 suppressed E2 production from days 8–12, regardless of whether FSH was added. The group cultured with 10 ng/mL BMP-4 without FSH showed the lowest P4 production among all groups for all culture periods. OCGCs that produced mature oocytes tended to secrete more E2 and less P4 than OCGCs that produced immature oocytes. In conclusion, until day 8 of the IVG culture, P4 production by OCGCs was suppressed by the addition of 10 ng/mL BMP-4 in the absence of FSH, without inhibiting E2 production. These conditions appear to mimic growing follicles until day 8 and mimic degenerating follicles from days 8–12 of culture.

Invitro anti-Candida activity of selective serotonin reuptake inhibitors against fluconazole-resistant strains and their activity against biofilm-forming isolates.

Recent research has shown broad antifungal activity of the classic antidepressants selective serotonin reuptake inhibitors (SSRIs). This fact, combined with the increased cross-resistance frequency of the genre Candida regarding the main treatment today, fluconazole, requires the development of novel therapeutic strategies. In that context, this study aimed to assess the antifungal potential of fluoxetine, sertraline, and paroxetine against fluconazole-resistant Candida spp. planktonic cells, as well as to assess the mechanism of action and the viability of biofilms treated with fluoxetine. After 24h, the fluconazole-resistant Candida spp. strains showed minimum inhibitory concentration (MIC) in the ranges of 20-160μg/mL for fluoxetine, 10-20μg/mL for sertraline, and 10-100.8μg/mL for paroxetine by the broth microdilution method (M27-A3). According to our data by flow cytometry, each of the SSRIs cause fungal death after damaging the plasma and mitochondrial membrane, which activates apoptotic signaling pathways and leads to dose-dependant cell viability loss. Regarding biofilm-forming isolates, the fluoxetine reduce mature biofilm of all the species tested. Therefore, it is concluded that SSRIs are capable of inhibit the growth invitro of Candida spp., both in planktonic form, as biofilm, inducing cellular death by apoptosis.

Sheep Isolated Secondary Follicles Are Able to Produce Metaphase II Oocytes After Vitrification and Long-Term In Vitro Growth.

The vitrification of preantral follicles followed by in vitro growth (IVG) could be valuable to produce fertilizable oocytes. However, the meiotic resumption rates of oocytes cultured from vitrified secondary follicles (SF) have been reported as suboptimal. This study aimed to verify two base media (alpha modification of minimum essential medium, α-MEM, and tissue culture medium 199, TCM199) on vitrified SF regarding different requirements during IVG. Sheep ovarian fragments were divided in six groups: (1) Fresh groups (Control α-MEM and TCM199): SF without vitrification; (2) Follicle-Vitrified (Follicle-Vit α-MEM and TCM199): SF vitrified after isolation; and (3) Tissue-Vitrified (Tissue-Vit α-MEM and TCM199): SF vitrified enclosed in ovarian fragments and, subsequently, isolated. The isolated SF were submitted to IVG for 18 days. Thereafter, the recovered cumulus-oocyte complexes (COCs) underwent in vitro maturation (IVM) and evaluation of chromatin configuration. Follicular granulosa cells were analyzed for their gene expression of Bax, Bcl2, and Connexins (CX) 37 and 43. COCs from in vivo antral follicles were used as in vivo control. Data were analyzed by analysis of variance, Tukey, and chi-square tests. Differences were considered significant if p-value is <0.05. Follicle-Vit groups had higher (p < 0.05) percentage of antrum formation compared with Tissue-Vit groups. Vitrification did not affect (p > 0.05) oocyte diameter postmaturation. Oocytes from Follicle-Vit in α-MEM reached metaphase II stage after IVM. Gene expression for CX37, CX43, and Bax was lower in Tissue-Vit groups. For Bcl2, the gene expression was the opposite. In conclusion, during IVG for 18 days, maximal oocyte meiotic resumption was not negatively impacted by vitrification and was greatest for isolated SF using α-MEM as a medium.

Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration

Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth invitro and regeneration invivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.

Ellagic acid inhibits human glioblastoma growth invitro and invivo.

Ellagic acid (EA) is present in various fruits and plants and has recently been found to possess anticarcinogenic effects. The objective of this study was to investigate the anti‑glioblastoma effect of EA and its mechanisms invitro and invivo. We first studied the anticancer activity of EA in U87 and U118 human glioblastoma cell lines. The cell viability and cell proliferation were examined by Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine staining respectively. The cell cycle was detected with propidium iodide staining method by flow cytometry and the DNA damage of the cells caused by EA exposure was evaluated by detection of γ-H2AX foci. Then we examined the effect of EA on tumor growth in glioblastoma xenografted mice, and expression of Akt and Notch signaling and their target gene products were detected by immunohistochemistry and western blot analysis. As a result, we found that the cell viability and proliferation of glioblastoma cells treated with EA were significantly suppressed compared with the control; EA significantly increased the proportion of cells in the Sphase accompanied by a decrease in the population in the G1 and G2/M phase in both cell lines. Meanwhile, the level of DNA damage in the EA-treated group was significantly higher than that of the control. Treatment of glioblastoma in xenografted mice by EA led to a significant suppression in tumor growth. EA upregulated the expression of E-cadherin and inhibited the expression of Snail, matrix metalloproteinase (MMP)-2 and MMP-9. EA also inhibited the expression of Bcl-2, cyclinD1, cyclin-dependent kinase (CDK)2 and CDK6 in U87 xenograft tissues. In addition, significant suppression of Akt and Notch was found in the xenografts of the tumor-bearing mice treated with EA. These data indicate that EA can suppress glioblastoma proliferation and invasion by inhibiting the Akt and Notch signaling pathways, which suggests that EA may be beneficial for the treatment of glioblastoma.

Supplement of cilostamide in growth medium improves oocyte maturation and developmental competence of embryos derived from small antral follicles in pigs

This study was conducted to evaluate the effects of cyclic AMP (cAMP) modulator cilostamide (CIL) and forskolin (FSK) treatment during in vitro growth (IVG) on growth, maturation, and embryonic development of cumulus-oocyte complexes (COCs) derived from small antral follicles < 3 mm in diameter (SAFCOCs). SAFCOCs were untreated (control) or treated with 20 μM CIL and/or 50 μM FSK for 2 days for IVG. Next, IVG oocytes were cultured for maturation and then induced for parthenogenesis (PA) or used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation of oocytes was significantly lower in the control (49.6 ± 9.3%) group than in other groups (67.2 ± 5.0–79.8 ± 7.9%). The cumulus expansion score after IVG-IVM was significantly higher in the control and CIL group than in the FSK and CIL + FSK groups. CIL significantly increased mean diameter SAF-derived oocytes (120.0 ± 0.5 μm) compared to the control, FSK, and CIL + FSK (114.8 ± 0.5–116.7 ± 0.6 μm) and showed a comparable level of intracellular glutathione (GSH) contents (0.84 ± 0.07 pixels/oocyte) to medium antral follicle (MAF)-derived oocytes (1.00 ± 0.08 pixels/oocyte), but was higher than those of oocytes treated with FSK and CIL + FSK (0.29 ± 0.05 and 0.37 ± 0.05 pixels/oocyte, respectively). CIL treatment significantly increased blastocyst formation (55.1 ± 4.7%) after PA relative to the control (29.4 ± 6.4%), FSK (34.8 ± 7.1%), and CIL + FSK (41.1 ± 5.2%). A higher proportion of oocytes treated with CIL, FSK, and CIL + FSK (73.3 ± 1.7–82.8 ± 9.1%) remained at the germinal vesicle stage after IVG culture than control oocytes (40.0 ± 5.0%). Following SCNT, blastocyst formation of SAFCOCs treated with CIL (22.4 ± 6.3%) was higher than that of oocytes (0–10.4 ± 5.3%) in control, FSK, and CIL + FSK, but similar to that (25.3 ± 3.5%) of MAF-derived COCs not cultured for IVG. The cAMP level of SAFCOCs before IVG was 0.1 ± 0.03 fmol/oocyte. After 2 days of IVG culture, cAMP level was increased significantly by treatment with FSK and CIL + FSK (3.0 ± 0.57 and 12.1 ± 0.62 fmol/oocyte, respectively) relative to the control and CIL treatment (0.1 ± 0.03 and 0.3 ± 0.04 fmol/oocyte, respectively). Our results demonstrate that CIL treatment during IVG improves the low developmental competence of SAFCOCs to levels comparable to MAFCOCs by allowing oocyte growth while inhibiting premature meiotic maturation, probably via maintenance of cAMP concentrations at appropriate levels.