Ionizing radiation­induced modification of nialamide as an anti­inflammatory agent against lipopolysaccharide­induced RAW 264.7 and DH82 cells.

Nialamide is a non-selective monoamine oxidase inhibitor that was widely used as an antidepressant. However, it has been prohibited for decades in the depressive medicine market due to the adverse hepatotoxic side effects. The re-use of drugs that have been withdrawn from the market represents a promising approach for the development of novel incrementally modified drugs and, in this context, ionizing radiation can serve as a powerful tool for producing new drug candidates. The present study exposed nialamide to γ radiation at 50 kGy to obtain the novel cyclized benzylamide, nialaminosin (compound 2), along with five known compounds, 3-amino-N-benzylpropanamide (compound 3), 3-methoxy-N-benzylpropanamide (compound 4), 3-hydroxy-N-benzylpropanamide (HBPA; compound 5), N-benzylpropanamide (compound 6) and isonicotinamide (compound 7). Among the isolated compounds, HBPA was established to inhibit the lipopolysaccharide-induced overproduction of pro-inflammatory mediators, including nitric oxide (NO) and prostaglandin E2 and cytokines including TNF-α, IL-6 and IL-10, without causing cytotoxicity to both RAW 264.7 and DH82 cells. Furthermore, HBPA was found to reduce the protein expression of inducible NO synthase and cyclooxygenase-2 in macrophages and compared with nialamide, it was established to have more potent radical scavenging activity. The present study therefore suggested the application of HBPA for the improvement of anti-inflammatory properties using ionizing radiation technology on the withdrawn drug nialamide.

Characteristics and biological control functions of Bacillus sp. PM8313 as a host-associated probiotic in red sea bream (Pagrus major) aquaculture.

Host-associated probiotics (HAPs) are bacteria originally isolated from rearing water or the host's gastrointestinal tract in order to enhance the host's growth and health. This study investigated the HAP potential of Bacillus sp. PM8313, isolated from wild red sea bream (Pagrus major), through characterization and feeding trials. Results based on in vitro tests showed that PM8313 is safe, confirming its hemolytic, cytotoxic, and antibiotic resistance. In addition, PM8313 showed advantages as a probiotic with high viability in the gastrointestinal model and a high cell adhesion rate. Whole-genome sequencing demonstrated that PM8313 has a 4,615,871 bp single circular chromosome and a guanine-cytosine content of 45.25%. It also showed the absence of genes encoding virulence factors, such as cytotoxin, enterotoxin, hemolysin, sphingomyelinase, and phospholipase. In the feeding trial, a supplemental diet of 1 × 108 CFU/g PM8313 positively altered the weight gain, digestive enzyme activity, and intestinal microbiota composition of red sea bream. Analysis of nonspecific immune parameters and immune-related gene expression, and a challenge test showed that PM8313 supplementation increases immunity and pathogenic bacteria resistance. Our findings suggest that PM8313 should be considered for application as a novel HAP to red sea bream aquaculture.

Effect of Bacillus sp. Supplementation Diet on Survival Rate and Microbiota Composition in Artificially Produced Eel Larvae (Anguilla japonica).

This study was performed to investigate the effect of microbial supplementation diet on the survival rate and microbiota composition of artificially produced eel larvae. Microorganisms supplemented in the diet were isolated from wild glass eel intestines and identified as Bacillus sp. through 16S rRNA sequencing analysis. In vitro tests confirmed that the strain had no hemolytic activity and virulence genes. Microbial supplemental feeding significantly increased the survival rate of artificially produced eel larvae for 30 days post-hatchling compared with that of the control group. It also caused changes in the α-diversity, ß-diversity, and relative abundance of the bacterial communities. Analysis via phylogenetic investigation of communities by reconstruction of unobserved states predicted that these microbial community changes would significantly increase the carbohydrate metabolism, membrane transport, and cellular community pathway of the microbial supplementation group. Therefore, microbial supplementation feeding for eel aquaculture could increase the viability of artificially produced eel larvae and alter the microbial composition to induce metabolic changes.

Follistatin supplementation during in vitro embryo culture improves developmental competence of bovine embryos produced using sex-sorted semen.

Using sex-sorted semen to produce offspring of desired sex is associated with reduced developmental competence in vitro and lower fertility rates in vivo. The objectives of the present study were to investigate the effects of exogenous follistatin supplementation on the developmental competence of bovine embryos produced with sex-sorted semen and possible link between TGF-ß regulated pathways and embryotrophic actions of follistatin. Effects of follistatin on expression of cell lineage markers (CDX2 and Nanog) and downstream targets of SMAD signaling (CTGF, ID1, ID2 and ID3) and AKT phosphorylation were investigated. Follistatin was supplemented during the initial 72 h of embryo culture. Exogenous follistatin restored the in vitro developmental competence of embryos produced with sex-sorted semen to the levels of control embryos produced with unsorted semen, and comparable results were obtained using sorted semen from three different bulls. The mRNA abundance for SMAD signaling downstream target genes, CTGF (SMAD 2/3 pathway) and ID2 (SMAD 1/5 pathway), was lower in blastocysts produced using sex-sorted versus unsorted semen, but mRNA levels for CDX2, NANOG, ID1 and ID3 were similar in both groups. Follistatin supplementation restored CTGF and ID2 mRNA in blastocysts produced using sex-sorted semen to levels of control embryos. Moreover, levels of phosphorylated (p)AKT (Ser-473 and Thr-308) were similar in embryos derived from sex-sorted and unsorted semen, but follistatin treatment increased pAKT levels in both groups. Taken together, results demonstrated that follistatin improves in vitro development of embryos produced with sex-sorted semen and such effects are associated with enhanced indices of SMAD signaling.

A green chemical oligomerization of phloroglucinol induced by plasma as novel α-glucosidase inhibitors.

A new facile method was developed for simple green synthesis of methylene-bridged phloroglucinol oligomers using nonthermal dielectric barrier discharge (DBD) plasma in methanolic solution. The chemical structures of these newly generated oligomers 2-5 were determined by interpretation of the spectroscopic data, and the inhibitory activity toward α-glucosidase of all isolates was evaluated. The unusual phloroglcuinol pentamer 5 connected by four methylene linkages showed a much higher potential inhibitory effect against α-glucosidase than the other generated oligomers 2-4 and appeared to be a promising lead for development as a potential antidiabetic agent. Abbreviations: T2DM, type2 diabetes mellitus; DBD, dielectric barrier discharge; HPLC, high-performance liquid chromatography; IC50, 50% inhibition concentration; NMR, nuclear magnetic resonance; FABMS, fastatom bombardment mass spectrometry.

Follistatin Rescues Blastocyst Development of Poor Quality Porcine Cumulus-Oocyte Complexes by Delaying Meiotic Resumption With Decreased cGMP.

Mammalian oocytes resume maturation when removed from follicles and cultured in vitro. During folliculogenesis, oocytes are bathed in follicular fluid (FF), which provides an important and specialized microenvironment for oocyte competence. Follistatin (FST) is one component of FF that may play a role in oocyte maturation and embryo development. This study was conducted to examine possible effects of FST on porcine oocyte competence and embryo development. Exogenous FST in oocyte maturation medium for 22 or 44 hours did not improve nuclear maturation and had no effect on good quality cumulus-oocyte complexes (COCs). However, FST improved blastocyst rates in embryos derived from oocytes with less than 2 layers of cumulus. Follistatin treatment of the poor quality COC group increased transcript levels for genes indicative of oocyte quality. Transcript levels were also altered for cumulus expansion-related genes in response to FST when measured during the germinal vesicle breakdown stage. Interestingly, high-quality oocytes remained at germinal vesicle stage much longer than low-quality oocytes, FST treatment induced temporary blockage of spontaneous meiotic resumption when added during culture of both good and poor quality COCs, and levels of cyclic guanosine monophosphate (cGMP) were higher in FST-treated versus untreated groups for both good and poor quality oocytes. In conclusion, FST treatment of porcine oocytes during in vitro maturation can rescue competency of poor quality oocytes to develop to blastocyst stage following in vitro fertilization. Beneficial effects of addition of FST to culture medium may be mediated by inhibiting degradation of cGMP and temporarily delaying nuclear maturation.

Effect of Acteoside as a Cell Protector to Produce a Cloned Dog.

Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT involves the reprogramming a somatic nucleus by injecting a somatic cell into a recipient oocyte whose nucleus has been removed. Therefore, the nucleus donor cells are considered as a crucial factor in SCNT. Cell cycle synchronization of nucleus donor cells at G0/G1 stage can be induced by contact inhibition or serum starvation. In this study, acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for inducing cell cycle synchronization, cytoprotection, and improving SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 µM) for various time periods (24, 48 and 72 hours). Cell cycle synchronization at G0/G1 stage did not differ significantly with the method of induction: acteoside treatment, contact inhibition or serum starvation. However, of these three treatments, serum starvation resulted in significantly increased level of reactive oxygen species (ROS) (99.5 ± 0.3%) and apoptosis. The results also revealed that acteoside reduced ROS and apoptosis processes including necrosis in canine fetal fibroblasts, and improved the cell survival. Canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos using nucleus donor cells treated with acteoside produced a healthy cloned dog, but not the embryos produced using nucleus donor cells subjected to contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT because of its cytoprotective effects.

Effect of acteoside on the re-localization and abnormal morphology of mitochondria in porcine oocytes during in vitro maturation.

PURPOSE: The aim of this study is to investigate the effect of acteoside, an antioxidant, on in vitro maturation (IVM) of oocytes to improve early parthenogenetic embryonic developmental competence. METHODS: Porcine immature oocytes (total 770) were cultured in IVM medium with acteoside at various concentrations, 0 (control), 10, 30, and 50 µM. Each group was assessed for maturation and subsequent development rates, reactive oxygen species (ROS) level (15 oocytes per group and four independent experiments performed), ultrastructure observation (15 oocytes per group), mitochondrial activity (30 oocytes per groups and three independent experiments performed), and expression patterns of apoptosis-related genes (100 expended parthenogenetic embryos per group and three independent experiment performed). Main outcome measures were the rates of IVM, blastocyst formation, ROS, mitochondria, and expression of apoptosis-related genes in oocytes treated with acteoside. RESULT(S): Addition of acteoside during IVM did not change the maturation efficiency of oocytes but improved the rate of blastocyst formation with significantly decreased ROS level. Moreover, in acteoside-treated oocytes, cytoplasmic maturation was improved with morphologically uniform distribution of mitochondria and lipid droplets in cytoplasm. Acteoside supplementation also increased the mRNA expression levels of antiapoptotic genes and reduced those of pro-apoptotic genes. CONCLUSION(S): Acteoside supplementation in IVM medium improves the oocyte quality and subsequent development of pre-implantation embryos that would eventually contribute to produce embryos with high embryonic development competence.

Evidence supporting a role for SMAD2/3 in bovine early embryonic development: potential implications for embryotropic actions of follistatin.

The TGF-beta-SMAD signaling pathway is involved in regulation of various aspects of female reproduction. However, the intrinsic functional role of SMADs in early embryogenesis remains poorly understood. Previously, we demonstrated that treatment with follistatin, an activin (TGF-beta superfamily ligand)-binding protein, is beneficial for bovine early embryogenesis and specific embryotropic actions of follistatin are dependent on SMAD4. Because SMAD4 is a common SMAD that can bind both SMAD2/3 and SMAD1/5, the objective of this study was to further determine the intrinsic role of SMAD2/3 in the control of early embryogenesis and delineate if embryotropic actions of follistatin in early embryos are SMAD2/3 dependent. By using a combination of pharmacological and small interfering RNA-mediated inhibition of SMAD2/3 signaling in the presence or absence of follistatin treatment, our results indicate that SMAD2 and SMAD3 are both required for bovine early embryonic development and stimulatory actions of follistatin on 8- to 16-cell and that blastocyst rates, but not early cleavage, are muted when SMAD2/3 signaling is inhibited. SMAD2 deficiency also results in reduced expression of the bovine trophectoderm cell-specific gene CTGF. In conclusion, the present work provides evidence supporting a functional role of SMAD2/3 in bovine early embryogenesis and that specific stimulatory actions of follistatin are not observed in the absence of SMAD2/3 signaling.

Expression of TGFß superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development.

Brilliant cresyl blue (BCB) is a super-vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocyte's developmental potential, and the transcript abundance for select TGFß-superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus-cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low-quality oocytes, based on BCB staining, was also determined. Cumulus-oocyte complexes (COCs) from abattoir-derived ovaries were subjected to BCB staining, and germinal-vesicle-stage oocytes and cumulus cells were harvested from control, BCB+, and BCB- (low-quality oocyte) groups for real-time PCR or Western-blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB- oocytes. Western-blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB- oocytes. Embryo-culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY-1 treatment, improve the developmental competence of BCB- oocytes. These results contribute to a better understanding of molecular indices of oocyte competence.

Transcriptomic diversification of developing cumulus and mural granulosa cells in mouse ovarian follicles.

Cumulus cells and mural granulosa cells (MGCs) have functionally distinct roles in antral follicles, and comparison of their transcriptomes at a global and systems level can propel future studies on mechanisms underlying their functional diversity. These cells were isolated from small and large antral follicles before and after stimulation of immature mice with gonadotropins, respectively. Both cell types underwent dramatic transcriptomic changes, and differences between them increased with follicular growth. Although cumulus cells of both stages of follicular development are competent to undergo expansion in vitro, they were otherwise remarkably dissimilar with transcriptomic changes quantitatively equivalent to those of MGCs. Gene ontology analysis revealed that cumulus cells of small follicles were enriched in transcripts generally associated with catalytic components of metabolic processes, while those from large follicles were involved in regulation of metabolism, cell differentiation, and adhesion. Contrast of cumulus cells versus MGCs revealed that cumulus cells were enriched in transcripts associated with metabolism and cell proliferation while MGCs were enriched for transcripts involved in cell signaling and differentiation. In vitro and in vivo models were used to test the hypothesis that higher levels of transcripts in cumulus cells versus MGCs is the result of stimulation by oocyte-derived paracrine factors (ODPFs). Surprisingly ∼48% of transcripts higher in cumulus cells than MGCs were not stimulated by ODPFs. Those stimulated by ODPFs were mainly associated with cell division, mRNA processing, or the catalytic pathways of metabolism, while those not stimulated by ODPFs were associated with regulatory processes such as signaling, transcription, phosphorylation, or the regulation of metabolism.

Requirement of the transcription factor USF1 in bovine oocyte and early embryonic development.

Upstream stimulating factor 1 (USF1) is a basic helix-loop-helix transcription factor that specifically binds to E-box DNA motifs, known cis-elements of key oocyte expressed genes essential for oocyte and early embryonic development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated that USF1 mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8-16-cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown of USF1 in germinal vesicle stage oocytes did not affect meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA-mediated depletion of USF1 in presumptive zygote stage embryos demonstrated that USF1 is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 implies a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development, which also functions in regulation of JY1, GDF9, and FST genes associated with oocyte competence.

Evidence supporting a functional requirement of SMAD4 for bovine preimplantation embryonic development: a potential link to embryotrophic actions of follistatin.

Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent.

Temporal regulation of mRNAs for select bone morphogenetic proteins (BMP), BMP receptors and their associated SMAD proteins during bovine early embryonic development: effects of exogenous BMP2 on embryo developmental progression.

BACKGROUND: We previously demonstrated embryotrophic actions of maternal (oocyte-derived) follistatin during bovine early embryogenesis. Classical actions of follistatin are attributed to inhibition of activity of growth factors including activins and bone morphogenetic proteins (BMP). However, temporal changes in BMP mRNA in early bovine embryos and the effects of exogenous BMP on embryo developmental progression are not understood. The objectives of present studies were to characterize mRNA abundance for select BMP, BMP receptors and BMP receptor associated SMADs during bovine oocyte maturation and early embryogenesis and determine effects of addition of exogenous BMP protein on early development. METHODS: Relative abundance of mRNA for BMP2, BMP3, BMP7, BMP10, SMAD1, SMAD5, ALK3, ALK6, ALK2, BMPR2, ACVR2A and ACVR2B was determined by RT-qPCR analysis of germinal vesicle (GV) and in vitro matured metaphase II (MII) oocytes and in vitro produced embryos collected at pronuclear, 2-cell (C), 4C, 8C, 16C, morula and blastocyst stages. Effects of addition of recombinant human BMP2 (0, 1, 10 and 100 ng/ml) during initial 72 h of embryo culture on early cleavage (within 30 h post insemination), total cleavage, development to 8C-16C and blastocyst stages and blastocyst mRNA abundance for markers of inner cell mass (NANOG) and trophectoderm (CDX2) were also determined. RESULTS: Abundance of mRNA for BMP2, BMP10, SMAD1, SMAD5, ALK3, ALK2, BMPR2 and ACVR2B was elevated in MII oocytes and/or pronuclear stage embryos (relative to GV) and remained elevated through the 8C -16C stages, whereas BMP3, BMP7 and ALK2 mRNAs were transiently elevated. Culture of embryos to the 8C stage in the presence of α-amanitin resulted in increased abundance for all of above transcripts examined relative to untreated 8C embryos. Effects of addition of exogenous BMP2 on early cleavage rates and rates of development to 8C-16C and blastocyst stages were not observed, but BMP2 treatment increased blastocyst mRNA for CDX2 and NANOG. CONCLUSIONS: Abundance of maternally derived mRNAs for above BMP system components are dynamically regulated during oocyte maturation and early embryogenesis. Exogenous BMP2 treatment does not influence progression to various developmental endpoints, but impacts characteristics of resulting blastocysts. Results support a potential role for BMPs in bovine early embryogenesis.

The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy.

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases.

Functional role of the bovine oocyte-specific protein JY-1 in meiotic maturation, cumulus expansion, and subsequent embryonic development.

Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine early embryogenesis. Given that JY-1 is present in oocytes throughout folliculogenesis, and oocyte-derived JY-1 mRNA is temporally regulated postfertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 small interfering RNA was microinjected into cumulus-enclosed germinal vesicle-stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation (IVM), was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion, and embryonic development after in vitro fertilization. Depletion of JY-1 protein during IVM effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8- to 16-cell stage, and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion, and early cleavage, but did not rescue development to 8- to 16-cell and blastocyst stages. However, effects of JY-1 depletion postfertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion, and subsequent embryonic development.

Neuronal cell differentiation of mesenchymal stem cells originating from canine amniotic fluid.

The amniotic fluid contains mesenchymal stem cells (MSCs) and can be readily available for tissue engineering. Regenerative treatments such as tissue engineering, cell therapy, and transplantation show potential in clinical trials of degenerative diseases. Disease presentation and clinical responses in the Canis familiaris not only are physiologically similar to human compared with other traditional mammalian models but is also a suitable model for human diseases. The aim of this study was to investigate whether canine amniotic-fluid-derived mesenchymal stem cells (cAF-MSCs) can differentiate into neural precursor cells in vitro when exposed to neural induction reagent. During neural differentiation, cAF-MSCs progressively acquire neuron-like morphology. Messenger RNA (mRNA) expression levels of neural-specific genes, such as NEFL, NSE, and TUBB3 (ßIII-tubulin) dramatically increased in the differentiated cAF-MSCs after induction. In addition, protein expression levels of nestin, ßIII-tubulin, and tyrosine hydroxylase remarkably increased in differentiated cAF-MSCs. This study demonstrates that cAF-MSCs have great potential for neural precursor differentiation in vitro. Therefore, amniotic fluid may be a suitable alternative source of stem cells and can be applied to cell therapy in neurodegenerative diseases.

Glutathione and cysteine enhance porcine preimplantation embryo development in vitro after intracytoplasmic sperm injection.

Because intracytoplasmic sperm injection (ICSI) had been introduced to animal science, not only reproductive biology of domestic animals, but also medicine to treat infertility has been developed. This assisted reproductive technology is beneficial for generating transgenic animals, especially pigs, because polyspermy is the greatest hurdle in porcine IVF when researchers make highly qualified preimplantation embryos. However, ICSI-derived embryos expressed high level of reactive oxygen species (ROS), which are known to cause serious dysfunction during preimplantation development. The objective of this study was to investigate the developmental competence, ROS level, and apoptosis index when glutathione (GSH) or cysteine was supplemented into the in vitro culture medium for ICSI-derived porcine embryos. First, we evaluated the effect of different concentrations of GSH or cysteine on developmental ability of porcine ICSI-derived embryos. The cleavage rate (79.6%) and the blastocyst formation rate (20.9%) were significantly improved in culture medium supplemented with 1 mmol/L GSH compared with other concentrations or no supplementation. Also, 1.71 mmol/L cysteine showed a significantly higher proportion of cleavage (80.7%) and blastocyst formation (22.5%) than other cysteine-supplemented groups. Next, we confirmed that intracellular ROS level was significantly reduced in the group of blastocysts cultured with GSH or cysteine after ICSI compared with the no supplementation group. Finally, we found that terminal uridine nick-end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased in blastocysts when ICSI-derived embryos were cultured with supplementation of 1.71 mmol/L cysteine or 1 mmol/L GSH. Taken together, these results strongly indicate that GSH or cysteine can improve the developmental competence of porcine ICSI-derived embryos by reducing intracellular ROS level and the apoptosis index.

Embryotropic actions of follistatin: paracrine and autocrine mediators of oocyte competence and embryo developmental progression.

Despite several decades since the birth of the first test tube baby and the first calf derived from an in vitro-fertilised embryo, the efficiency of assisted reproductive technologies remains less than ideal. Poor oocyte competence is a major factor limiting the efficiency of in vitro embryo production. Developmental competence obtained during oocyte growth and maturation establishes the foundation for successful fertilisation and preimplantation embryonic development. Regulation of molecular and cellular events during fertilisation and embryo development is mediated, in part, by oocyte-derived factors acquired during oocyte growth and maturation and programmed by factors of follicular somatic cell origin. The available evidence supports an important intrinsic role for oocyte-derived follistatin and JY-1 proteins in mediating embryo developmental progression after fertilisation, and suggests that the paracrine and autocrine actions of oocyte-derived growth differentiation factor 9, bone morphogenetic protein 15 and follicular somatic cell-derived members of the fibroblast growth factor family impact oocyte competence and subsequent embryo developmental progression after fertilisation. An increased understanding of the molecular mechanisms mediating oocyte competence and stage-specific developmental events during early embryogenesis is crucial for further improvements in assisted reproductive technologies.

Develop to term rat oocytes injected with heat-dried sperm heads.

This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term.