The present study was designed to investigate the comprehensive function of maternal factors of primordial germ cell 7 (PGC7) and POU5F1-POU class 5 homeobox 1 (OCT4), as well as the epigenetic modification roles on the mitosis for the extrusion of first polar body (PB1) in pig maturated oocytes. First, the common distribution of histone modifications, including H3K4me2, H3K27me3, H3K9me2, and H4K12ac and DNA methylation, were detected at the high level in the nucleus. However, only one part of the chromosome was higher methylated or acetylated when the mitosis happened to extrude the PB1. When the mitosis was inhibited by the cytochalasin B (CB) treatment, the expression of PGC7, OCT4, DNA methyltransferase1 (DNMT1), DNA methyltransferase3b (DNMT3b), tet methylcytosine dioxygenase 1 (TET1), tet methylcytosine dioxygenase 2 (TET2), and tet methylcytosine dioxygenase 3 (TET3) could be inhibited (p < 0.01), and no concentrated expression of the PGC7 and OCT4 was observed on the chromosome, but the levels of H3K9me2 and H4K12ac were higher. In addition, when the trichostatin A was performed on the in vitro maturation, the extrusion of the PB1 was inhibited too. And the histone methylation (H3K9me2 and H3K27me3) could be detected all the time with relative higher level and no demethylation could be observed. However, the expression of PGC7 and OCT4 was lower in the chromosome. It might indicate that the maternal factor of PGC7 and histone modification that included H4K12ac and H3K9me2 could regulate the extrusion of the PB1 and play an important role in the maturation of pig oocytes.

The histone variant H3.3 is an important maternal factor in fertilization of oocytes and reprogramming of somatic cell nuclear transfer (SCNT) embryos. As a crucial replacement histone, maternal H3.3 is involved in chromatin remodeling and zygote genome activation. Litte is, however, known about the replacement of H3.3 in the bovine SCNT embryos. In this study, the maternal H3.3 in mature ooplasm was labeled with HA tag and the donor cells H3.3 was labeled with Flag tag, in order to observe the replacement of H3.3 in the bovine SCNT embryos. Meanwhile, maternal H3.3 knockdown was performed by microinjecting two different interfering fragments before nucleus transfer. It was showed that the dynamic replacement between maternal- and donor nucleus-derived H3.3 was detected after SCNT. And it could be observed that the blastocyst development rate of the cloned embryos decreased from 22.3% to 8.2-10.3% (P < 0.05), the expression of Pou5f1 and Sox2 was down-regulated and the level of H3K9me3 was increased in the interfered embryos. In summary, H3.3 replacement impacted on the process of reprogramming, including embryonic development potential, activation of pluripotency genes and epigenetic modification in bovine SCNT embryos.

Keywords: Bovine; H3.3 replacement; H3k9me3; Pluripotency genes; SCNT embryo.

In many mammalian species, surface markers have been used to obtain enriched populations of spermatogonial stem cells (SSCs) for assisted reproduction and other applications; however, little is known about the expression patterns of feline SSCs. In this study, we assessed expression of the SSC surface markers commonly used in other species, KIT, ITGA6, CD9, GFRalpha1, ADGRA3, and THY1, in addition to the less frequently used pluripotent markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA-4 in SSCs of both prepubertal and adult domestic cats (Felis catus). To further characterize cat SSCs, we sorted cells using SSC-specific markers and evaluated the expression of the pluripotent transcription factors NANOG, POU5F1, and SOX2 and the proto-oncogene MYC within these populations. We concluded that SSC surface markers used in other mammalian species were not specific for identifying cat SSCs. However, the pluripotent markers we evaluated were more specific to cat spermatogonia, and the presence of SSEA-1 and SSEA-4 in fewer and primarily individual cells suggests that these two markers may be used for enrichment of cat SSCs. The expression of pluripotent transcription factors at mRNA level by single-stained cells positive for SSEA-4 and by dual-stained cells positive for both GFRalpha1 and SSEA-4 reflects the undifferentiated stage of cat SSCs. The absence of transcription factors in double-stained cells positive for only one marker implies the loss of the stem cell-like identity with the loss of either GFRalpha1 or SSEA-4. Further investigation is warranted to elucidate the biological characteristics of these spermatogonial subpopulations.

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture.

There is no consensus as to how a precursor lesion, germ cell neoplasia in situ (GCNIS), develops into the histologic types of testicular germ cell tumor type II (TGCT). The present meta-analysis examined RNA expressions of 24 candidate genes in three datasets. They included 203 samples of normal testis (NT) and histologic types of TGCT. The Fisher's test for combined p values was used for meta-analysis of the RNA expressions in the three datasets. The histologic types differed in RNA expression of PRAME, KIT, SOX17, NANOG, KLF4, POU5F1, RB1, DNMT3B, and LIN28A (p < 0.01). The histologic types had concordant differences in RNA expression of the genes in the three datasets. Eight genes had overlap with a high RNA expression in at least two histologic types. In contrast, only seminoma (SE) had a high RNA expression of KLF4 and only embryonal carcinoma (EC) had a high RNA expression of DNMT3B. In conclusion, the meta-analysis showed that the development of the histologic types of TGCT was driven by changes in RNA expression of candidate genes. According to the RNA expressions of the ten genes, TGCT develops from NT over GCNIS, SE, EC, to the differentiated types of TGCT.

Keywords: RNA expression; biomarkers; genes for pluripotency; meta-analysis; oncogenes; pathogenesis; testicular neoplasms; tumor suppressor genes.

The expression of pluripotency factors is a key regulator of tumor differentiation status and cancer stem cells. The purpose of this study was to examine the expression of pluripotency factors and differentiation status of human mesothelioma and the role of mitochondria in their regulation. We tested the expression of OCT4/POU5F1, NANOG, SOX2, PI3K-AKT pathway and BCL2 genes and proteins in 65 samples of human mesothelioma and 19 samples of normal mesothelium. Mitochondrial membrane potential, reactive oxygen species (ROS) generation and expression of pluripotency factors were also tested in human mesothelioma cell line. Human mesothelium and mesothelioma expressed SOX2, NANOG, PI3K and AKT genes and proteins and POU5F1 gene, whereby NANOG, SOX2 and phosphorylated (activated) AKT were upregulated in mesothelioma. NANOG protein expression was elevated in less differentiated samples of human mesothelioma. The expression of genes of PI3K-AKT pathway correlated with pluripotency factor genes. Mesothelioma cells had functional, but depolarized mitochondria with large capacity to generate ROS. Mitochondrial ROS upregulated NANOG and mitoTEMPO abrogated it. In conclusion, human mesothelioma displays enhanced expression of NANOG, SOX2 and phosphorylated AKT proteins, while elevated NANOG expression correlates with poor differentiation of human mesothelioma. Mitochondria of mesothelioma cells have a large capacity to form ROS and thereby upregulate NANOG, leading to dedifferentiation of mesothelioma.

Keywords: mesothelioma; mitochondria; pluripotency factors; reactive oxygen species; reprogramming.

Background: Increasing evidence suggests the involvement of cancer stem cells (CSCs) in both oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Among the various CSC markers, aldehyde dehydrogenase (ALDH) 1, B cell-specific Moloney murine leukaemia virus integration site 1 (Bmi1), and octamer-binding protein 4 (OCT4) have been noted to increase in OSCC. The aim of the study was to analyze ALDH1, Bmi1, and OCT4 expression in OED and OSCC with clinicopathologic correlation and survival analysis.

Methods: A total of 40 cases each of OED and OSCC were retrieved from departmental archives. Expression of ALDH1, Bmi1, and OCT4 was analyzed using immunohistochemistry and was correlated with clinicopathological parameters. A follow-up ranging from 6 to 52 months was considered for Kaplan-Meier survival analysis. The log-rank test was performed to analyze significant difference in survival rates.

Results: The expression levels of ALDH1, Bmi1, and OCT4 increased significantly from OED through OSCC (P < .05). The expression of ALDH1 and OCT4 showed a significant correlation with lymph node metastasis. Positive cases of ALDH1 showed a significantly reduced survival rate compared to cases showing negative expression. Kaplan-Meier survival analysis showed a significant reduction of survival rate (P = .00) in patients showing a positive expression for all the 3 markers.

Conclusion: ALDH1 and OCT4 could be used as individual prognostic markers for assessing prognosis. ALDH1, Bmi1, and OCT4 could be used as a collective panel of markers to enable surgeons in predicting the prognosis of patients and thereby carry out prompt follow-up for such cases.

Keywords: Bmi1 protein; POU5F1 protein; aldehyde dehydrogenase 1; mouth neoplasms; neoplastic stem cells; survival analysis.

Previous studies have shown that the transforming growth factor β1 pathway plays an important role in breast cancer metastasis to the liver. However, the mechanism of this metastasis has not been fully clarified. Cancer stem cells are essential for the initiation and propagation of tumor metastasis. The objective of our current study was to define the role of cancer stem cells in transforming growth factor β1-mediated breast cancer hepatic metastases.

Hematoxylin and eosin staining was used to assess the formation of breast cancer liver metastases and local invasion. Cancer stem cells surface markers (CD44, CD24, and Epithelial cell adhesion molecule [EpCAM]), luminal/mesenchymal markers (keratin8 and alpha smooth muscle actin), and proliferation markers (Ki-67 and cyclinD1) were detected by immunohistochemistry assays. Flow cytometry was used to evaluate the effect of transforming growth factor β1 on the CD44+/CD24- cancer stem cell population. Quantitative real-time polymerase chain reaction was employed to assess the gene expression of the stem cell self-renewal markers nanog, pou5f1 (coding for Oct4), and sox2.

Transforming growth factor β1 increased the formation of liver metastases by the MDA-MB231 (MDA) breast cancer cell line but did not affect the liver metastasis of CD44+/CD24+ noncancer stem cells. Transforming growth factor β1 treatment did not significantly affect tumor proliferation in vitro or in vivo. Transforming growth factor β1 promoted mammary tumor local invasion. Furthermore, the CD44high/CD24- cancer stem cell population was also significantly increased by transforming growth factor β1 treatment. Besides, the gene expression of the stem cell self-renewal markers (nanog, pou5f1, and sox2) and another stem cell surface marker (EpCAM) was increased by transforming growth factor β1 treatment. Finally, clusters of CD44-positive breast cancer cells were observed in the livers of mice from the control and transforming growth factor β1 pretreatment groups.

Our results indicate that transforming growth factor β1 increases the local invasive capacity and liver metastasis of breast cancer cells by inducing the CD44high/CD24- cancer stem cell population.

Induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) differentiated into hepatocyte-like cells (HLCs) provide a defined and renewable source of cells for drug screening, toxicology and regenerative medicine. We previously reprogrammed human fetal foreskin fibroblast cells (HFF1) into iPSCs employing an episomal plasmid-based integration-free approach, this iPSC-line and the hESC lines H1 and H9 were used to model hepatogenesis in vitro. Biochemical characterisation confirmed glycogen storage, ICG uptake and release, urea and bile acid production, as well as CYP3A4 activity. Microarray-based transcriptome analyses was carried out using RNA isolated from the undifferentiated pluripotent stem cells and subsequent differentiation stages- definitive endoderm (DE) hepatic endoderm (HE) and HLCs. K-means identified 100 distinct clusters, for example, POU5F1/OCT4 marking the undifferentiated stage, SOX17 the DE stage, HNF4α the HE stage, and ALB specific to HLCs, fetal liver and primary human hepatocytes (PHH). This data descriptor describes these datasets which should be useful for gaining new insights into the molecular basis of hepatogenesis and associated gene regulatory networks.

OCT4 (also known as POU5F1) plays an essential role in reprogramming. It is the only member of the POU (Pit-Oct-Unc) family of transcription factors that can induce pluripotency despite sharing high structural similarities to all other members. Here, we discover that OCT6 (also known as POU3F1) can elicit reprogramming specifically in human cells. OCT6-based reprogramming does not alter the mesenchymal-epithelial transition but is attenuated through the delayed activation of the pluripotency network in comparison with OCT4-based reprogramming. Creating a series of reciprocal domain-swapped chimeras and mutants across all OCT factors, we clearly delineate essential elements of OCT4/OCT6-dependent reprogramming and, conversely, identify the features that prevent induction of pluripotency by other OCT factors. With this strategy, we further discover various chimeric proteins that are superior to OCT4 in reprogramming. Our findings clarify how reprogramming competences of OCT factors are conferred through their structural components.

Epigenetic modifications operate in concert to maintain cell identity, yet how these interconnected networks suppress alternative cell fates remains unknown. Here, we uncover a link between the removal of repressive histone H3K9 methylation and DNA methylation during the reprogramming of somatic cells to pluripotency. The H3K9me2 demethylase, Kdm3b, transcriptionally controls DNA hydroxymethylase Tet1 expression. Unexpectedly, in the absence of Kdm3b, loci that must be DNA demethylated are trapped in an intermediate hydroxymethylated (5hmC) state and do not resolve to unmethylated cytosine. Ectopic 5hmC trapping precludes the chromatin association of master pluripotency factor, POU5F1, and pluripotent gene activation. Increased Tet1 expression is important for the later intermediates of the reprogramming process. Taken together, coordinated removal of distinct chromatin modifications appears to be an important mechanism for altering cell identity.

Regarding that undifferentiated mesenchymal stem cells, as donor cells, require less epigenetic reprogramming, possibility of using bovine adipose tissue-derived stem cells (BASCs) with low level of DNMTs and HDACs expression was evaluated.

In this experimental study, we examined gene expression of epigenetic modifiers including DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and histone deacetylases (HDAC1-3), as well as protein levels of histone H3 acetylation at lysine 9 (H3K9ac) and POU5F1 (also known as OCT4) at two stages of preimplantation development among in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) groups.

The results revealed that developmental competence of IVF embryos was higher than SCNT embryos (P<0.05). In the PA and SCNT groups, DNMT1, HDAC2 and HDAC3 mRNA were overexpressed (P<0.05), and proteins levels of H3K9ac and POU5F1 were reduced at 6-8 cells and blastocyst stages compared to IVF (P<0.05). The mRNA expression of DNMT1 and HDAC1 and proteins levels of POU5F1 and H3K9ac were significantly different between SCNT and PA groups (P<0.05) in both developmental stages (except HDAC1 in blastocyst stage).

The SCNT embryos derived from BASCs have endured considerable nuclear reprogramming during early embryo development. Comparison of PA and SCNT blastocysts demonstrated that HDAC1 and DNMT1 may attribute to developmental competence variability of bovine embryos.

We generated eight induced pluripotent stem cell (iPSC) lines from Parkinson's disease (PD) patients with different familial mutations using non-integrating episomal plasmids. All iPSC lines have a normal karyotype, express pluripotent genes including POU5F1, NANOG, and show alkaline phosphatase activity, as well as the ability to differentiate into all three germ layers. These PD iPSC lines can be used for disease modeling to identify PD mechanisms and for the development or stratification of new drugs.

Human embryonic stem cell (hESC) lines have been derived from the inner cell mass of blastocysts. Five hESC lines have been derived from 32 discarded blastocysts in Taiwan, and these lines have since been continuously cultured on mitotically inactivated mouse embryonic fibroblasts as feeder in the hESC medium for more than 44 passages and underwent freezing/thawing processes. All of five hESC lines expressed characteristic undifferentiated hESC markers such as SSEA-4, TRA-1-81, alkaline phosphatase, TERT, transcription factors POU5F1 (OCT4), and NANOG. The hESC lines T1 and T3 possess normal female karyotypes, whereas lines T4 and T5 are normal male, but line T2 is male trisomy 12 (47XY,+12). The hESC lines T1, T2, T3, and T5 were able to produce teratomas in SCID mice, and line T4 could only form embryoid bodies in vitro. Global gene expression profiles of single colonies of these five hESC lines were analyzed using Affymetrix human genome U133 plus 2.0 GeneChip. The results showed that 4,145 transcripts, including 19% of unknown functions, were detected in all five hESC lines. Comparison of the 4,145 genes commonly expressed in the five hESC lines with those genes expressed in teratoma produced by hESC line T1 and placenta revealed 40 genes exclusively expressed in all five hESC lines. These 40 genes include the previously reported stemness genes such as POU5F1 (OCT4), NANOG, TDGF1 (CRIPTO), SALL4, LECT1, and BUB1 responsible for self-renewal and pluripotent differentiation. The global gene expression analysis also indicated that the TGFβ/activin branch components inhibin BC, ACVR2A, ACVR1 (ALK2), TGFBR1 (ALK5), and SMAD2 were found to be highly expressed in undifferentiated states of these five hESC lines and decreased upon differentiation. The epigenetic states and expression of 32 known imprinted genes in these five hESC lines and/or differentiated derivatives were also investigated. In short, the hESC nature of these five hESC lines is supported by the undifferentiated state, extensive renewal capacity, and pluripotency, including the ability to form teratomas and/or embryoid bodies; and these cell lines will be useful for research on human embryonic stem cell biology and drug development/toxicity testing. The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.

Abstract.

Background/purpose: Although 2,3,5,4'-Tetrahydroxystilbene-2-O-beta-glucoside (THSG) reportedly has anti-inflammatory properties, its role in inducing the dedifferentiation of human dental pulp stem cells (DPSC) into pluripotent-like stem cells remains to be determined. The purpose of this study is to evaluate the effects of THSG on the pluripotent-like possibility and mechanism of DPSC.

Materials and methods: DPSCs were treated with THSG, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTS) assay. Real-time polymerase chain reaction was used to analyze the mRNA expression levels of pluripotency-associated genes and oncogenes and to detect telomerase activity in the cells. Embryoid body formation assay was conducted, and pluripotency-related proteins were identified using Western blotting. Data were analyzed using one-way analysis of variance.

Results: Cell viability, telomerase activity, and embryoid body formation were enhanced in THSG-treated DPSCs. The mRNA expression levels of pluripotent-like genes (including Nanog homeobox [NANOG], SRY-box 2 [SOX2], and POU class 5 homeobox 1 [POU5F1/OCT4]) significantly increased after THSG treatment. The expression levels of pluripotency-related genes (Janus kinase-signal transducer 2 [JAK2] and signal transducer and activator of transcription 3 [STAT3]) increased, whereas those of oncogenes (Ras, SRC, HER2, and C-sis) decreased. Furthermore, the expression levels of the phosphorylated JAK2 and STAT3 proteins significantly increased after THSG treatment.

Conclusion: THSG treatment may enhance the pluripotent-like possibility of DPSC through the JAK2/STAT3 axis. Hence, it may be used as an alternative cell-based therapeutic strategy in regenerative dentistry.

Keywords: 2,3,5,4′-tetrahydroxystilbene-2-O-b-glucoside; Dental pulp stem cells; Pluripotency.

This study investigated the effect of the antioxidant dieckol, a component of Ecklonia cava, on maturation and developmental competence of porcine oocytes exposed to oxidative stress in vitro. Oocytes were matured in in vitro maturation (IVM) medium containing various concentrations of dieckol. The blastocyst formation rate was highest in the 0.5 μM dieckol-treated (0.5 DEK) group. The reactive oxygen species level was decreased, and the level of glutathione and expression of antioxidant genes (NFE2L, SOD1, and SOD2) at metaphase II were increased in the 0.5 DEK group. Abnormal spindle organization and chromosome misalignment were prevented in the 0.5 DEK group. Expression of maternal markers (CCNB1 and MOS) and activity of p44/42 mitogen-activated protein kinase were increased in the 0.5 DEK group. After parthenogenetic activation, the total number of cells per blastocyst was increased and the percentage of apoptotic cells was decreased in the 0.5 DEK group. Expression of development-related genes (CX45, CDX2, POU5F1, and NANOG), antiapoptotic genes (BCL2L1 and BIRC5), and a proapoptotic gene (CASP3) were altered in the 0.5 DEK group. These results indicate that the antioxidant dieckol improves IVM and subsequent development of porcine oocytes and can be used to improve the quality of oocytes under peroxidation experimental conditions.

Keywords: IVM; dieckol; embryo; oxidative stress; porcine.

Stem cells show the capability to proliferate in an undifferentiated state with long-term self-renewal, which gives the cells advantages for use as bioactive material (BM) for embryo culture in vitro. The objective of this experiment was to investigate the effect of two BMs-human adipose tissue-derived mesenchymal stem cell BM (hAT-MSC-BM) and human embryonic stem cell-derived BM (hESC-BM)-on porcine embryo development compared to commonly used bovine serum albumin (BSA) or serum treatment groups. In vitro-fertilized (IVF) embryos were cultured in PZM-5 with 4 mg/mL BSA until day 4 and equally divided into four groups. Starting from day 4 (until day 6), each group was treated with the following protein additives: 4 mg/mL BSA (control), 10% fetal bovine serum (FBS), 10% hAT-MSC-BM, or 10% hESC-BM. Our results show FBS- and two other BM-treated groups showed significant increases in blastocyst formation rate, hatching rate, and total cell number compared with the control group (p<0.05). The hAT-MSC-BM and hESC-BM treatment groups presented better-quality embryo development, especially from the middle expanding stage to hatching. In particular, the hAT-MSC-BM-treated group showed the highest developmental potential of all groups and formed the most expanding-stage blastocysts. The relative expression of reprogramming-related transcription factor (POU5F1, SOX2, DPPA5, and CDH1), antioxidant (PRDX5), and apoptosis (BCL2L1 and BIRC5) genes also increased in two types of BMs compared to the control. In addition, we investigated the protein synthesis of the tight junction- and gap junction-related genes, connexin 43 and zonula occludens-1 (ZO-1); these increased more than in the control. These results demonstrate that stem cell-derived BMs accelerate porcine preimplantation embryo development and that the BMs would be helpful in the development of preimplantation embryos.

Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study, buffalo spermatogonial stem-like cells were isolated, cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubules were enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin, were determined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining in buffalo testes and buffalo spermatogonial stem-like cells, respectively. In the following, when the isolated buffalo buffalo spermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derived neurotrophic factor medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-like cells, and accelerating the generation of genetic modified buffaloes.

In this study, a modified method of handmade cloning (m-HMC), which had been originally developed in sheep, was used for somatic cell nuclear transfer (SCNT) in the dromedary camel. The unique feature of m-HMC over current SCNT methods lies in the use of a simple device (a finely drawn micropipette made of Pasteur pipette) for chemically-assisted enucleation of oocytes under a stereomicroscope with improved efficiency and ease of operation. Using this system, the throughput of cloned embryo reconstitution was increased over 2-fold compared to the control SCNT method (c-NT). Stepwise measurement of reactive oxygen species (ROS) revealed that method, steps, and duration of SCNT all influenced oxidative activity of oocytes, but their impact were not similar. Specifically, UV-assisted oocyte enucleation was identified as the major source of ROS production, which explained significantly higher total ROS of reconstituted embryos in c-NT compared to m-HMC. Fusion efficiency (95.3±3.3 vs. 75.4±7.6%) and total efficiency of blastocyst development (22.5±3.0 vs. 14.1±4.3%) were significantly higher in m-HMC compared to c-NT, respectively, and blastocysts of transferable quality were obtained in similar rates (41.9±8.2 vs. 48.0±15.2%, respectively). Significance differences were observed in total cell number (155.3±13.6 vs. 123.6±19.5) and trophectoderm (145±9.5 vs. 114.3±15.2), but not inner cell mass (10.3±4.1 vs. 9.3±5.3) counts between blastocysts developed in c-NT compared to m-HMC, respectively. However, expression of key developmental genes (POU5F1, KLF4, SOX2, MYC, and CDX2) was comparable between blastocysts of both groups. The introduced m-HMC method might be a viable approach for efficient production of dromedary camel clones for research and commercial utilization.

The therapeutic protocols for treatment of germinomas and non-germinomatous germ cell tumors (NGGCTs) are completely different, so it is important to distinguish pure germinomas from NGGCTs. As it can be difficult to diagnose by morphology alone, immunohisto-chemistry (IHC) has been widely used as an ancillary test to improve diagnostic accuracy. However, IHC has limitations due to the misinterpretation of results or the aberrant loss of immunoreactivity. However, real-time RT-PCR has certain advantages over IHC, including its quantitative nature. The aim of our study was to evaluate the usefulness of real-time RT-PCR on formalin-fixed paraffin-embedded (FFPE) tissue blocks for the diagnosis of germ cell tumors of the central nervous system. We selected eight markers of germ cell tumors using a literature search, and validated them using real-time RT-PCR. Among them, POU5F1, NANOG and TGFB2 were statistically significant (P=0.05) in multiple comparisons (MANOVA) of three groups (pure germinomas, mature teratomas and malignant germ cell tumors). Two-group (pure germinomas and NGGCTs) discriminant analysis achieved a 70.0% success rate in cross-validation. We concluded that real-time RT-PCR using FFPE tissue has adequate validating power comparable to IHC in the diagnosis of central nervous system germ cell tumors; therefore, when IHC is not available, not conclusive or not informative, RT-PCR is a potential alternative to a repeat biopsy.

Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

OBJECTIVE

Gonocytes differentiate into spermatogonial stem cells, which make it possible to maintain spermatogenesis continuously throughout life. We previously reported attenuated spermatogonial stem cell activity in cryptorchid testes, which resulted in altered spermatogenesis and affected fertility. However, few groups have examined the differentiation process from gonocytes to spermatogonial stem cells. To clarify the underlying mechanisms comprehensively we performed microarray analysis to assess differential expression of transcripts between normal and undescended testes in juvenile rats.

METHODS

Using microarray analysis we compared whole mRNA expression of normal and cryptorchid testes in a rat model. We subsequently validated differential expression of candidate genes by real-time reverse transcriptase-polymerase chain reaction and performed immunohistochemistry. We also investigated the methylation status of histone H3K4 in cryptorchid testes and the GC-1 spermatogonial cell line.

RESULTS

We detected 24 up-regulated and 39 down-regulated genes. Of these genes Kdm5a expression was significantly higher in undescended testes. Immunohistochemistry showed that Kdm5a was localized in the nuclei of gonocytes, spermatogonia and spermatocytes. H3K4me2/me3 expression levels were decreased in undescended testes at 9 days postpartum. Furthermore, Kdm5a over expression in GC-1 cells led to increased expression of Esr2, Neurog3, Pou5f1, Ret and Thy1.

CONCLUSIONS

Recent investigations revealed that not only genetic but also epigenetic regulation has a role in spermatogenesis. Kdm5a is likely involved in the transformation of gonocytes into spermatogonial stem cells by transcriptional regulation of specific genes via H3K4 histone modification. To our knowledge this is the first report of epigenetic analysis of germ cell differentiation during early spermatogenesis.