Effects of pyruvate and dimethyl-α-ketoglutarate, either alone or in combination, on pre- and post-implantation development of mouse zygotes cultured in vitro.

PURPOSE: Dimethyl α-ketoglutarate (dm-α-KG) promotes in vitro development to blastocysts of C57BL/6J X C3He F1 mouse zygotes cultured in medium lacking pyruvate. Here, we examined the effects of pyruvate and dm-α-KG on in vitro development to blastocysts of ICR mouse zygotes and their post-implantation developmental ability. METHODS: Zygotes were cultured in medium with pyruvate at 0-0.2 mmol/L in the presence or absence of 1 mmol/L dm-α-KG for 96 hours and evaluated for blastocyst formation rates. The resultant blastocysts were non-surgically transferred to surrogates and evaluated for birth rates. RESULTS: In medium lacking pyruvate, zygotes could not develop beyond the two-cell stage, in the presence or absence of dm-α-KG. However, the blastocyst formation rate in medium with 0.01 mmol/L pyruvate (12%) was markedly increased with addition of dm-α-KG (49%). Around 80% of embryos developed to blastocysts in medium with 0.2 mmol/L pyruvate, in the presence or absence of dm-α-KG. Importantly, birth rate was markedly improved by treatment with 0.2 mmol/L pyruvate and dm-αKG (31.0%), compared with those with pyruvate treatment alone (16.3%). CONCLUSIONS: Pyruvate and dm-α-KG synergistically work during in vitro culture to markedly improve the blastocyst formation rate and post-implantation developmental ability of the resultant blastocysts in ICR mice.

Mild hypothermia promotes the viability of in vitro-produced bovine blastocysts and their transcriptional expression of the cold-inducible transcription factor Rbm3 during in vitro culture.

In this study, we evaluated the effects of holding in vitro-produced bovine blastocysts under mild hypothermia (33°C or 35°C), by examining viability and hatching rates of day 7 blastocysts (day 0: in vitro fertilization) cultured for 6 days and transcriptional expression of cold-inducible transcription factors Cirp and Rbm3, implicated in mild hypothermia-induced cellular protection against various types of stress. In the normothermic control (38.5°C), viability of the embryos decreased rapidly after day 10, and most samples were degenerated on day 13. However, mild hypothermia, particularly at 33°C, resulted in maintenance of high embryonic survival rates until day 13 (77.1% on day 13) and significant increases in transcriptional expression of Rbm3 in day 11 embryos compared with those at 38.5°C. Thus, our results suggested that upregulation of Rbm3 may occur in response to mild hypothermia in many bovine embryos, providing insights into the effects of mild hypothermia on embryo quality.

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

Long-term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes.

Undifferentiated spermatogonia eventually differentiate in the testis to produce haploid sperm. Within this cell population, there is a small number of spermatogonial stem cells (SSCs). SSCs are rare cells in the testis, and their cellular characteristics are poorly understood. Establishment of undifferentiated cell line would provide an indispensable tool for studying their biological nature and spermiogenesis/spermatogenesis in vitro. However, there have been few reports on the long-term culture of undifferentiated spermatogonia in species other than rodents. Here, we report the derivation and long-term in vitro culture of undifferentiated spermatogonia cell lines from immature and adult bovine testes. Cell lines from immature testes were maintained in serum-free culture conditions in the presence of glial-cell-line-derived neurotropic factor (GDNF) and bovine leukemia inhibitory factor (bLIF). These cell lines have embryonic stem (ES)-like cell morphology, express pluripotent-stem-cell-specific and germ-cell-specific markers at the protein and mRNA levels, and contributed to the inner cell mass (ICM) of embryos in the blastocyst stage. Meanwhile, cell lines established from adult testes were maintained in low-serum media in the presence of 6-bromoindirubin-3'-oxime (BIO). These cell lines have characteristics resembling those of previously reported male mouse germ cell lines as confirmed by their botryoidally aggregated morphology, as well as the expression of germ-cell-specific markers and pluripotent stem cell markers. These findings could be useful for the development of long-term culture of undifferentiated spermatogonia, which could aid in conservation of species and improvement of livestock production through genome editing technology.

Immobilized pH in culture reveals an optimal condition for somatic cell reprogramming and differentiation of pluripotent stem cells.

Aim: One of the parameters that greatly affects homeostasis in the body is the pH. Regarding reproductive biology, germ cells, such as oocytes or sperm, are exposed to severe changes in pH, resulting in dramatic changes in their characteristics. To date, the effect of the pH has not been investigated regarding the reprogramming of somatic cells and the maintenance and differentiation of pluripotent stem cells. Methods: In order to investigate the effects of the pH on cell culture, the methods to produce induced pluripotent stem cells (iPSCs) and to differentiate embryonic stem cells (ESCs) into mesendoderm and neuroectoderm were performed at each medium pH from 6.6 to 7.8. Using the cells of the Oct4-GFP (green fluorescent protein) carrying mouse, the effects of pH changes were examined on the timing and colony formation at cell reprogramming and on the cell morphology and direction of the differentiation of the ESCs. Results: The colony formation rate and timing of the reprogramming of the somatic cells varied depending on the pH of the culture medium. In addition, mesendodermal differentiation of the mouse ESCs was enhanced at the high pH level of 7.8. Conclusion: These results suggest that the pH in the culture medium is one of the key factors in the induction of the reprogramming of somatic cells and in the differentiation of pluripotent stem cells.

Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules.

Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.

Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors.

Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3/4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERRß, and IFN-τ) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy-related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle.

Factors supporting long-term culture of bovine male germ cells.

Spermatogonial stem cells (SSCs) are unipotent in nature, but mouse SSCs acquire pluripotency under the appropriate culture conditions. Although culture systems are available for rodent and human germ-cell lines, no proven culture system is yet available for livestock species. Here, we examined growth factors, matrix substrates and serum-free supplements to develop a defined system for culturing primitive germ cells (gonocytes) from neonatal bovine testis. Poly-L-lysine was a suitable substrate for selective inhibition of the growth of somatic cells and made it possible to maintain a higher gonocyte:somatic cell ratio than those maintained with gelatin, collagen or Dolichos biflorus agglutinin (DBA) substrates. Among the serum-free supplements tested in our culture medium, knockout serum replacement (KSR) supported the proliferation and survival of gonocytes better than the supplements B-27 and StemPro-SFM after sequential passages of colonies. Under our optimised culture conditions consisting of 15% KSR supplement on poly-L-lysine-coated dishes, the stem-cell and germ-cell potentials of the cultured gonocytes were maintained with normal karyotype for more than 2 months (over 13 passages). The proposed culture system, which can maintain a population of proliferating bovine germ stem cells, could be useful for studying SSC biology and germline modifications in livestock animals.

Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors.

Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naïve-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRRß, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naïve-type biPSCs showed the hallmarks of naïve mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naïve-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naïve mouse PSCs and a demonstrated potential to contribute to chimeras.

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos.

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.

The role of signaling pathways on proliferation and self-renewal of cultured bovine primitive germ cells.

Purpose: Gonocytes are primitive male germ cells residing in the neonatal testes and are unipotent in nature, but also have pluripotent stem cell ability in mice under appropriate culture conditions. This study was performed to elucidate the molecular mechanisms of self-renewal and survival of cultured bovine gonocytes. Methods: Gonocytes were isolated from neonatal bull calves and were cultured in DMEM/F12 supplemented with 15 % knock-out serum replacement (KSR) and glial cell-derived neurotrophic factor (GDNF). Cells were analyzed six days after culturing for cell-signaling molecular markers. Results: Colony formation was observed 3-4 days after being cultured. Addition of GDNF enhanced mitogen-activated protein kinase 1/2 (MAPK1/2) phosphorylation and activated the MAPK signaling pathway. Inhibition of MAPK signaling reduced cell proliferation and abolished colony formation. However, inhibition of phosphoinositide 3-kinase-AKT (PI3K-AKT) signaling, a dominant pathway for self-renewal of mouse germ cells, did not show any effects on cultured bovine gonocytes. Expression of cell cycle-related regulators cyclin D2 and cyclin-dependent kinase 2 (CDK2) was downregulated with inhibition of MAPK signaling. Conclusions: These results indicate activation of MAPK plays a critical role in self-renewal and survival of bovine gonocytes via cyclin D1 and CDK2.

Effects of extracellular matrices and lectin Dolichos biflorus agglutinin on cell adhesion and self-renewal of bovine gonocytes cultured in vitro.

Surface molecules of primitive male germ cells, gonocytes, are essential components for regulating cell adhesion and maintaining self-renewal in mammalian species. In domestic animals, the stage-specific glycan epitope α-N-acetylgalactosamine (GalNAc) is recognised by the lectin Dolichos biflorus agglutinin (DBA) and is found on the surface of gonocytes and spermatogonia. Gonocytes from bovine testis formed mouse embryonic stem-like cell colonies on plates that had been coated with DBA or extracellular matrix (ECM) components, such as gelatin (GN), laminin (LN) and poly-L-lysine (PLL). The number of colonies on the DBA-coated plate was significantly higher than that on the GN-, LN- and PLL-coated plates. Pretreating gonocytes with DBA to neutralise the terminal GalNAc residues strongly suppressed colony formation. Furthermore, expression of a germ cell-specific gene and pluripotency-related transcription factors was increased considerably on the DBA-coated plates. These results suggest that the GalNAc residues on gonocytes can recognise precoated DBA on plates and the resulting GalNAc-DBA complexes support germ cell and stem cell potentials of gonocytes in vitro. These glycan complexes, through the GalNAc epitope, may provide a suitable microenvironment for the adhesion and cell proliferation of gonocytes in culture.

Midkine and cytoplasmic maturation of mammalian oocytes in the context of ovarian follicle physiology.

UNLABELLED: Midkine (MK) was originally characterized as a member of a distinct family of neurotrophic factors functioning in the CNS. However, it was later discovered that MK is abundantly expressed in ovarian follicles. Since then, the physiological roles of this molecule in the ovary have been steadily investigated. During the in vitro maturation (IVM) of oocytes MK was shown to promote the cytoplasmic maturation of oocytes, as indicated by post-fertilization development. This effect of MK could be mediated via its pro-survival (anti-apoptotic) effects on the cumulus-granulosa cells that surround oocytes. The oocyte competence-promoting effects of MK are discussed in the context of the recently discovered involvement of MK in the full maturation of ovarian follicles. MK was at the frontline of a new paradigm for neurotrophic factors as oocytetrophic factors. MK may promote the developmental competence of oocytes via common signalling molecules with the other neurotrophic factor(s). Alternatively or concomitantly, MK may also interact with various transmembrane molecules on cumulus-granulosa cells, which are important for ovarian follicle growth, dominance and differentiation, and act as a unique pro-survival factor in ovarian follicles, such that MK promotes oocyte competence. MK, along with other ovarian neurotrophic factors, may contribute to the optimization of the IVM system. LINKED ARTICLES: This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4.

Valproic acid treatment from the 4-cell stage improves Oct4 expression and nuclear distribution of histone H3K27me3 in mouse cloned blastocysts.

We examined effects of treatment with valproic acid (0, 0.2, 1 or 2 mM, VPA), an inhibitor of class I and IIa histone deacetylases (HDACs), of mouse somatic cell nuclear transfer (SCNT) embryos for 24 h from 48 h (4-cell stage), 24 h (2-cell stage) or immediately after oocyte activation on blastocyst formation rates and qualities of the resultant blastocysts. Blastocyst formation rates (33.4-37.0%) were not improved by VPA treatments compared with the untreated control (35.1-36.4%). However, immunofluorescence staining revealed that Oct4 expression levels, evaluated from percentages of embryos expressing Oct4 strongly and having more than 10 Oct4-positive cells, in blastocysts from SCNT embryos treated with 1 mM VPA for 24 h from the 4-cell stage (VPA-4C) were highest among all the groups and that the proportion of cells with a normal nuclear distribution of histone H3 trimethylated at lysine 27 (H3K27me3), a marker of the state of X-chromosome inactivation, significantly increased in the VPA-4C group (36.6%) compared with the control group (12.4%, P<0.05). Treatments with scriptaid and sodium butyrate, inhibitors of class I and IIa/b HDACs, for 24 h from the 4-cell stage also had beneficial effects on SCNT blastocysts. These findings indicate that treatment with 1 mM VPA from the 4-cell stage improves the Oct4 expression and nuclear distribution of H3K27me3 in mouse SCNT blastocysts and suggest that the inhibition of class I and IIa HDACs from the 4-cell stage plays an important role in these effects.

Simple and efficient method for generation of induced pluripotent stem cells using piggyBac transposition of doxycycline-inducible factors and an EOS reporter system.

PiggyBac (PB) transposition of reprogramming factors (Oct3/4 (O), Sox2 (S), Klf4 (K) and c-Myc) is a safe, nonviral method for generating induced pluripotent stem cells (iPSCs). However, compared with retroviral methods, the reprogramming efficiency of the PB-mediated methods is relatively low. In this study, we describe a simple and efficient system for generating high-quality iPSCs by a single transfection of multiple plasmids that does not require the use of a virus, special instruments or skilled techniques. To improve reprogramming efficiency, we modified the components of the polycistronic 2A vectors used in this study and also investigated the combination of another reprogramming-related factor (L-Myc). By simultaneous transposition of multiple PB vectors containing an EOS (early transposon promoter and Oct3/4 and Sox2 enhancers) reporter and modified polycistronic doxycycline (Dox)-inducible factors, we reprogrammed mouse somatic cells with an efficiency higher than is usually obtained with retroviral methods and we established some iPSC lines that contributed highly to chimeras. By using the Dox-inducible system, we also showed that the appropriate elimination of exogenous-factor expression at appropriate time accelerated the induction of Oct3/4 when a combination of OKS and c-Myc vectors were used.

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos.

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.

Characterization and in vitro culture of male germ cells from developing bovine testis.

The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. In this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stem cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stem cell potential in vitro.

Structural and functional changes linked to, and factors promoting, cytoplasmic maturation in mammalian oocytes.

In most mammals, oocyte maturation is the final process of oogenesis, from the prophase of the first meiosis (germinal vesicle stage) to the metaphase of the second meiosis (MII), during which the oocyte acquires fertilizable competence as well as post-fertilization development competence. The nuclear and cytoplasmic maturation processes occur in synchrony but independently. Cytoplasmic maturation entails biochemical and structural changes in the cytoplasm, which give rise to oocytes capable of being fertilized and developing into embryos. Herein we review the literature and results from our own experiments on the structural and molecular events regulating cytoplasmic maturation in oocytes, concentrating on (1) the appropriate reorganization of active mitochondria and the endoplasmic reticulum, a structural and functional feature of cytoplasmic maturation, and (2) factors involved in regulatory mechanisms such as cumulus cell-oocyte gap junctional signaling, cumulus cell-oocyte bidirectional paracrine signaling, and the complex interactions of these signaling processes and follicular fluid constituents in the follicle environment.

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo.

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (-/-) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (-/-) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.

Multipotential ability of primitive germ cells from neonatal pig testis cultured in vitro.

Gonocytes are progenitor-type germ cells that arise from primordial germ cells and differentiate further into spermatogonia, thereby initiating spermatogenesis. In the present study, freshly isolated gonocytes were found to have either weak or no expression of pluripotency determining transcription factors, such as POU5F1, SOX2 and C-MYC. Interestingly, the expression of these transcription factors, as well as other vital transcription factors, such as NANOG, KLF4 and DAZL, were markedly upregulated in cultured cells. Cells in primary cultures expressed specific germ cell and pluripotency markers, such as lectin Dolichos biflorus agglutinin (DBA), KIT, ZBTB16, stage-specific embryonic antigen (SSEA-1), NANOG and POU5F1. Using a monoclonal antibody to specifically identify porcine germ cells, the stem cell potential of fresh and cultured cells was determined with a testis xenotransplantation assay. Colonised porcine germ cells were detected only in mouse testes that were either transplanted with fresh testicular cells or with cells from primary cultures. Interestingly, testes transplanted with cells from primary cultures showed colonisation of germ cells in the interstitial space, reflecting their tumourigenic nature. The formation of teratomas with tissues originating from the three germinal layers following the subcutaneous injection of cells into nude mice from primary cultures confirmed their multipotency. The results of the present study may provide useful information for the establishment of multipotent germ stem cell lines from neonatal pig testis.