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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.

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

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.

Nuclear transfer has been regarded as the only reliable tool for studying nuclear reprogramming of mammalian somatic cells by oocytes. However, nuclear transfer is not well suited for biochemical analyses of the molecular mechanisms of reprogramming. A cell-free system from oocytes is an attractive alternative way to mimic reprogramming in vitro, since a large number of cells can be treated and analyzed. Nevertheless, a cell-free system using oocytes has not been developed in mammals. Here, cell extracts from porcine oocytes were prepared and their ability to induce nuclear reprogramming was evaluated. Extracts from metaphase II (MII) oocytes erased the machinery for regulating gene expression in reversibly permeabilized somatic cells. For example, the extracts caused histone deacetylation and the disappearance of TATA box-binding protein from the nuclei. However, MII-extract-treated cells did not show any obvious changes after cell culture. In contrast, extracts from germinal vesicle (GV) oocytes activated pluripotent marker genes, especially NANOG, and induced partial dedifferentiation after cell culture. The activation of pluripotent marker genes by GV extracts was associated with histone acetylation that was induced during extract treatment. These results indicate that GV- and MII-oocyte extracts have different roles on nuclear reprogramming. Furthermore, both oocyte extracts induced site-specific demethylation in the upstream region of NANOG. These results indicate that cell-free extracts derived from GV- and MII-oocytes could be useful for studying the mechanisms involved in nuclear reprogramming.

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P

It is known that differentiated cells can be reprogrammed to an undifferentiated state in oocyte cytoplasm after nuclear transfer. Recently, some reports suggested that Xenopus egg extracts have the ability to reprogram mammalian somatic cells. Reprogramming events of mammalian cells after Xenopus egg extract treatment and after cell culture of extract-treated cells have not been elucidated. In this experiment, we examined reprogramming events in reversibly permeabilized or nonpermeabilized porcine fibroblast cells after Xenopus egg extract treatment. The Xenopus egg-specific histone B4 was assembled on porcine chromatin and nuclear lamin LIII was incorporated into nuclei. Deacetylation of histone H3 at lysine 9 in extract-treated cells was detected in nonpermeabilized cells, suggesting that a part of reprogramming may be induced even in nonpermeabilized cells. Following culture of extract-treated cells, the cells began to express the pluripotent marker genes such as POU5F1 (OCT4) and SOX2 and to form colonies. Reactivation of the OCT4 gene in extract-treated cells was also confirmed in bovine fibroblasts transformed with an OCT4-EGFP construct. These results suggest that nuclei of mammalian cells can be partially reprogrammed to an embryonic state by Xenopus egg extracts and the remodeled cells partly dedifferentiate after cell culture. A system using egg extracts may be useful for understanding the mechanisms and processes of dedifferentiation and reprogramming of mammalian somatic cells after nuclear transfer. Mol. Reprod. Dev. 74: 1268–1277, 2007. © 2007 Wiley-Liss, Inc.

Spermatogonial stem cells (SSC) self-renew and differentiate into spermatocytes to produce haploid sperm. Because SSC are a small population of adult stem cells in the testis, numerous studies have been reported to derive cell lines from cultured SSC. It has been reported that neonatal and adult mouse SSC can be cultured in vitro over the long term. Male germline stem (GS) cells, embryonic stem (ES)-like cells, and multipotent male germline stem (MGS) cells were derivated from mouse SSC. However, in domestic species including cattle, information about in vitro culture of SSC is mainly available in the neonatal and immature animal. To our knowledge, there are no reports about long-term culture of SSC isolated from adult bovine testis. In this report, we established culture conditions to maintain SSC isolated from adult and immature testes. The SSC were isolated by 3-step enzymatic digestion and enriched by Percoll gradient centrifugation. For adult testicular cell suspensions, SSC were further enriched by differential plating on precoated gelatin dish. After Percoll gradient centrifugation, we found differential expression of SSC markers (GFRα-1 and UCHL-1) in the isolated cells from immature and adult testis. The RT-PCR results also confirmed the expression of differentiated spermatogonia markers (SYCP3 and STRA-8) in adult testicular cell suspensions. It suggests that isolated testicular germ cell population from adult testis are more heterogeneous than those of immature testis. The SSC isolated from adult testes were cultured in low-serum media containing 6-bromoindirubin-3′-oxime (BIO), an inhibitor of glycogen synthase kinase-3α (GSK3), and subsequently the cultures were maintained in the medium containing glial cell line-derived neurotropic factor (GDNF). The cell lines have characteristics resembling mouse GS cell lines as confirmed by their grape-like shape morphology, the expression of SSC markers (UCHL-1, DBA, and GFRa-1), and pluripotent stem cell markers (POU5F1, SOX2, KLF4). The SSC from immature testes were proliferated for more than 3 months in serum-free culture conditions in the presence of GDNF and bovine leukemia inhibitory factor (LIF). The cell lines had ES-like cell morphology, expressed pluripotent stem cell markers and SSC-specific markers. They differentiated in vitro into 3 germ layers confirmed by the expression of ectoderm (NESTIN), mesoderm (BMP4), and endoderm (GATA-6) markers by RT-PCR and neuron like-cells confirmed by the expression of glial fibrillary acidic protein (GFAP) by immunofluorescence analysis. In conclusion, these findings indicate an efficient method to enrich SSC without cell sorting method and different long-term culture systems subsequently established to maintain SSC from adult and immature testes. Furthermore, our data would be useful for further studies that aim to preserve endangered species and improve livestock production through genome editing technology.

In the present study, we cloned bovine midkine (bMK) cDNA by RT- and RACE-PCR, and determined its nucleotide sequence. The nucleotide and deduced amino acid sequences of bMK showed a high degree of homology to those of mouse and human MK. Moreover, a large amount of recombinant bMK (rbMK) was produced using a baculovirus expression system and the protein was purified to homogeneity by heparin affinity chromatography. To examine whether treatment with rbMK during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes affects their nuclear maturation and postfertilization development to the blastocyst stage, bovine cumulus-enclosed oocytes obtained from slaughterhouse-derived ovaries were cultured for 24 hr in IVM medium without (control) or with various concentrations (50-400 ng/ml) of rbMK, followed by in vitro fertilization (IVF) and culture. Although rbMK treatment during IVM did not affect the rates of nuclear maturation and postfertilization cleavage of oocytes, rbMK at all experimental concentrations significantly (P < 0.05) increased the blastocyst yields per tested and per cleaved oocyte compared to the control. Next, it was examined whether heparitinase (HTN) treatment of cumulus-enclosed oocytes would affect the enhancing activity of rbMK during IVM on the developmental competence of oocyte after IVF. The enhancing activity of rbMK was completely suppressed by HTN (1.0 mU/ml) treatment. These results indicate that the presence of rbMK during IVM of bovine cumulus-enclosed oocytes can enhance their developmental competence to the blastocyst stage after IVF and suggest that heparan sulfate chains on the cell surface of cumulus cells and/or oocytes are required for bMK to exert its effect.

We have cloned a novel 100-kDa mammalian protein, which was recognized by an anti-peptide antibody against an epitope-containing nuclear localization signal of NF-κB p65 subunit. Predicted amino acid sequence of the protein is similar to those of yeast splicing factors, Prp1p/Zer1p of Schizosaccharomyces pombe and Prp6p of Saccharomyces cerevisiae . Among these proteins, tetratrico peptide repeat (TPR) motif, which mediates protein–protein interactions, is conserved, whereas leucine zipper motif is found only in the 100-kDa protein. Indirect immunofluorescent staining showed that the 100-kDa protein localized in the nucleus in HeLa cells.

We found that there are at least five subclasses of N-acetylglucosaminyltransferase I (GnT-I; EC 2.4.1.101) mRNA with different 5′-untranslated regions in rat brain. These five subclasses were also expressed in many tissues with distinct tissue-specific patterns. Moreover, they were regulated differently in response to acute-phase inflammation. The expression of the most abundant subclass of GnT-I mRNA in rat liver decreased 2.5-fold in response to inflammation, concomitantly with a significant decrease in the total amount of GnT-I mRNA. In contrast, one of the minor subclasses of GnT-I mRNA was induced 10-fold by inflammation.

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.

Gonocytes are primitive germ cells that are present in the neonatal testis and are committed to male germline development. Gonocytes differentiate to spermatogonia, which establish and maintain spermatogenesis in the postnatal testis. However, it is unknown whether large animal species have pluripotency-specific proteins in the testis. Nanog and Pou5f1 (Oct3/4) have been identified as transcription factors essential for maintaining pluripotency of embryonic stem cells in mice. Here, we show that NANOG protein was expressed in the germ cells of neonatal pig testes, but was progressively lost with age. NANOG was expressed in most of the lectin Dolichos biflorus agglutinin- and ZBTB16-positive gonocytes, which are known gonocyte-specific markers in pigs. NANOG was also expressed in Sertoli and interstitial cells of neonatal testes. Interestingly, POU5F1 expression was not detected at either the transcript or the protein level in neonatal pig testis. In the prepubertal testis, NANOG and POU5F1 proteins were primarily detected in differentiated germ cells, such as spermatocytes and spermatids, and rarely in undifferentiated spermatogonia. By using a testis transplantation assay, we found that germ cells from 2- to 4-day-old pigs could colonize and proliferate in the testes of the recipient mice, suggesting that primitive germ cells from neonatal pig testes have stem cell potential.

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.

We have previously found toxic effects of iron chelate, Fe-NTA on cultured normal rat liver epithelial cells (RL34). In the present study, when RL34 cells were exposed to 50 micrograms/ml iron of Fe-NTA for 15 days, besides the expected cytolytic effects in most cells, the appearance of resistant cells was observed. The resistant cells showed drastic morphological transformation, grew in soft agar, and induced hepatocellular carcinomas when transplanted into syngeneic newborn rats in a short period of time. Since DNA instability in the transformed cells was ascertained by differential AO staining, it is suggested that DNA damage by Fe-NTA is of a critical importance for extremely rapid neoplastic transformation of normal epithelial cells.

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, ESRRβ, 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., ウシ体細胞から全能性を有するiPS細胞株の樹立. 京都大学プレスリリース. 2015-08-20.

Structural proteins of simian virus 40 (SV40), Vp2 and Vp3 (Vp2/3) and Vp1, carry individual nuclear targeting signals, Vp3(198-206) (Vp2(316-324) and Vp1(1-8), respectively, which are encoded in different reading frames of an overlapping region of the genome. How signals coordinate nuclear targeting during virion morphogenesis was examined by using SV40 variants in which there is only one structural gene for Vp1 or Vp2/3, nuclear targeting-defective mutants thereof, Vp2/3(202T) and Vp1 delta N5, or nonoverlapping SV40 variants in which the genes for Vp1 and Vp2/3 are separated, and mutant derivatives of the gene carrying either one or both mutations. Nuclear targeting was assessed immunocytochemically following nuclear microinjection of the variant DNAs. When Vp2/3 and Vp1 mutants with defects in the nuclear targeting signals were expressed individually, the mutant proteins localized mostly to the cytoplasm. However, when mutant Vp2/3(202T) was coexpressed in the same cell along with wild-type Vp1, the mutant protein was effectively targeted to the nucleus. Likewise, the Vp1 delta N5 mutant protein was transported into the nucleus when wild-type Vp2/3 was expressed in the same cells. These results suggest that while Vp1 and Vp2/3 have independent nuclear targeting signals, additional signals, such as those defining protein-protein interactions, play a concerted role in nuclear localization along with the nuclear targeting signals of the individual proteins.

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.

How the DNA tumor virus, simian virus 40, reaches the nucleus is unknown. In this report we have tested the affinity of simian virus 40 toward the nucleus by microinjecting virion particles into the cytoplasm under conditions in which cell-surface-mediated viral infection was blocked. Subcellular localization of viral structural proteins Vp1, Vp2, and Vp3, large tumor antigen, and virion particles was followed immunocytochemically and ultrastructurally. Both virion particles and viral structural proteins localized in the nucleus within 1-2 hr after cytoplasmic injection and subsequently expressed large tumor antigen, which was detected in the nucleus as early as 3 hr after cytoplasmic injection. Vp1 and large tumor antigen nuclear accumulation, as well as virion nuclear entry, were blocked by wheat germ agglutinin and an anti-nucleoporin monoclonal antibody, mAb 414. Virion particles were visualized in the vicinity of nuclear pores and in the cytoplasm with this agent. We conclude that virion particles are karyophilic and enter through nuclear pores. This study suggests that virion structural proteins facilitate virion import into the nucleus and viral gene expression.

Gonocytes are primitive germ cells that reside in neonatal testis and are believed to be progenitor-type stem cells that differentiate into spermatogonial stem cells. Because of their self-renewal ability, gonocytes may be one of the targets for cryopreservation of genetic resources in domestic animals and in endangered species. However, there are only a few reports regarding the preservation of gonocytes and spermatogonial stem cells isolated from the testis. In this experiment, porcine gonocytes were used as a model for preservation of genetic resources. Porcine testes were collected at 2–6 days after birth. They were divided into the 5 experimental groups for storage: (1) DMEM/F12 medium, (2) DMEM/F12 + 15 mm HEPES, (3) PBS, (4) PBS + 15 mm HEPES, and (5) Liquid-Free, and stored at 4�C for 24 h. The testes were minced by scissors and digested with 2-step enzyme treatments. The gonocytes were isolated by Percoll density gradients and recovered from the fraction between 50 and 60%. The viability of cells was assessed using trypan blue dye exclusion. To determine optimum cryopreservation conditions for gonocytes, 10% DMSO, 10% glycerol, and 0.07 mm sucrose were used as cryoprotectants. The isolated gonocytes were suspended in DMEM/F12 + 10% FBS containing cryoprotectant at 4�C, kept at –80�C overnight, and finally immersed in liquid nitrogen. After freezing and thawing of gonocytes, cells were examined for viability and then cultured in DMEM/F12 + 10% FBS in 5% CO2, 95% air at 37�C in humidified atmosphere. Identification of gonocytes was performed using a specific marker of gonocytes, a lectin Dolichos biflorus agglutinin (DBA; Goel et al. 2007 Biol. Reprod. 77, 127–137). The gonocytes were recovered from testes at the purity level of around 70%. Cell viability in average after storage of testes at 4�C was significantly higher in DMEM/F12 + HEPES (95.3%) and PBS + HEPES (89.8%) than in DMEM/F12 (73.9%), PBS (79.7%), and Liquid-Free (72.2%) (P < 0.05; ANOVA). The addition of HEPES in storage medium seemed to be effective for improving cell viability. The use of 10% DMSO and 0.07 mm sucrose as cryoprotectants supported high cell viability (74.4%) of gonocytes after freezing and thawing. The addition of glycerol had an adverse effect on cell viability after freezing (18.3%). When cells were cultured, gonocytes started to form colonies after 3 days and continued to proliferate for at least 7 days in culture. These colonies showed DBA affinity and maintained their nature as gonocytes. The viability of gonocytes can be maintained in the testis at 4�C for at least 24 h and after freezing and thawing. The stored gonocytes successfully proliferated in culture for at least 7 days. In conclusion, these results may provide useful information for short-term storage of primitive germ cells and preservation of genetic resources in domestic animals and in endangered species. It may also have implications for assisted reproductive technology in humans.

Livestock transgenesis is primarily directed towards improving productive traits or for biomedical research. Transgenic pig is of immense interest because it could provide tissues and organs for xenotransplantation to humans. Conventional methods like pronuclear microinjection of zygote and, more recently, somatic cell nuclear transfer (SCNT) have been used for generating genetically modified pigs. These methods are, however, inefficient and have numerous shortcomings. Male germline stem cells have enormous potential, because they could serve as an alternative approach to generate genetically modified pigs, in which the homologous recombination technique could be applied. However, this could not be achieved due to lack of knowledge of culture conditions and markers that can distinguish germ cells from somatic cells, enabling their isolation and identification in culture. Lectin dolichos biflourus agglutinin (DBA), which has specific affinity for ?-d-N-acetyl-galactosamine, reacts with the large round primordial germ cells of the pig genital ridge (Takagi et al. 1997 Mol. Reprod. Dev. 46, 567–580). Immunohistochemical analysis of testicular samples from different age groups revealed that DBA binds to primitive germ cells in neonatal pig testis like gonocytes and pre-spermatogonia. Variable DBA affinity among germ cells and its progressive loss with age suggested that DBA bound strongly to gonocytes, weakly to undifferentiated spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase activity in germ cells from neonatal pig testis further confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were isolated using 2-step enzymatic digestion and discontinuous Percoll density gradient. Approximately 70% gonocytes were present in the fraction 5 (50–60% gradient) that were identified by their DBA affinity. Isolated cells were cultured in DMEM/F12 supplemented with 10 �g mL-1 of insulin, 10 �g mL-1 of apo-transferrin, 100 U mL-1 of penicillin, 50 �g mL-1 of streptomycin, 40 �g mL-1 of gentamycin sulphate, 1 mM pyruvate, 1� non-essential amino acid solution, and 10% fetal bovine serum at 37�C in 5% CO2 in air in a humidified atmosphere. Initially, gonocytes grew as adherent cells that formed foci of flat colonies. These colonies grew in size during the culture period, and DBA-positive cells were observed. Flat colonies transformed to ES-like colonies by 6–7 days of culture, which were still positive for DBA binding. Proliferation of germ cells was evaluated by double immunostaining with DBA and anti-bromodeoxyuridine (BrdU) antibody following BrdU incorporation, suggesting that 53–55% DBA-positive cells were in S phase after 7 days of culture. In conclusion, lectin DBA is a marker for porcine primitive germ cells in neonatal pig testis including gonocytes that could be used for their identification during isolation and in culture. Isolated gonocytes could proliferate in vitro without specific growth factor supplementation.

Revealing an adequate cell state for nuclear reprogramming is essential to achieve efficient production of cloned embryos and animals. Previous reports suggest that nuclei from undifferentiated cells such as blastomeres or embryonic stem cells can support efficient development of cloned embryos to term. In recent years, differentiated somatic cells are frequently used for donor cells because of ease of preparation and application for genetic modification. The efficiency of the somatic cell nuclear transfer (SCNT) is still extremely low. We hypothesized that somatic cells that had been reprogrammed to dedifferentiated states before SCNT might support higher developmental ability of SCNT embryos. To test this hypothesis, porcine fibroblast cells were treated with Xenopus egg extracts, and the extract-treated cells (ETCs) were used as donor cell for SCNT to examine their ability to support early embryonic development. Xenopus egg extracts were prepared from activated S-phase eggs. Porcine fibroblast cells (106/mL) were permeabilized by 500 ng mL-1 of Streptolysin O and were incubated in the egg extracts with the energy-regenerating system for 2 hours at 23�C. After the extract treatment, permeabilized membranes were resealed in DMEM containing 2 mM CaCl2. The ETCs were fused with porcine enucleated oocytes and simultaneously activated. The reconstructed embryos were cultured in PZM-3 medium for 7 days. All statistical differences were analyzed by ANOVA. Reprogramming of ETCs was evaluated on changes of chromatin states and gene expression. Chromatin-binding proteins of ETCs were separated and analyzed on SDS-PAGE. Some proteins were incorporated onto and/or released from chromatins after the extract treatment. Especially, Xenopus egg-specific linker histone B4 was assembled on chromatins. Non-permeabilized control cells did not show these protein exchanges. Deacetylation of histone H3 lysine9 was detected in half number of ETCs in an ATP-dependent manner. In contrast, a high population of histone H3-acetylated cells was observed in buffer-treated cells as well as cells before the extract treatment. The pluripotent marker gene expression, such as OCT4 and SOX2, was also observed in ETCs after culture. The gene expression of these genes was not detected in non-treated cells. These results indicate that the extract treatment induces or triggers a part of dedifferentiation of somatic cells. These ETCs were used as donor cell for SCNT, and reconstructed cloned embryos were cultured. SCNT embryos showed no significant difference in cleavage rates and developmental rates to the blastocyst stage (25%) compared with non-treated control cells (26%). However, the total cell number of embryos at the blastocyst stage was significantly higher in SCNT embryos from ETCs compared with those of control cells (62 � 7 vs. 43 � 2, respectively; P < 0.05). These results indicate that the extract treatment before nuclear transfer may stimulate cell proliferation of SCNT embryos but not improve early development. More studies, however, are needed to investigate their developmental ability to term.

Much evidence indicates that somatic cells can be reprogrammed in an oocyte cytoplasm. The nuclear reprogramming consists of many unknown processes, and mechanisms underlying these processes still remain to be elucidated. Recently some reports noted that Xenopus oocytes or eggs can induce some of the reprogramming events in mammalian cells. We investigated the processes of nuclear reprogramming of porcine fibroblast cells by Xenopus egg extracts to understand how egg extracts trigger the reprogramming and/or dedifferentiation of cells. Unfertilized Xenopus eggs were collected from mature females. After removal of the jelly coat, activation was routinely achieved by calcium ionophore A23187. The eggs were immediately centrifuged and the cytoplasmic fraction was used as egg extracts. Porcine fibroblast cells were permeabilized by streptolysin O and incubated in the egg extracts under the ATP-generating system (1 mM ATP, 5 mM phosphocreatine, and 20 U/mL creatine kinase) for 30 min at 37�C or 2 h at 23�C. The incorporation of Xenopus-specific linker histone B4 into porcine fibroblasts was examined by immunofluorescence and immunobloting analysis. After collection of cells from the extracts, permeabilized membranes of the cells were resealed in culture medium containing 2 mM CaCl2 for 2 h. The cells were then incubated in DMEM with 10% fetal bovine serum (FBS) or porcine zygote medium-3 (PZM-3: Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) containing 5.55 mM glucose and 5% FBS. RNAs were extracted from the cells in each culture dish and Oct-4 expression was examined by RT-PCR analysis every day until Day 8. The primers were designed to span the 99 base-pair intron region of porcine Oct-4 gene for recognizing both spliced and unspliced transcripts. The incorporation of histone B4 from Xenopus egg extracts was observed at the nuclear region of the porcine fibroblasts under both the 37�C and the 23�C conditions. Because the histone B4 incorporation was inhibited by addition of Apyrase, an ATPase, a part of reprogramming might be an ATP-dependent process. When treated cells were incubated in DMEM or PZM-3, Oct-4 expression was detected in the cells cultured in DMEM, but not in PZM-3. However, the transcripts of Oct-4 were mainly obtained in unspliced form at the earlier stage of culture (after Day 1 to Day 4 of culture), suggesting that a part of reprogramming processes by the egg extracts involves induction of dedifferention of cells or activation of a pluripotent marker gene such as Oct-4. Xenopus egg extract may provide a system to investigate the processes involving nuclear reprogramming and the pluripotent state of mammalian cells in vitro.

The cell cycle of donor cells is one of the essential factors for the success of somatic cell nuclear transfer (SCNT), and G0/G1-phase cells have been widely used as donor cells. However, cells synchronized at the G0/G1-phase also have a population of cells with cell cycles other than G0/G1-phase, and we cannot precisely know the cell cycle of donor cells being used for SCNT. In this experiment, we reconstructed SCNT embryos from donor cells that were synchronized at the G1-phase or at the G0/G1-phase and compared their developmental ability in two different culture media. Immature oocytes were isolated from ovaries collected from domestic gilts at a local slaughterhouse and were co-cultured with follicle shells for in vitro maturation (Hoshino et al. 2003 Theriogenology 59, 260). Donor cells were collected from fibroblast cells of miniature Potbelly pigs. Cells synchronized at the G1-phase were prepared shortly after dividing M-phase cells that had been synchronized using 2-methoxyestradiol, as described by Urakawa et al. (2004 Theriogenology 62, 714–728). The G0/G1 cells were also prepared from a fully confluent culture of cells. Donor cells were fused with enucleated oocytes and simultaneously activated by two electric pulses. Reconstructed embryos were cultured in two different media [Whitten and Biggers medium supplemented with 0.5 mg/mL hyaluronic acid sodium salt (WM) and porcine zygote medium-3 (PZM-3, Yoshioka et al. 2002 Biol. Reprod. 66, 112–119)] under 5% CO2 in air. Cleavage rate and development rate to the blastocyst stage were assessed after 48 and 168 hr of culture, respectively. The results are summarized in Table 1. Developmental rate to the blastocyst stage of SCNT embryos reconstructed from G1 cells and cultured in PZM-3 (40%) was significantly higher than that of embryos cultured in WM (25%). The SCNT embryos of the G1 cells showed significantly lower cleavage rate (51%) than that of the G0/G1 cells (69%). However, the developmental rates to the blastocyst stage per cleaved embryo in WM were significantly higher in G1 cells (50%) compared with G0/G1 cells (32%). In addition, the total cell number of the SCNT blastocysts was comparable between the cultures in WM (58 ± 4) and PZM-3 (46 ± 5), although the ratio of inner cell mass cells to the total cell number was significantly higher in PZM-3 (32%) compared with WM (14%). These results suggest that PZM-3 may fit with the culture of SCNT embryos, and that the G1 synchronized cells could be stably reprogrammed for early embryonic development in SCNT embryos and be useful as donor cells for analyzing the processes of nuclear reprogramming. Table 1. Development of nuclear transfer embryos reconstructed from G1 or G0/G1 cells in different culture media

It has been found that inorganic phosphate (P) at concentrations as low as 10 microM markedly inhibits in vitro development of early rat embryos at the 1-cell or 2-cell stage to the blastocyst stage, although P is present at concentrations of 0.37-1.19 mM in oviductal fluid, in which the development of early embryos occurs. We show here that fractions (PTF, 50-250 microg/ml) of rat oviductal extracts (OVEs) passed through a Blue CL6B affinity column have the ability to overcome the inhibitory effects of P on the development of 2-cell rat embryos in a dose-dependent manner, whereas 250 microg/ml OVE or 250 microg/ml of the bound fractions (BF) induced degenerative changes in the embryos at the 2-cell stage. Moreover, PTF at concentrations of >/=100 microg/ml stimulated the hatching of blastocysts both in medium with and without P, although none of the blastocysts in medium without PTF hatched from their zona pellucida.

An epithelial cell line, designated as SGE1, has been established from isolated rat renal glomeruli. SGE1 cells are able to grow continuously in a serum-free medium at similar growth rates to those in the medium containing serum. Quantitative studies of the cells in the serum-free condition demonstrated that SGE1 cells required a collagen type I or IV, fibronectin, or laminin-coated substratum for adhesion and growth, and among them, collagen type I and IV were most effective. Essential medium supplements for the adhesion and growth were epidermal growth factor and transferrin, respectively, and both effects were noticeably enhanced with linoleic acid. Morphological observation found that in a monolayer culture, SGE1 cells formed domes, and in a collagen embedding culture, they formed cystic spheres having features of a simple cuboidal epithelium, polarized formation of microvilli and tight junctions as well as a lateral cell membrane with cytoplasmic projections. In addition, SGE1 cells expressed Fx1A antigens, which are nephritogenic antigens on their microvilli.