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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.

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., ウシ精巣に由来する多能性生殖幹細胞株の樹立 --精子になるはずのものが多能性細胞に--. 京都大学プレスリリース. 2018-03-08.

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.

Background and aims: Panax ginseng C.A. Meyer is a medicinal herb widely used in Asian countries. Many of its pharmacological actions are attributed to ginsenosides (saponin). However, the pharmacological effects or functions of ginsenosides on mammalian spermatogenesis are unclear.

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.

[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., First online: 19 August 2014

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

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

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.

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.

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.

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.

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.

Plasma membranes can be reversibly permeabilized by Streptolysin O. The permeabilized cells can be reprogrammed and partially dedifferentiated in the cell-free system from egg extracts. However, the permeabilizing activity of Streptolysin O is not stable, and therefore it is difficult to control its activity. An alternative method for reversible permeabilization is useful for establishing a cell-free system. Here, we used a nonionic detergent, digitonin, for permeabilization. A low concentration of digitonin induced reversible permeabilization of the plasma membrane in bovine, mouse, and porcine somatic cells. The permeabilized cells were treated with Xenopus laevis egg extracts. The treated cells showed exchange of nuclear proteins from extracts such as incorporation of Xenopus-specific histone B4 and Lamin LIII into nuclei. After resealing of the membrane, the cells showed upregulation of OCT4, SOX2, and NANOG expression. Our results suggest that reversible permeabilization with digitonin can be used to induce nuclear reprogramming and to activate pluripotent genes by a cell-free system.

Aim Gonocytes are primitive germ cells in neonatal male testes. Germ cells from the neonatal testes of mice have a self-renewal activity and have pluripotential characteristics in established stem-cell lines. Therefore, these germ cells are reliable sources for the preservation of genetic resources of domestic animals and endangered species. The aim of the present study was to examine the cryopreservation of porcine gonocytes in liquid nitrogen (LN2) from neonatal testes that were freshly collected or stored at 4°C for 24 h.

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.

Background and aims Panax ginseng C.A. Meyer is a medicinal herb widely used in Asian countries. Many of its pharmacological actions are attributed to ginsenosides (saponin). However, the pharmacological effects or functions of ginsenosides on mammalian spermatogenesis are unclear.

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.

The developmental competence of domestic pig oocytes that were transferred to somatic cell nuclei of miniature pig was examined. A co-culture system of oocytes with follicle shells was used for the maturation of domestic pig oocytes in vitro. Co-cultured oocytes progressed to the metaphase II stage of meiosis more quickly and more synchronously than non co-cultured oocytes. Oocytes were enucleated and fused with fibroblast cells of Potbelly miniature pig at 48 h of maturation. The blastocyst formation rate of nuclear transfer (NT) embryos using cocultured oocytes (24%) was significantly higher (p0.05) than that of non-co-cultured oocytes (13%). Cleaved embryos at 48 h after nuclear transfer using co-cultured oocytes were transferred to the oviducts of 14 Göttingen miniature pigs and four Meishan pigs. Estrus of all Göttingens returned at around 20-31 days of pregnancy. Two of the four Meishans became pregnant. Three and two cloned piglets were born after modest number of embryo transfer (15 and 29 embryos transferred), respectively. These results indicated that oocytes co-cultured with follicle shells have a high developmental competence after nuclear transfer and result in full-term development after embryo transfer.

Vitamin A is one of the micronutrients which have been implicated in cattle reproduction. In cattle, ingested vitamin A, mainly as beta-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells.

The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.

EDTA saturated with Ca(2+), Fe(3+) or Cu(2+) can induce parthenogenetic activation of pig oocytes at the germinal vesicle stage, whereas EDTA saturated with Zn(2+), which is unable to chelate Zn(2+), does not, indicating that chelation of Zn(2+) with EDTA saturated with Ca(2+) (Ca-EDTA) in maturing pig oocytes plays a pivotal role in the induction of parthenogenetic activation of oocytes. In the present study, the involvement of Zn(2+) chelation in the induction of parthenogenetic activation of pig oocytes at the germinal vesicle stage was confirmed first by examining the effects of concomitant addition of Zn(2+), Cu(2+) or Ni(2+) at various concentrations together with 1 mmol Ca-EDTA l(-1) to the maturation medium. The titration experiments revealed that the pronuclear formation induced by 1 mmol Ca-EDTA l(-1) was completely inhibited by the addition of > 30 micromol Zn(2+) l(-1) to the medium, but not by the addition of Cu(2+) and Ni(2+) at any concentration examined. Second, bovine and mouse oocytes at the germinal vesicle stage were cultured in medium with or without 1 mmol Ca-EDTA l(-1) for 48 h to examine the effects of Ca-EDTA treatment on these oocytes during maturation culture. Most (70-86%) of the bovine oocytes that underwent germinal vesicle breakdown matured to the MII stage via the MI phase, regardless of whether Ca-EDTA was present for the first 24 h of culture. However, 61% of oocytes that had been cultured with Ca-EDTA for 48 h formed a pronucleus without a second polar body, whereas oocytes cultured in the absence of Ca-EDTA were not observed to form a pronucleus at any time during culture. However, even when mouse oocytes at the germinal vesicle stage were cultured for up to 48 h in maturation medium containing Ca-EDTA, pronuclear formation was not observed. Finally, when bovine oocytes that had been cultured with 1 mmol Ca-EDTA l(-1) for 48 h from the germinal vesicle stage were cultured further in medium without Ca-EDTA that was supplemented with 5% fetal calf serum, only 26% of the oocytes developed to the cleaved stage, and none could develop further.

Changes in the activities of nitric oxide synthase (NOS) during embryonic development, and the distribution of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) isoforms were examined in unfertilized mouse oocytes at the second meiotic metaphase (MII) stage and in fertilized mouse embryos during preimplantation development. In addition, the effects of NOS inhibitors on mouse preimplantation development in vitro were investigated. The activities of NOS in MII oocytes and fertilized embryos during the preimplantation period were determined by NADPH-diaphorase staining. Although NOS activity was detected in unfertilized MII oocytes, the intensity of staining was much weaker than that of fertilized embryos at the one-cell stage. There was a decrease in NOS activity in embryos from the four-cell to the eight-cell stage; however, NOS activity increased again in embryos at the morula stage, particularly in the inner cell population. In the expanded blastocysts, staining was confined to the inner cell mass. Immuno-cytochemical staining showed that eNOS and iNOS were expressed in the cytoplasm of oocytes and embryos during the preimplantation period, and eNOS was also distributed in the nuclei of the embryos. When one-cell embryos were treated with 1 mmol N(omega)-nitro-L-arginine methyl ester (L-NAME) l(-1), their development in vitro was arrested at the two-cell stage. This inhibition of development was overcome by the addition of 1 mmol L-arginine l(-1) to the medium. These observations indicate that nitric oxide plays an important role as a diffusible regulator of cell proliferation and differentiation, especially at the developmental transition from the two-cell to the four-cell stage during preimplantation development of mice.

The present study showed that treatment with a cell membrane-impermeable metal ion chelator, EDTA, of porcine oocytes at the germinal vesicle (GV) stage collected from follicles 2‐6 mm in diameter induced artificial activation followed by formation of a pronucleus (PN). When the oocytes were cultured for 48 h in medium containing 0.1 to 2 mM EDTA disodium salt (Na-EDTA), they were activated to form PN, and the maximum PN formation rate (63%, n 5 68) was achieved in oocytes cultured with 1 mM Na-EDTA. More than 90% of oocytes activated by 1 mM Na-EDTA treatment formed 1 PN without emission of the first and the second polar bodies (PB). This result suggests that EDTA at 1 mM may force the maturing (meiosis I) oocytes to form a PN without chromosome segregation. When oocytes at the GV stage that had been cultured with 1 mM Na-EDTA for 48 h were further cultured in 0.4% BSA-containing NCSU23 medium for 144 h, blastocysts that appeared to be morphologically normal were formed at the rate of 10%, whereas no blastocysts were formed from oocytes that had not been cultured with Na-EDTA. Next we examined the effects of Ca 21 ,Z n 21 ,F e 31 ,o r Cu 21 -saturated EDTA (Ca-EDTA, Zn-EDTA, Fe-EDTA, and Cu-EDTA, respectively), and a Ca 21 -specific chelator, EGTA, at a concentration of 1 mM. The Ca-EDTA, FeEDTA, and Cu-EDTA, but not Zn-EDTA or EGTA, had the ability to activate the oocytes. From these results, it is suggested that extracellular chelation of Zn 21 with EDTA of maturing (meiosis I) porcine oocytes results in parthenogenetic activation of the oocytes, which induces PN formation followed by development to blastocysts. developmental biology, implantation/early development, oocyte development

Midkine (MK) is known to be a member of a new family of heparin-binding growth/differentiation factors, together with pleiotrophin, and to be quite rich in bovine follicular fluid. To examine whether treatment with MK during in vitro maturation (IVM) of bovine granulosa-enclosed oocytes affects their nuclear maturation and postfertilization development to the blastocyst stage, bovine granulosa-enclosed oocytes obtained from slaughterhouse-derived ovaries were cultured for 24 h in IVM medium without (control) or with various concentrations (1-500 ng/ml) of MK, followed by in vitro fertilization (IVF) and culturing. Although the MK treatment during IVM did not affect the rate of nuclear maturation or the postfertilization cleavage of oocytes, MK at > or = 10 ng/ml significantly (P: < 0.05) increased the blastocyst yields per tested and per cleaved oocyte compared with the case of the control. Next, the effects of various glycosaminoglycans (heparin, heparan sulfate, chondroitin sulfate A and C, and hyaluronic acid) preincubated with MK at 50 ng/ml were examined. The enhancing activity of MK was completely suppressed by heparin at 600 ng/ml but not by the other compounds. The effects of MK during IVM were also tested on oocytes freed from granulosa cells (GCs). When the denuded oocytes were cultured in IVM medium, no blastocyst formation after IVF was observed, regardless of MK supplementation. However, coculture of the denuded oocytes with isolated GC pellets enhanced the cleavage rates and the blastocyst yield, and these effects were more pronounced with MK supplementation. These results indicate that the presence of MK during IVM of bovine granulosa-enclosed oocytes can enhance their developmental competence to the blastocyst stage after IVF and suggest that the enhancing effects might be mainly mediated by GCs.

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.

To elucidate the beneficial effects of follicular fluid (FF) added to maturation medium on nuclear maturation and postfertilization development of bovine oocytes in vitro, we attempted to separate largely bovine FF (bFF) collected from small follicles into heparin-binding and -nonbinding fractions (HBF and HNF, respectively) by heparin affinity chromatography. Each fraction was dialyzed (molecular weight cut off ？？1, 000 Da) and lyophilized, then dissolved in a half original bFF volume of modified synthetic oviduct fluid (mSOF). Experiment 1: Cumulus-oocyte-complexes (COCs) were matured in mSOF in the presence or absence (control) of whole bFF (wFF), HBF or HNF at a concentration of 10% (v/v). After in vitro fertilization, oocytes were freed from COCs and then cultured in mSOF with fetal calf serum until Day 8. Nuclear maturation rates were not different among wFF, HBF, HNF and control (69.8, 75.6, 63.6 and 72.9%, respectively). However, HBF favored postfertilization development, especially to the blastocyst stage (27.2%) compared with the control (19.0%), whereas HNF showed inhibitory effects, although wFF had no effect. Experiment 2: To determine whether the effects of HBF and HNF would become more evident by high-dose supplementation, HBF or HNF was added to in vitro maturation (IVM) medium at concentrations of 10, 20 or 40% (v/v). HBF at all concentrations examined resulted in significantly higher (p

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.

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.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.

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, ERRβ, 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.

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.

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.

In most mammals, oocyte maturation is the final process of oogenesis, from the prophase of the first meiosis to the metaphase of the second meiosis, during which the oocyte acquires fertilizable competence as well as post-fertilization development competence. Notably, cytoplasmic maturation is important for oocytes to acquire fertilizable and post-fertilization developmental competence during maturation, and mainly regulated by environmental factors in ovarian antral follicles including follicular fluids and intercellular communication between oocytes and follicle cells such as granulosa and cumulus cells.

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 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.

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.

The mechanism of puromycin aminonucleoside (PAN)-induced nephrosis has not yet been well defined. In the present study, we examined the protective effect of active oxygen scavengers on the PAN-induced injury of cultured rat glomerular epithelial cells (GECs) and the generation of active oxygen species in PAN-treated GECs. When exposed to PAN (greater than or equal to 25 micrograms/ml), cellular damage occurred in a time- and dose-dependent manner as evaluated by both the LDH release and MTT colorimetric assays. Concomitant addition of either the hydrogen peroxide (H2O2) scavenger, catalase, or the iron chelating agent, deferoxamine, to the culture medium caused a striking reduction of cellular injury. This suggested a role for H2O2 and for hydroxyl radicals (OH.) generated via the iron-catalyzed breakdown of H2O2 in PAN nephrosis. Using the scopoletin fluorescence assay, the release of H2O2 into the culture medium by GECs exposed to PAN (greater than or equal to 50 micrograms/ml) was shown to increase dose-dependently (greater than or equal to 57 +/- 11 pmol/4.4 x 10(6) cells per h, P less than 0.01) as compared with control cells (14 +/- 2 pmol/4.4 x 10(6) cells per h). These results strongly suggested that active oxygen species, especially H2O2 and OH., might play an important role in PAN-induced GEC injury in vitro as well as in vivo.

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.

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.

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.