Masayasu Yamada

Excessive concentration of glucose during in vitro maturation impairs the developmental competence of bovine oocytes after in vitro fertilization: relevance to intracellular reactive oxygen species and glutathione contents.

The effect of glucose (0, 1.5, 5.6 or 20.0 mM) in synthetic oviduct fluid supplemented with 20 amino acids (SOFaa) on the developmental competence of bovine oocytes after in vitro fertilization was investigated. Intracellular reactive oxygen species (ROS) and the glutathione content of bovine oocytes matured in SOFaa containing 0–20.0 mM glucose were also examined. When oocytes were matured in SOFaa without glucose, the nuclear maturation rate was lower than that in oocytes matured in glucose‐containing medium. The developmental competence to the blastocyst stage of oocytes matured in 1.5 mM glucose was higher than that of oocytes matured in 20.0 mM glucose. In addition, the intracellular ROS content of oocytes matured in 0, 1.5 or 5.6 mM glucose was lower than that of oocytes matured in 20.0 mM glucose. Furthermore, the intracellular glutathione content of oocytes matured in 0, 1.5 or 5.6 mM glucose was higher than that of oocytes matured in 20.0 mM glucose. These results show that excessive glucose in the medium for oocyte maturation impairs the development of bovine oocytes to the blastocyst stage, possibly due to the increase of ROS and the decrease in the intracellular glutathione content of bovine oocytes. Mol. Reprod. Dev. 56:520–526, 2000.

Low Oxygen Tension During In Vitro Maturation is Beneficial for Supporting the Subsequent Development of Bovine Cumulus-Oocyte Complexes

The effects of carbohydrates on meiotic maturation and ATP content of bovine oocytes under low oxygen tension (5%) were investigated. Furthermore, the developmental competence or intracellular H2O2 contents of the oocytes matured under 5% or 20% O2 was assessed. In vitro maturation of bovine cumulus–oocyte complexes was performed in synthetic oviduct fluid (SOF) containing 20 amino acids and hormones (SOFaa). The proportion of the oocytes that matured to the metaphase II stage in SOFaa containing 1.5 mM glucose, 0.33 mM pyruvate, and 3.3 mM lactate under 5% O2 was dramatically lower than that of oocytes matured under 20% O2 (P < 0.01). Similarly, the ATP content of the oocytes that matured under 5% O2 was much lower than that of oocytes matured under 20% O2 (P < 0.05). Under 5% O2 the proportion of metaphase II oocytes increased with increasing glucose concentration (0–20 mM) in SOFaa without pyruvate or lactate. In addition, the ATP content of oocytes cultured in 20 mM glucose was higher (P < 0.05) than that of oocytes cultured in 1.5 mM glucose. Two glucose metabolites (pyruvate and lactate) and a nonmetabolizable glucose analog (2‐deoxy‐glucose), however, had no noticeable effects on meiotic maturation under 5% O2. These results suggest that ATP production under 5% O2 is not dependent on the TCA cycle. Addition of iodoacetate, a glycolytic inhibitor, to SOFaa containing 20 mM glucose significantly reduced (P < 0.01) the proportion of metaphase II and ATP content. Moreover, the proportion of the development to the blastocyst stage of oocytes matured under 5% O2 was higher (P < 0.05) than that of oocytes matured under 20% O2. H2O2 contents of oocytes matured under 5% O2 was lower (P < 0.05) than that of oocytes matured under 20% O2. The results of the present study demonstrate that glucose plays important roles in supporting the completion of meiotic maturation in bovine cumulus–oocyte complexes under low oxygen tension and that low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine oocytes. Mol. Reprod. Dev. 57:353–360, 2000.

Identification, Isolation, and In Vitro Culture of Porcine Gonocytes

Abstract 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.

Cell-Free Extracts from Mammalian Oocytes Partially Induce Nuclear Reprogramming in Somatic Cells

Abstract 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.

Roles of active oxygen species in glomerular epithelial cell injury in vitro caused by puromycin aminonucleoside

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.

Effects of Midkine During In Vitro Maturation of Bovine Oocytes on Subsequent Developmental Competence

Abstract 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 ≥ 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.

Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis

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.

Reversible membrane permeabilization of mammalian cells treated with digitonin and its use for inducing nuclear reprogramming by Xenopus egg extracts.

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.

Dependence of DNA Synthesis and In Vitro Development of Bovine Nuclear Transfer Embryos on the Stage of the Cell Cycle of Donor Cells and Recipient Cytoplasts

Abstract 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.

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

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