JSAR

Abstracts of the presentations selected for
the 2003 JSAR Outstanding Presentation Award

Protein Tyrosine Phosphatase PTPepsilonM Induces Apoptosis in Porcine Ovarian Granulosa Cells

Mariko Tamura, Yosimi NakagAwa, Hidehisa Shimizu, Noriaki Yamada, Takashi Miyano¹ and Hitoshi Miyazaki

Gene Research Center, University of Tsukuba, Ibaraki 305-8572, Japan; and ¹the Graduate School of Science and Technology and the Faculty of Agriculture, Kobe University, Kobe, Japan

ABSTRACT. Protein tyrosine phosphatases (PTPs) in addition to protein tyrosine kinases (PTKs) play crucial roles in regulating various cellular responses including survival and apoptosis. We found that PTPepsilonM, a receptor-type PTP, is expressed in atretic as well as healthy porcine ovarian follicles, and examined its actions in porcine granulosa cells. Adenovirus-mediated overexpression of the wild type and a substrate-trapping DA mutant in which Asp313 of PTPepsilonM was mutated to Ala induced retraction of the cell body with the extension of processes. Cell adhesion to the substratum subsequently decreased with the induction of apoptosis even in the presence of serum. Both wild-type and mutated PTPepsilonM suppressed Rho GTPase activity, which is responsible for the formation of focal adhesions. The phosphorylation level of the positive and negative regulatory sites Tyr416 and Tyr529, respectively, of c-Src was significantly reduced by the wild type with increased c-Src kinase activity, but enhanced by the DA mutant, and c-Src coimmunoprecipitated with the phosphatase, suggesting that c-Src is a substrate for PTPepsilonM. The phopshorylation state of the residues Tyr576/577 and Tyr925 that were involved in the downstream signal transduction of focal adhesion kinase (FAK), was decreased by both the wild type and the mutant of the phosphatase. In contrast, the phosphorylation state of the autophosphorylation site Tyr397 was unaffected. Moreover, coimmunoprecipitation of FAK with PTPepsilonM suggested that FAK is also a substrate for PTPepsilonM. Together with the fact that c-Src phosphorylates Tyr576/577 and Tyr925 of FAK through binding to phosphorylated Tyr397 of FAK, these data suggest that PTPepsilonM negatively regulates the FAK/c-Src signaling pathway that is essential for cell adhesion, by directly dephosphorylating FAK. Thus, PTPepsilonM appears to induce the detachment of granulosa cells with the subsequent induction of apoptosis by down-regulating Rho GTPase activity, at least in part, through the dephosphorylation of FAK. These findings show that PTPepsilonM plays important roles in controlling follicular functions via actin cytoskeleton rearrangement in granulosa cells.

Tumor Necrosis Factor alpha System in the Bovine Oviduct: A Possible Mechanism for Embryo Transport

Missaka P.B. Wijayagunawardane, Christoph Gabler¹, Gary Killian¹ and Akio Miyamoto

Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine, and ¹J O Almquist Research Center, The Pennsylvania State University, University Park

ABSTRACT. [Objectives] Tumor necrosis factor alpha (TNFalpha) is an important physiological mediator involved in the control of reproductive function. The present study therefore focused the possible involvement of TNFalpha in the regulation of production and secretion of prostaglandin (PG) E2 and F2alpha, which are the predominant contraction-related substances in the cow oviduct. [Methods] In Experiment I, in vitro microdialysis system (MDS) was utilized to explore the role of TNFalpha on the real-time changes in the local release of PG. The lumens of oviductal segments implanted with MDS were infused with TNFalpha (100 ng/ml) for 4Ð8 h during 16 h incubation period. In Experiment II, Effects of TNFalpha and embryo on in vitro production of PG were investigated. Oviductal epithelial cells (OEC) in first passage were incubated for 24 h with TNFalpha (0.1, 1 or 10 ng/ml). To determine the effect of embryo on oviductal PG production, in vitro matured and fertilized 40 embryos at day 3 were co-cultured with OEC monolayer for 4 or 24 h. The levels of PG were measured using EIAs. In Experiment III, expression of mRNA for TNFalpha and its receptors (TNFalpha-R) was evaluated. [Results] Infusion of TNFalpha in MDS stimulated the secretion of PG during the follicular and postovulatory stages, but not during the luteal stage. Administration of TNFalpha significantly increased the production and secretion of both PG by OEC, The presence of embryo resulted in a 2-fold increase in PGE2 secretion by OEC at both 4 h and 24 h after incubation. Moreover, mRNA expression for TNFalpha, TNFalpha-R I and TNFalpha-R II was higher during the follicular and postovulatory stages than the luteal stage. This study provides the first direct evidence for maximum stimulation of PG by TNFalpha and high expression of TNFalpha and its receptors in the cow oviduct during the peri-ovulatory period. Although the origin of TNFalpha in the oviduct remains to be determined, TNFalpha may originate from immune cells present in the oviduct. Cyclic changes in these immune cells number have been reported, that may account for the stage differences in TNFalpha mRNA expression observed. In addition, the present study provide the first direct evidence that the developing embryo is capable of stimulating PGE2 production by OEC. The oviductal stage embryos produce minute quantities of TNFalpha that may act locally to enhance the production of PG in the oviduct and activate oviductal contraction in the micro-environment around the embryo. This finely-controlled local TNFalpha system in the oviduct may act to optimize the release of PG, and thereby may serve to transport the embryo into the uterus at optimal timing.

Generation of Chimeric Sheep with Monkey Hematopoiesis by the in Utero Transplantation of Embryonic Stem Cells

Yoshikazu Nagao¹, Kyoko Sasaki², Yoshihiro Kitano³, Satoshi Hayasi³, Hideaki Hasegawa^1,2, Kiyonori Harii⁴, Keiya Ozawa², Yutaka Hanazono²

¹Faculty of Agriculture, Utsunomiya University, ²Jichi Medical School, ³National Center for Child Health and Development, and ⁴Faculty of Medicine, University of Tokyo

ABSTRACT. Although blood formation from ES cells in vitro has been reported, achieving stable blood engraftment from ES cells in animals is an enormous challenge. The difficulty has been attributable to the developmental immaturity of ES-derived blood cells, which most closely resemble primitive embryonic yolk sac hematopoietic progenitors. Blood cells from ES cells do not engraft in irradiated mice unlike stem cells isolated from adult bone marrow. Here we show a new approach around this obstacle. We differentiated primate (cynomolgus macaque) ES cells in vitro to mesodermal cells, followed by in vivo transplantation into fetal liver microenvironment of sheep, resulting in long-term cynomolgus hematopoietic engraftment.
Cynomolgus ES cells were cultured on murine stromal OP9 cells in the presence of multiple cytokines. Cobblestone-like cells emerged at day 6. These cells likely represent a differentiated population of mesodermal cells. We transplanted the day 6 ES-derivative cells (average 4 x 10⁷ cells) into the liver of fetal sheep at the end of the first trimester (day 55-73, full term 147 days) by directly trans-amniotic injection or ultrasonographic-guided trans-uterus injection method. Continuous pregnancy was examined by ultrasonography at 14 days after transplantation. The presence of fetuses with heart beats were defined as a successful transplantation. Frequency of successful transplantation after trans-amniotic injection was 40% (4/10), and that after ultrasonographic-guided injection was 78% (7/9). The animals were delivered at full term. Cynomolgus CFU assessed by sensitive PCR were detected in bone marrow at a level of 1-2% in all four lambs. The longest follow-up to date was 6 months after birth (9 months after transplantation), and the levels of cynomolgus CFU remained stable. No teratoma was found in the lambs.
These results suggest that long-term hematopoietic engraftment from primate ES cells is possible following in vitro differentiation to mesodermal cells, followed by in vivo transplantation into the fetal liver microenvironment. The mechanism of such directed differentiation of ES cells remains to be elucidated, but this procedure should allow further investigation and development of clinical applications.

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Protein Tyrosine Phosphatase PTPepsilonM Induces Apoptosis in Porcine Ovarian Granulosa Cells
Mariko Tamura, Yosimi NakagAwa, Hidehisa Shimizu, Noriaki Yamada, Takashi Miyano¹ and Hitoshi Miyazaki
Gene Research Center, University of Tsukuba, Ibaraki 305-8572, Japan; and ¹the Graduate School of Science and Technology and the Faculty of Agriculture, Kobe University, Kobe, Japan

	ABSTRACT. Protein tyrosine phosphatases (PTPs) in addition to protein tyrosine kinases (PTKs) play crucial roles in regulating various cellular responses including survival and apoptosis. We found that PTPepsilonM, a receptor-type PTP, is expressed in atretic as well as healthy porcine ovarian follicles, and examined its actions in porcine granulosa cells. Adenovirus-mediated overexpression of the wild type and a substrate-trapping DA mutant in which Asp313 of PTPepsilonM was mutated to Ala induced retraction of the cell body with the extension of processes. Cell adhesion to the substratum subsequently decreased with the induction of apoptosis even in the presence of serum. Both wild-type and mutated PTPepsilonM suppressed Rho GTPase activity, which is responsible for the formation of focal adhesions. The phosphorylation level of the positive and negative regulatory sites Tyr416 and Tyr529, respectively, of c-Src was significantly reduced by the wild type with increased c-Src kinase activity, but enhanced by the DA mutant, and c-Src coimmunoprecipitated with the phosphatase, suggesting that c-Src is a substrate for PTPepsilonM. The phopshorylation state of the residues Tyr576/577 and Tyr925 that were involved in the downstream signal transduction of focal adhesion kinase (FAK), was decreased by both the wild type and the mutant of the phosphatase. In contrast, the phosphorylation state of the autophosphorylation site Tyr397 was unaffected. Moreover, coimmunoprecipitation of FAK with PTPepsilonM suggested that FAK is also a substrate for PTPepsilonM. Together with the fact that c-Src phosphorylates Tyr576/577 and Tyr925 of FAK through binding to phosphorylated Tyr397 of FAK, these data suggest that PTPepsilonM negatively regulates the FAK/c-Src signaling pathway that is essential for cell adhesion, by directly dephosphorylating FAK. Thus, PTPepsilonM appears to induce the detachment of granulosa cells with the subsequent induction of apoptosis by down-regulating Rho GTPase activity, at least in part, through the dephosphorylation of FAK. These findings show that PTPepsilonM plays important roles in controlling follicular functions via actin cytoskeleton rearrangement in granulosa cells.

Tumor Necrosis Factor alpha System in the Bovine Oviduct: A Possible Mechanism for Embryo Transport
Missaka P.B. Wijayagunawardane, Christoph Gabler¹, Gary Killian¹ and Akio Miyamoto
Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine, and ¹J O Almquist Research Center, The Pennsylvania State University, University Park

	ABSTRACT. [Objectives] Tumor necrosis factor alpha (TNFalpha) is an important physiological mediator involved in the control of reproductive function. The present study therefore focused the possible involvement of TNFalpha in the regulation of production and secretion of prostaglandin (PG) E2 and F2alpha, which are the predominant contraction-related substances in the cow oviduct. [Methods] In Experiment I, in vitro microdialysis system (MDS) was utilized to explore the role of TNFalpha on the real-time changes in the local release of PG. The lumens of oviductal segments implanted with MDS were infused with TNFalpha (100 ng/ml) for 4Ð8 h during 16 h incubation period. In Experiment II, Effects of TNFalpha and embryo on in vitro production of PG were investigated. Oviductal epithelial cells (OEC) in first passage were incubated for 24 h with TNFalpha (0.1, 1 or 10 ng/ml). To determine the effect of embryo on oviductal PG production, in vitro matured and fertilized 40 embryos at day 3 were co-cultured with OEC monolayer for 4 or 24 h. The levels of PG were measured using EIAs. In Experiment III, expression of mRNA for TNFalpha and its receptors (TNFalpha-R) was evaluated. [Results] Infusion of TNFalpha in MDS stimulated the secretion of PG during the follicular and postovulatory stages, but not during the luteal stage. Administration of TNFalpha significantly increased the production and secretion of both PG by OEC, The presence of embryo resulted in a 2-fold increase in PGE2 secretion by OEC at both 4 h and 24 h after incubation. Moreover, mRNA expression for TNFalpha, TNFalpha-R I and TNFalpha-R II was higher during the follicular and postovulatory stages than the luteal stage. This study provides the first direct evidence for maximum stimulation of PG by TNFalpha and high expression of TNFalpha and its receptors in the cow oviduct during the peri-ovulatory period. Although the origin of TNFalpha in the oviduct remains to be determined, TNFalpha may originate from immune cells present in the oviduct. Cyclic changes in these immune cells number have been reported, that may account for the stage differences in TNFalpha mRNA expression observed. In addition, the present study provide the first direct evidence that the developing embryo is capable of stimulating PGE2 production by OEC. The oviductal stage embryos produce minute quantities of TNFalpha that may act locally to enhance the production of PG in the oviduct and activate oviductal contraction in the micro-environment around the embryo. This finely-controlled local TNFalpha system in the oviduct may act to optimize the release of PG, and thereby may serve to transport the embryo into the uterus at optimal timing.

Generation of Chimeric Sheep with Monkey Hematopoiesis by the in Utero Transplantation of Embryonic Stem Cells
Yoshikazu Nagao¹, Kyoko Sasaki², Yoshihiro Kitano³, Satoshi Hayasi³, Hideaki Hasegawa^1,2, Kiyonori Harii⁴, Keiya Ozawa², Yutaka Hanazono²
¹Faculty of Agriculture, Utsunomiya University, ²Jichi Medical School, ³National Center for Child Health and Development, and ⁴Faculty of Medicine, University of Tokyo

	ABSTRACT. Although blood formation from ES cells in vitro has been reported, achieving stable blood engraftment from ES cells in animals is an enormous challenge. The difficulty has been attributable to the developmental immaturity of ES-derived blood cells, which most closely resemble primitive embryonic yolk sac hematopoietic progenitors. Blood cells from ES cells do not engraft in irradiated mice unlike stem cells isolated from adult bone marrow. Here we show a new approach around this obstacle. We differentiated primate (cynomolgus macaque) ES cells in vitro to mesodermal cells, followed by in vivo transplantation into fetal liver microenvironment of sheep, resulting in long-term cynomolgus hematopoietic engraftment. Cynomolgus ES cells were cultured on murine stromal OP9 cells in the presence of multiple cytokines. Cobblestone-like cells emerged at day 6. These cells likely represent a differentiated population of mesodermal cells. We transplanted the day 6 ES-derivative cells (average 4 x 10⁷ cells) into the liver of fetal sheep at the end of the first trimester (day 55-73, full term 147 days) by directly trans-amniotic injection or ultrasonographic-guided trans-uterus injection method. Continuous pregnancy was examined by ultrasonography at 14 days after transplantation. The presence of fetuses with heart beats were defined as a successful transplantation. Frequency of successful transplantation after trans-amniotic injection was 40% (4/10), and that after ultrasonographic-guided injection was 78% (7/9). The animals were delivered at full term. Cynomolgus CFU assessed by sensitive PCR were detected in bone marrow at a level of 1-2% in all four lambs. The longest follow-up to date was 6 months after birth (9 months after transplantation), and the levels of cynomolgus CFU remained stable. No teratoma was found in the lambs. These results suggest that long-term hematopoietic engraftment from primate ES cells is possible following in vitro differentiation to mesodermal cells, followed by in vivo transplantation into the fetal liver microenvironment. The mechanism of such directed differentiation of ES cells remains to be elucidated, but this procedure should allow further investigation and development of clinical applications.