In situ assessment of mitochondrial respiratory activity and lipid metabolism of mouse oocytes using resonance Raman spectroscopy.

This study aimed to develop a method to determine the degree of oocyte maturation in metaphase II in situ based on the balance between mitochondrial respiratory activity and lipid metabolism using resonance Raman spectroscopy. A decrease in the respiratory activity of overmatured oocytes was indicated by the reduced intensities of the resonance Raman bands corresponding to reduced cytochrome c in the cytoplasm. Moreover, the increased lipid concentration in overmature oocytes indicated lower lipid metabolism with a decreased mitochondrial function. New indexes were defined in terms of the ratios of the representative Raman peak intensities of reduced cytochrome c (750 and 1127 cm-1) to those of lipids (1438 cm-1 ) and they successfully classify the oocytes into groups based on their quality, which varied with their maturation degree. The high development rate of embryos that were fertilized in vitro after laser irradiation showed that laser irradiation was noninvasive to oocytes. The evaluation of two factors in situ, the active respiration and lipid metabolism, means to catch the most fundamental biochemical reactions of life activities. Our results demonstrate the potential application of resonance Raman spectroscopy as a new, noninvasive, and universal cell evaluation technology, for not only oocytes but also more general cells such as somatic cells and iPS cells.

Phosphoric acid and phosphorylation levels are potential biomarkers indicating developmental competence of matured oocytes.

Here, we aimed to identify biomarkers for mice oocyte maturation in metaphase II in vivo and in situ using Raman spectroscopy. Principal component analysis of 324 Raman data points of oocytes at Phase I, II, III, and IV showed that the phosphoric acid concentration uniformly increased in oocytes with higher developmental competence than in oocytes at other maturation stages, and proteins were more phosphorylated. The maturation phases were successfully predicted by linear discriminant analysis with high accuracy (90.7%) using phosphoric molecular information mentioned above. Furthermore, detections of higher concentration of unsaturated fatty acids in overmatured oocytes indicated that a decline in metabolic activity due to overmaturation induced a surplus of these lipid components. Upon assessing invasiveness by laser irradiation, about 50% irradiated oocytes progressed to morula and blastocyst stages in good conditions. Thus, Raman spectroscopy holds promise in evaluating oocyte maturation and quality based on molecular information in infertility treatment.

Updating the markers for oocyte quality evaluation: intracellular temperature as a new index.

BACKGROUND: The developmental competence of an embryo is principally dictated by the oocyte. Usually, oocyte selection is based on morphological properties; however, all morphological criteria that are currently used for the grading and screening of oocytes are not able to eliminate the subjectivity. Despite recent studies of the molecular factors related to oocyte quality, it is technically difficult to develop an index based on these factors, and new indices that reflect intracellular conditions are necessary. METHODS: Morphological and molecular factors influencing developmental competence were comprehensively reviewed, and intracellular temperature was evaluated as a new marker of oocyte quality. MAIN FINDINGS: The intracellular temperature of mature oocytes was high in fresh oocytes and decreased with time after polar body release. Under the same conditions, the intracellular temperature and its distribution differed among oocytes, suggesting that temperature represents the state of each oocyte. CONCLUSION: Intracellular temperature is advantageous as an objective and quantitative indicator of oocyte quality. Further studies should evaluate the link between temperature and cellular phenomena to establish its use as an indicator of quality.

Expression of Six Proteins Causes Reprogramming of Porcine Fibroblasts Into Induced Pluripotent Stem Cells With Both Active X Chromosomes.

In this study, we created porcine-induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA-Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537-553, 2017. © 2016 Wiley Periodicals, Inc.

Localization and quantitative analysis of Cx43 in porcine oocytes during in vitro maturation.

Many studies of the main gap junction protein, Cx43, have been conducted in porcine oocyte research, but they have been limited to investigations of cumulus-oocyte complexes (COCs). In this study, we verified Cx43 not in COCs, but in porcine oocytes during maturation, and conducted a quantitative time course analysis. The location and dynamics of Cx43 were examined by immunocytochemistry and western blotting, respectively. COCs were cultured in NCSU23 medium and processed for immunocytochemistry and western blotting at 0, 14, 28, and 42 h after denuding. A Cx43 signal was detected on oolemmas, transzonal projections and the surface of zona pellucidae. Western blotting showed that Cx43 band density increased from 0 to 14 h, and gradually decreased thereafter. Our results clarified that Cx43 is localized in the ooplasmic membrane through zona pellucidae and its level changes over time during culture in porcine oocytes.

Comparison of the effects of BPA and BPAF on oocyte spindle assembly and polar body release in mice.

Bisphenol AF (BPAF), a homolog of bisphenol A (BPA), is a widely used environmental chemical that has adverse effects on reproduction. The aim of this study was to analyse the effects of BPA and BPAF exposure on oocyte maturation in vitro. Oocytes were cultured in the presence of BPA or BPAF (2, 20, 50 or 100 µg/ml) for 18 h. At concentrations of 50 and 100 µg/ml, BPA and BPAF inhibited oocyte maturation, with BPAF treatment causing a sharp decrease in the number of oocytes reaching maturity. Oocytes were exposed to BPA or BPAF at 2 µg/ml and cultured for different durations (6, 9, 12, 15 or 18 h). Both BPAF and BPA caused a cell cycle delay under these conditions. Oocytes cultured in the presence of BPA or BPAF (50 µg/ml) for 21 h were tested for the localization of α-tubulin and MAD2 using immunofluorescence. High concentrations of BPAF induced cell cycle arrest through the activation of the spindle assembly checkpoint. After 12 h of culture in BPAF (50 µg/ml), oocytes were transferred to control medium for 9 h. Only 63.3% oocytes treated in this manner progressed to metaphase II (MII). Oocytes exposed to high doses of BPA experienced a cell cycle delay, but managed to progress to MII when the culture period was prolonged. In addition, MAD2 was localized in the cytoplasm of these oocytes. In conclusion, both BPAF and BPA exposure affected oocyte maturation, however BPAF and BPA have differential effects on SAC activity.

Roles of epidermal growth factor (EGF)-like factor in the ovulation process.

Luteinizing hormone (LH) surge stimulates preovulatory follicles to induce the ovulation process, including oocyte maturation, cumulus expansion, and granulosa cell luteinization. The matured oocytes surrounded by an expanded cumulus cell layer are released from follicles to the oviduct. However, LH receptors are dominantly expressed in granulosa cells, but less in cumulus cells and are not expressed in oocytes, indicating that the secondary factors expressed and secreted from LH-stimulated granulosa cells are required for the induction of the ovulation process. Prostaglandin and progesterone are well-known factors that are produced in granulosa cells and then stimulate in both granulosa and cumulus cells. The mutant mice of prostaglandin synthase (Ptgs2KO mice) or progesterone receptor (PRKO mice) revealed that the functions were essential to accomplish the ovulation process, but not to induce the ovulation process. To identify the factors initiating the transfer of the stimuli of LH surge from granulosa cells to cumulus cells, M. Conti's lab and our group performed microarray analysis of granulosa cells and identified the epidermal growth factor (EGF)-like factor, amphiregulin (AREG), epiregulin (EREG), and ß-cellulin (BTC) that act on EGF receptor (EGFR) and then induce the ERK1/2 and Ca2+-PLC pathways in cumulus cells. When each of the pathways was down-regulated using a pharmacological approach or gene targeting study, the induction of cumulus expansion and oocyte maturation were dramatically suppressed, indicating that both pathways are inducers of the ovulation process. However, an in vitro culture study also revealed that the EGFR-induced unphysiological activation of PKC in cumulus cells accelerated oocyte maturation with low cytostatic activity. Thus, the matured oocytes are not arrested at the metaphase II (MII) stage and then spontaneously form pronuclei. The expression of another type of EGF-like factor, neuregulin 1 (NRG1), that does not act on EGFR, but selectively binds to ErbB3 is observed in granulosa cells after the LH surge. NRG1 supports EGFR-induced ERK1/2 phosphorylation, but reduces PKC activity to physiological level in the cumulus cells, which delays the timing of meiotic maturation of oocytes to adjust the timing of ovulation. Thus, both types of EGF-like factor are rapidly induced by LH surge and then stimulate cumulus cells to control ERK1/2 and PKC pathways, which results in the release of matured oocytes with a fertilization competence.

Expression of focal adhesion kinase in mouse cumulus-oocyte complexes, and effect of phosphorylation at Tyr397 on cumulus expansion.

We investigated the expression of focal adhesion kinase (FAK) in mouse cumulus-oocyte complexes (COCs), as well as the role of FAK phosphorylation at Tyr397 during oocyte maturation. The effect of inhibiting FAK phosphorylation at Tyr397 during in vitro maturation (IVM) on subsequent fertilization and preimplantation embryo development was also examined. Western blotting analyses revealed that total and Tyr397-phosphorylated FAK were expressed in vivo in both cumulus cells and oocytes. Immunocytochemical studies localized this kinase throughout the cytoplasm of cumulus cells and oocytes; in particular, Tyr397-phosphorylated FAK tended to accumulate in regions where cumulus cells contact each other. Interestingly, the in vivo level of Tyr397 phosphorylation in cumulus cells was significantly lower after compared to before cumulus expansion. Addition of FAK inhibitor 14, which specifically blocks phosphorylation at Tyr397, stimulated oocyte meiotic maturation and cumulus expansion during IVM in the absence of follicle-stimulating hormone (FSH). Reverse-transcriptase PCR showed that the mRNA expression of hyaluronan synthase 2 (Has2), a marker of cumulus expansion, was significantly induced in cumulus cells. Subsequent in vitro fertilization and culture showed that more oocytes developed to the blastocyst stage when they were treated with FAK inhibitor 14 during IVM, although the blastocyst total cell number was lower than in oocytes stimulated with FSH. These results indicate that FAK is involved in the maturation of COCs; specifically, phosphorylation at Tyr397 may regulate cumulus expansion via the expression of Has2 mRNA in cumulus cells, which could affect the developmental competence of oocytes.

C-type natriuretic peptide inhibits porcine oocyte meiotic resumption.

C-type natriuretic peptide (CNP) is a recently identified meiotic inhibitor in mice. However, it has not been investigated in porcine oocytes to date. This study aimed to demonstrate the inhibitory effect of CNP against germinal vesicle breakdown (GVBD) in porcine oocyte meiotic resumption. Immunohistochemical analysis revealed intense natriuretic peptide receptor 2 (NPR2) immunoreactivity in the oocyte surrounded cumulus cells in the follicles. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) analysis showed the expression of npr2 mRNA only in cumulus cells but not in oocytes, suggesting that cumulus cells are the targets of CNP. When cumulus-oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with various concentrations of CNP (10, 50, 100, 500, and 1,000 nM), inhibitory effect was observed in the COC group, but not in the DO group, confirming that CNP indirectly inhibits GVBD via cumulus cells. This evidence is the first indication that the CNP-NPR2 pathway is involved in meiotic arrest in porcine oocytes. Furthermore, we investigated the effect of oocyte-derived paracrine factor (ODPF) on npr2 mRNA expression level in cumulus cells by evaluating changes in mRNA expression in oocytectomised COCs (OXCs) by real-time PCR. A significant decrease in npr2 mRNA expression level was observed in OXCs, whereas mRNA expression level was restored in OXCs with DOs, indicating that ODPF participates in the regulation of npr2 expression in porcine cumulus cells.

Quantitative analysis in LC3-II protein in vitro maturation of porcine oocyte.

Microtubule-associated protein light chain 3 (LC3)-II is a marker of autophagosome. In this study, LC3-II expression was used to identify autophagy, during the in vitro maturation of porcine oocytes. In a time-course experiment, cumulus-oocyte complexes (COCs) were cultured in NCSU23 medium for 0 h, 14 h, 28 h or 42 h. The cumulus cells were removed and denuded oocytes were processed for western blotting or immunostaining. Western blotting showed that the LC3-II levels changed over time, with maximum levels observed at 14 h and minimum levels at 42 h. Immunostaining of LC3 showed the signals with dot shapes and ring shapes in oocytes at every group that probably represent autophagosomes. To ascertain whether autophagic induction and degradation were occurring, we treated the cultures with autophagic inhibitors. Lysosomal protease inhibitor E64d and pepstatin A increased the LC3-II levels and wortmannin, inhibitor of autophagic induction, decreased the LC3-II levels. Western blotting and immunostaining demonstrated that LC3-II is present in porcine oocytes cultured in vitro. The decreased LC3-II levels after wortmannin treatment suggest that it is newly generated in porcine oocytes, a phenomenon that represents autophagic induction. Furthermore, increased LC3-II levels after E64d and pepstatin A addition imply that LC3-II is degraded by lysosomal proteases, an indication of autophagic degradation. Our results suggest that autophagy, which is a dynamic process whereby autophagosomes are newly generated and subsequently degraded, is probably occurring in porcine oocytes during in vitro maturation.

Distribution of tubulointerstitial nephritis antigen-like 1 and structural matrix proteins in mouse embryos during preimplantation development in vivo and in vitro.

Summary Tubulointerstitial nephritis antigen-like 1 (TINAGL1) is a novel matricellular protein that interacts with structural matrix proteins and promotes cell adhesion and spreading. We have previously reported unique localization of TINAGL1 to the trophectoderm (TE) of mouse blastocysts. TINAGL1 was found to be upregulated in implantation-competent blastocysts after estrogen treatment using progesterone-treated delayed-implantation models. Moreover, colocalization of TINAGL1 and extracellular matrix (ECM) protein laminin 1 was detected in the Reichert membrane on embryonic days 6.5 and 7.5. Although these data suggested a role for TINAGL1 in the embryo development at postimplantation, its relevance to other ECM proteins during preimplantation development is not clear. In this study, we examined the expression of TINAGL1 and its relevance to other ECM proteins fibronectin (FN) and collagen type IV (ColIV) during in vivo development of preimplantation embryos, particularly at blastocyst stage in detail. Localizations of TINAGL1, FN, and ColIV were similar. In 1-cell to 8-cell embryos, they were expressed in cytoplasm of blastomeres, and in morulae they were localized in the outer cells. FN and ColIV were expressed primarily on outer surface of the cells. In blastocysts, FN and ColIV were distributed in the cytoplasm of TE, but, just prior to implantation, they became localized uniquely to the blastocoelic surface of TE. In in vitro fertilized (IVF) blastocysts, expression levels of TINAGL1 and FN were lower than in in vivo blastocysts. These results suggest that, during preimplantation development, TINAGL1 may be involved in roles of structural matrix proteins, whose expression in blastocysts may be affected by in vitro culture.

Effect of single-oocyte culture system on in vitro maturation and developmental competence in mice.

Purpose: To investigate whether single-culture systems influence the quality of in vitro-matured oocytes, we examined the maturation and developmental competence of oocytes obtained by grouped in vitro maturation (IVM) or single IVM. Methods: In vitro-matured oocytes were obtained using the culture drop (CD) method for the grouped IVM experiments, and the CD and hanging drop (HD) method for the single IVM experiments. To evaluate oocyte developmental competence, we performed in vitro fertilization and culture, and counted the number of blastocysts. To evaluate the oocyte cytoplasmic maturation, we measured the maturation promoting factor (MPF) expression levels. Results: Oocytes cultured singly had lower maturity and developmental competence than the grouped IVM oocytes. However, enhanced oocyte fertility and blastocyst quality was achieved by the HD single IVM method. Additionally, the MPF activity level increased in all culture methods, compared to the control; however, it lagged behind nuclear maturation. Conclusions: These results suggest that the HD method is efficient for single IVM.

Immune-mediated necrotizing myopathy with concomitant development of Kikuchi-Fujimoto disease.

Immune-mediated necrotizing myopathy (IMNM) is a distinct type of idiopathic inflammatory myositis, pathologically characterized by myofiber necrosis and degeneration in the absence of lymphocyte infiltration. Herein, we present a case of IMNM with concomitant development of Kikuchi-Fujimoto disease (KFD), characterized by histiocytic necrotizing lymphadenitis, in a 36-year-old woman who had a treatment history for rheumatoid arthritis (RA). Treatment with oral prednisolone and tacrolimus as immunosuppressants resulted in the remission of the skeletomuscular involvement and lymphadenopathy. To the best of our knowledge, this is the first report of IMNM and KFD developing concomitantly during the clinical course of RA.

Distribution and association of mTOR with its cofactors, raptor and rictor, in cumulus cells and oocytes during meiotic maturation in mice.

Mammalian target of rapamycin (mTOR), a Ser/Thr protein kinase, is the catalytic component of two distinct signaling complexes, mTOR-raptor complex (mTORC1) and mTOR-rictor complex (mTORC2). Recently, studies have demonstrated mitosis-specific roles for mTORC1, but the functions and expression dynamics of mTOR complexes during meiotic maturation remain unclear. In the present study, to evaluate the roles of respective mTOR complexes in maternal meiosis and compare them with those in mitosis, we sought to elucidate the spatiotemporal immunolocalization of mTOR, the kinase-active Ser2448- and Ser2481-phosphorylated mTOR, and raptor and rictor during cumulus-cell mitosis and oocyte meiotic maturation in mice. mTOR principally accumulated around the chromosomes and on the spindle. Phosphorylated mTOR (Ser2448 and Ser2481) exhibited elevated fluorescence intensities in the cytoplasm and punctate localization adjacent to the chromosomes, on the spindle poles, and on the midbody during mitotic and meiotic maturation, suggesting functional homology of mTOR between the two cell division systems, despite their mechanistically distinctive spindles. Raptor colocalized with mTOR during both types of cell division, indicating that mTORC1 is predominantly associated with these events. Mitotic rictor uniformly distributed through the cytoplasm, and meiotic rictor localized around the spindle poles of metaphase-I oocytes, suggesting functional divergence of mTORC2 between mitosis and female meiosis. Based on the general function of mTORC2 in the organization of the actin cytoskeleton, we propose that mTORC1 controls spindle function during mitosis and meiosis, while mTORC2 contributes to actin-dependent asymmetric division during meiotic maturation in mice.

Selection of in vitro-matured porcine oocytes based on localization patterns of lipid droplets to evaluate developmental competence.

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.

Frontal Encephalocele Plus Epilepsy: A Case Report and Review of the Literature.

An encephalocele is a pathological brain herniation caused by osseous dural defects. Encephaloceles are known to be regions of epileptogenic foci. We describe the case of a 44-year-old woman with refractory epilepsy associated with a frontal skull base encephalocele. Epilepsy surgery for encephalocele resection was performed; however, the epilepsy was refractory. A second epilepsy surgery for frontal lobectomy using intraoperative electroencephalography was required to achieve adequate seizure control. Previous reports have shown that only encephalocele resection can result in good seizure control, and refractory epilepsy due to frontal lobe encephalocele has rarely been reported. To the best of our knowledge, this is the first report of frontal encephalocele plus epilepsy in which good seizure control using only encephalocele resection was difficult to achieve. Herein, we describe the possible mechanisms of encephalocele plus epilepsy and the surgical strategy for refractory epilepsy with encephalocele, including a literature review.

Interaction between PKR and PACT mediated by LPS-inducible NF-κB in human gingival cells.

The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double-stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 µg/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF-κB to the nucleus after 30 min, and inhibition of NF-κB decreased the PACT-PKR interaction induced by LPS. The level of pro-inflammatory cytokine mRNA, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro-inflammatory cytokines was not affected by RNAi-mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF-κB decreased it. These results indicated that LPS induces PKR phosphorylation and the PACT-PKR association in Sa3 cells. Our results also suggest that NF-κB is involved in the PACT-PKR interaction and the production of pro-inflammatory cytokines in periodontitis.

Distribution and Y397 phosphorylation of focal adhesion kinase on follicular development in the mouse ovary.

Several protein tyrosine kinases (PTKs) are identified as follicle survival factors that suppress apoptosis in granulosa cells. Focal adhesion kinase (FAK/PTK2) interacts with numerous signaling partners and is important for cell adhesion, survival and other vital processes in which FAK autophosphorylation at Y397 (pY397 FAK) is critical for activating signaling pathways. Despite its important roles in apoptosis, the expression and function of FAK in the ovaries remain unknown. Here, we describe FAK expression, including pY397 FAK, in normal healthy mouse ovaries and its association with follicular development and/or atresia. Normal healthy mouse ovaries were used for western blot (n > 60) and immunohistochemical (n > 180) analyses. Western blot results in immature and mature mice revealed that total FAK and pY397 FAK were highly expressed in the ovary and immunohistochemistry results in 3-week-old mice showed they were localized to granulosa cells of ovarian follicles, especially preantral follicles. In 3-week-old mice treated with 5 IU pregnant mare serum gonadotropin (for obtaining homogenous populations of growing or atretic follicles), western blotting revealed that follicular atresia progression involved decreased phosphorylation of Y397 at 72 and 96 h after treatment, particularly in granulosa cells of atretic follicles, as shown by immunohistochemistry results at 72 h after treatment. Moreover, immunostaining patterns of FAK and cleaved caspase-3 were negatively correlated in serial sections of 3-week-old mouse ovaries. These results suggest that FAK is most active in ovarian follicle granulosa cells and that its phosphorylation at Y397 is histologically meaningful in follicular development in normal healthy ovaries.

Efficient establishment of pig embryonic fibroblast cell lines with conditional expression of the simian vacuolating virus 40 large T fragment.

The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.

[Two cases of pulmonary embolism in spinal surgeries].

We experienced two cases of pulmonary embolism (PE) in the perioperative period. Although the incidence of perioperative PE is low, it may lead to a critical outcome. The first case is a 59-year-old man without risk factors of PE, scheduled for laminectomy. The end tidal CO2 of 25 mmHg and Pa(CO2) of 48 mmHg developed at the same time during the operation, suggesting PE. He was diagnosed as PE by pulmonary perfusion scan later. The second case was a 71-year-old woman with hypertension and diabetes mellitus, scheduled for laminectomy. Although there were no events during the surgery, she complained of chest pain and dyspnea after the operation. Blood gas analysis showed Pa(O2) of 55 mmHg (FI(O2) 0.4). She was also diagnosed as PE by pulmonary perfusion scan. Both patients made satisfactory progress by appropriate diagnosis and treatment. PE may occur in spite of prevention, and it is important to find out the signs of PE and to prepare for the occurrence of PE.