Antioxidant sericin averts the disruption of oocyte-follicular cell communication triggered by oxidative stress.

Antioxidants are free radical scavengers that increase oocyte quality and improve female fertility by suppressing oxidative stress. However, the related mechanisms remain unclear. The present study was designed to examine whether a reduction of oxidative stress from using the antioxidant sericin led to expanded cumulus cell (CC)-oocyte communication and oocyte developmental acquisition in a bovine model. We found that cumulus-oocyte complexes (COCs) matured in the presence of sericin showed a significantly increased oocyte meiotic maturation rate (P < 0.01) and accelerated subsequent blastocyst formation, as more blastocysts were found at the hatched stage (P < 0.05) compared to that in the control group. In contrast to the control group, sericin suppressed H2O2 levels in COCs, resulting in a markedly enhanced CC-oocyte gap junction communication index and number of transzonal projections, which were preserved until 18 h of oocyte maturation. These findings indicate that sericin reduces disruption of oocyte-follicular cell communication induced by oxidative stress. Sericin consistently increased intra-oocyte glutathione (GSH) levels and reduced oocyte H2O2 levels (P < 0.05), both of which were ablated when GSH synthesis was inhibited by buthionine sulfoximide (an inhibitor of GSH synthesis). Furthermore, the inhibition of GSH synthesis counteracted the positive effects of sericin on subsequent embryo developmental competence (P < 0.01). Intra-oocyte GSH levels were positively associated with blastocyst development and quality. These outcomes demonstrate new perspectives for the improvement of oocyte quality in assisted reproductive technology and may contribute to developing treatment strategies for infertility and cancer.

Effect of E-64 Supplementation during In Vitro Maturation on the Developmental Competence of Bovine OPU-Derived Oocytes.

Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.

Gene expression of bovine endometrial epithelial cells cultured in matrigel.

Glandular epithelial cells (GE) in the endometrium are thought to support the elongation and survival of ruminant embryos by secreting histotrophs. In the present study, the gene expression of bovine endometrial epithelial cells cultured in matrigel was analyzed and examined whether it could be an in vitro model of GE. Bovine endometrial epithelial cells (BEE) and stromal cells (BES) were isolated from the slaughterhouse uteri and cultured in DMEM/F12 + 10% FBS. BEE showed the gland-like structure morphological changes when cultured in 15% matrigel but could not be identified in higher concentrations of the matrigel (30% or 60%). The expression of typical genes expressed in GE, SERPINA14 and GRP, was substantially high in matrigel-cultured BEE than in monolayer (P  <  0.05). P4 and INFα have no significant effect on the SERPINA14 expression of BEE cultured in matrigel without co-culture with BES. On the other hand, when BEE were co-cultured with BES in matrigel culture, the expression of FGF13 was increased by the P4 treatment (P  <  0.05). Furthermore, SERPINA14 and TXN expressions were increased by P4 + IFNα treatment (P  <  0.05). These results demonstrate the appropriate conditions for BEE to form glandular structures in matrigel and the effect of co-culture with BES. The present study highlighted the possible use of matrigel for the culture of BEE to investigate the expression of cell-specific glandular epithelial genes as well as P4 and type-I IFN as factors controlling endometrial function during the implantation period.

Study of Excited States and Electron Transfer of Semiconductor-Metal-Complex Hybrid Photocatalysts for CO2 Reduction by Using Picosecond Time-Resolved Spectroscopies.

A semiconductor-metal-complex hybrid photocatalyst was previously reported for CO2 reduction; this photocatalyst is composed of nitrogen-doped Ta2 O5 as a semiconductor photosensitizer and a Ru complex as a CO2 reduction catalyst, operating under visible light (>400 nm), with high selectivity for HCOOH formation of more than 75 %. The electron transfer from a photoactive semiconductor to the metal-complex catalyst is a key process for photocatalytic CO2 reduction with hybrid photocatalysts. Herein, the excited-state dynamics of several hybrid photocatalysts are described by using time-resolved emission and infrared absorption spectroscopies to understand the mechanism of electron transfer from a semiconductor to the metal-complex catalyst. The results show that electron transfer from the semiconductor to the metal-complex catalyst does not occur directly upon photoexcitation, but that the photoexcited electron transfers to a new excited state. On the basis of the present results and previous reports, it is suggested that the excited state is a charge-transfer state located between shallow defects of the semiconductor and the metal-complex catalyst.

Charge Trapping Process in Photoexcited Nitrogen-Doped Titanium Oxides.

We present a first-principles study on the structural changes induced by charge trapping that occurs after photoexcitation in nitrogen-doped titanium oxide (N-TiO2). The charge trapping site and the corresponding K edge EXAFS spectra of Ti atoms were predicted and compared with those obtained by an experiment under ultraviolet (UV) light excitation. The results indicate that charge trapping occurs in the neighborhood of the oxygen vacancy (O-vac) sites. Furthermore, our calculations show that the O-vac site significantly affects the EXAFS spectra, while substitutional nitrogen doping for an oxygen site in the vicinity of the O-vac site is insensitive in the EXAFS spectra. Based on this observation combined with the knowledge from previous experiments, we propose a charge trapping process where the UV light-excited electron migrates at the O-vac site in bulk (∼300 ps) while the visible light-excited electron (N 2p → Ti 3d) is immediately trapped at the O-vac site neighboring the N site (∼1 ps).

Clinical feasibility and safety of transoesophageal motor-evoked potential monitoring.

OBJECTIVES: Canine experiments have shown that transoesophageal motor-evoked potential monitoring is feasible, safe and stable, with a quicker response to ischaemia and a better prognostic value than transcranial motor-evoked potentials. We aimed to elucidate whether or not these findings were clinically reproducible. METHODS: A bipolar oesophageal electrode mounted on a large-diameter silicon tube and a train of 5 biphasic wave stimuli were used for transoesophageal stimulation. Results of 18 patients (median age 74.5 years, 13 males) were analysed. RESULTS: There were no mortalities, spinal cord injuries or complications related with transoesophageal stimulation. Transcranial motor-evoked potential could not be monitored up to the end of surgery in 3 patients for unknown reasons, 2 of whom from the beginning. Transoesophageal motor-evoked potential became non-evocable after manipulation of a transoesophageal echo probe in 2 patients. Strenuous movement of the upper limbs during transoesophageal stimulation was observed in 3 patients. In 14 patients who successfully completed both monitoring methods up to the end of surgery (11 thoraco-abdominal and 3 descending aortic repair), the final results were judged as false positives in 6 by transcranial stimulation and in 1 by transoesophageal stimulation. The stimulation intensity was significantly lower and the upper limb amplitude was significantly higher by transoesophageal stimulation, while the lower limb amplitude was comparable. CONCLUSIONS: Transoesophageal motor-evoked potential monitoring is clinically feasible and safe with a low false positive rate. A better electrode design is required to avoid its migration by transoesophageal echo manipulation. Further studies may be warranted. CLINICAL REGISTRATION NUMBER: UMIN000022320.

Endoplasmic reticulum stress attenuation promotes bovine oocyte maturation in vitro.

We have previously reported that regulation of endoplasmic reticulum (ER) stress during in vitro culture acutely increases bovine embryo developmental rate and cryotolerance; these data indicate that ER stress is a critical factor reducing the quality of in vitro-produced embryos. In the current follow-up study, we examined whether ER stress attenuation during in vitro maturation influences meiotic maturation, oocyte quality, and subsequent embryonic development. Bovine cumulus oocyte complexes (COCs) derived from slaughterhouse ovaries were matured with or without tauroursodeoxycholic acid (TUDCA), a selective inhibitor of ER stress (0, 50, 100, and 200 µM) for 22 h followed by in vitro fertilization, and zygotes were cultured for 8 days. Of the different doses of TUDCA, 100 µM TUDCA significantly increased the maturation rate, and decreased reactive oxygen species in denuded oocytes, and appeared lower number of apoptotic cells in matured COCs. Subsequently, treatment of TUDCA (100 µM) decreased the localization and amount of GRP78/BIP protein level as well as ER stress (GRP78/BIP, PERK, IER1, ATF4, and XBP1) and apoptosis (CHOP and BAX)-related gene expression, while it increased the anti-apoptotic gene BCL2 level in matured COCs. Moreover, addition of TUDCA (100 µM) during IVM significantly improved the blastocyst formation rate (43.6 ± 1.8% vs 49.7 ± 1.3%) and decreased the number of apoptotic cells (7.7 ± 1.1% vs 5.03 ± 0.6%) in blastocysts. These findings suggest that the presence of ER stress during maturation impairs the developmental competence of bovine COCs and that this process can be reversed by TUDCA.

Heterogeneous water oxidation photocatalysis based on periodic mesoporous organosilica immobilizing a tris(2,2'-bipyridine)ruthenium sensitizer.

A periodic mesoporous organosilica (PMO) containing 2,2'-bipyridine groups (BPy-PMO) has been shown to possess a unique pore wall structure in which the 2,2'-bipyridine groups are densely and regularly packed. The surface 2,2'-bipyridine groups can function as chelating ligands for the formation of metal complexes, thus generating molecularly-defined catalytic sites that are exposed on the surface of the material. We here report the construction of a heterogeneous water oxidation photocatalyst by immobilizing several types of tris(2,2'-bipyridine)ruthenium complexes on BPy-PMO where they function as photosensitizers in conjunction with iridium oxide as a catalyst. The Ru complexes produced on BPy-PMO in this work were composed of three bipyridine ligands, including the BPy in the PMO framework and two X2bpy, denoted herein as Ru(X)-BPy-PMO where X is H (2,2'-bipyridine), Me (4,4'-dimethyl-2,2'-bipyridine), t-Bu(4,4'-di-tert-butyl-2,2'-bipyridine) or CO2Me (4,4'-dimethoxycarbonyl-2,2'-bipyridine). Efficient photocatalytic water oxidation was achieved by tuning the photochemical properties of the Ru complexes on the BPy-PMO through the incorporation of electron-donating or electron-withdrawing functionalities. The reaction turnover number based on the amount of the Ru complex was improved to 20, which is higher than values previously obtained from PMO systems acting as water oxidation photocatalysts.

Bipolar transesophageal thoracic spinal cord stimulation: A novel clinically relevant method for motor-evoked potentials.

OBJECTIVE: Although transesophageal motor-evoked potential elicited by monopolar cervical cord stimulation is more stable and rapid in response to ischemia than transcranial motor-evoked potential in canine experiments, direct cervical alpha motor neuron stimulation precludes clinical application. We evaluated a novel stimulation method using a bipolar esophageal electrode to enable thoracic cord stimulation. METHODS: Twenty dogs were anesthetized. For bipolar transesophageal stimulation, the interelectric pole distance was set at 4 cm. Changes in amplitude in response to incremental stimulation intensity (100-600 V) were measured to evaluate stability. Spinal cord ischemia was induced by aortic balloon occlusion at the T8 to T10 level for 10 minutes to evaluate response time or at the T3 to T5 level for 25 minutes to evaluate prognostic value. Neurological function was evaluated using the Tarlov score at 24 and 48 hours postoperatively. RESULTS: Bipolar transesophageal stimulation was successful in all animals and their forelimb waveforms were identical to those after transcranial stimulation. The minimum stimulation intensity to produce >90% of the maximum amplitude was significantly lower in both monopolar and bipolar transesophageal stimulation than in transcranial stimulation (n = 5). Time to disappearance and recovery (>75%) of the hindlimb potentials were significantly shorter by both monopolar and bipolar transesophageal stimulation than by transcranial stimulation (n = 5). Correlation with neurological outcomes was comparable among all stimulation methods (n = 10). CONCLUSIONS: Motor-evoked potential can be elicited by bipolar transesophageal thoracic cord stimulation without direct cervical alpha motor neuron stimulation, and its stability and response time are comparable to those elicited by monopolar stimulation.

Role of endoplasmic reticulum stress on developmental competency and cryo-tolerance in bovine embryos.

Endoplasmic reticulum (ER) stress, a dysfunction in protein folding capacity of the ER, is involved in many physiological responses including mammalian reproductive systems. Studies have shown that ER stress interferes with the developmental process of in vitro oocyte maturation and embryo development; however, little is known about its effects on bovine preimplantation embryonic development. In this study, we examined the effects of ER stress during IVC on developmental competency and cryo-tolerance in bovine embryos. IVF-derived zygotes were cultured in CR1aa medium supplemented with tauroursodeoxycholic acid (TUDCA) and/or tunicamycin (TM), which are ER stress-inhibitory and stress-inducing agents, respectively, for 8 days. TM treatment decreased the blastocyst developmental rate and increased the percentage of apoptotic cells compared to that in the control group (10.2 ±â€¯2.3% vs. 39.75 ±â€¯1.3% and 17.8 ±â€¯1.2% vs. 3.6 ±â€¯1.1%, respectively; P < 0.01). However, the blastocyst developmental rate was increased and the percentage of apoptotic cells was decreased by addition of TUDCA in IVC medium compared to that in the control group (50.9 ±â€¯0.9% vs. 39.75 ±â€¯1.3% and 1.13 ±â€¯1.0% vs. 3.6 ±â€¯1.1%, respectively; P < 0.01). Importantly, in the group treated with TM plus TUDCA, the developmental rate and the percentage of apoptotic cells in blastocysts were similar to that in the control group, indicating that TUDCA ameliorates the adverse effects of TM alone on embryo development. In addition, TUDCA treatment significantly reduced the reactive oxygen species, expression of ER stress (GRP78, ATF4, ATF6, IER1, and sXBP1) and pro-apoptotic (CHOP and BAX) genes, while it increased anti-apoptotic BCL2 gene expression and glutathione levels. Moreover, TUDCA improved blastocyst cryo-tolerance as marked by a significantly increased hatching rate and decreased the number of apoptotic cells recorded at 48 h after a post-warming. Therefore, in concordance with a previous report in mice or pig, we showed that TUDCA supplementation during IVC increases the developmental competency of bovine in vitro-derived embryos. Additionally, we found that the presence of TUDCA in IVC medium improves the cryo-tolerance of bovine embryos. These results suggest that modulation of ER stress during IVC contributes to the production of high-quality bovine embryos in terms of cryo-tolerance.

Efficient in vitro embryo production using in vivo-matured oocytes from superstimulated Japanese Black cows.

We examined whether the use of in vivo-matured oocytes, collected by ovum pick-up (OPU) from superstimulated Japanese Black cows, can improve the productivity and quality of in vitro produced embryos. The cows were superstimulated by treatment with progesterone, GnRH, FSH and prostaglandin F2α according to a standardized protocol. The resulting in vivo-matured oocytes were collected by OPU and used subsequently for the other experiments. The immature oocytes from cows in the non-stimulated group were collected by OPU and then subjected to maturation in vitro. We found that the rate of normally distributed cortical granules of the matured oocyte cytoplasm in the superstimulated group was significantly higher than that in the non-stimulated group. The normal cleavage rate (i.e., production of embryos with two equal blastomeres without fragmentation) and freezable blastocyst rate were significantly higher in the superstimulated group than in the non-stimulated group. Among the transferable blastocysts, the ratio of embryos from normal cleavage was also significantly higher in the superstimulated group than in the non-stimulated group. For in vivo-matured oocytes, it was observed that the pregnancy rates were significantly higher when normally cleaved embryos were used for transfer. Taken together, these results suggest that high-quality embryos with respect to developmental kinetics can be efficiently produced with the use of in vivo-matured oocytes collected by OPU from superstimulated Japanese Black cows.

Japanese perspective in surgery for thoracoabdominal aortic aneurysms.

OBJECTIVE: Operative mortality and morbidity after thoracoabdominal aortic surgery remain high. We report our strategy and outcomes, especially those of spinal cord protection. METHODS: Outcomes of 178 patients (age: 26-88 years) who underwent thoracoabdominal aortic replacement were retrospectively analyzed. 65 had aortic dissection, 14 had infected aneurysms, and 22 presented with rupture. Operations were non-elective in 24 and redo through re-thoracotomy in 21. Extent of replacement was Crawford-I in 39, II in 26, III in 78, and IV in 35. Staged repair was recently preferred, which resulted in decrease in extent II repair and increase in redo since 2009. Operations were performed under distal aortic perfusion and multi-segmental sequential repair to maximize collateral blood flow, and deep hypothermic circulatory arrest was preserved for those requiring open aortic anastomosis (n = 20). A total of 166 separate grafts were used for intercostal reconstruction in 88 patients, which was guided by preoperative feeding artery localization. Their patency was studied by postoperative MD-CT in 74 patients for 145 grafts. RESULTS: There were 3.9% hospital mortality and 5.1% spinal cord injury. Preoperative feeding artery localization resulted in reduced number of reconstruction and improved patency, and grafts connecting to the feeding artery were patent in 92%. Results of redo operations were not different (no mortality and spinal cord injury) from the de novo operations. CONCLUSIONS: Our concept of spinal cord protection, which was based on selective intercostal reconstruction while maximizing spinal cord collateral blood flow, seems justified.

Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos.

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non-cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non-cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non-cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.

Sericin enhances the developmental competence of heat-stressed bovine embryos.

We investigated the effects of sericin on the developmental competence of bovine embryos exposed to heat stress (HS). Putative zygotes were cultured with sericin and subjected to HS (40.5°C for 6 hr) on Day 2 or 7 followed by continuous culture at 38.5°C until Day 8. Day 2 HS significantly decreased blastocyst development on Day 8 as well as mitochondrial activity, and significantly increased the amount of intracellular reactive oxygen species and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive cells, whereas Day 7 HS only significantly decreased mitochondrial activity and increased the number of TUNEL-positive cells in Day 8 blastocysts. These detrimental effects were neutralized by sericin supplementation. Next, to investigate the potential production of blastocysts with high viability in terms of thermotolerance, embryos were cultured with sericin until Day 7, and then exposed to HS in the sericin-free medium. TUNEL-positive cell numbers were significantly lower in blastocysts produced by sericin culture than in control blastocysts. Transcript abundance for HSPA1A and BAX was significantly decreased but IFNT2 levels were increased in blastocysts produced by sericin culture. In conclusion, these findings demonstrate the anti-oxidative and anti-apoptotic activities of sericin, and the potential use of sericin to produce embryos with high viability in vitro.

Heat-shock-induced cathepsin B activity during IVF and culture compromises the developmental competence of bovine embryos.

Heat stress can cause significant reproductive dysfunction in mammals and previous studies report that expression and activity of cathepsin B (CTSB), a lysosomal cysteine protease, is negatively correlated with the developmental competence of bovine oocytes and embryos. However, the relationship between heat shock (HS) and CTSB remains largely unknown. Here, we investigated the effects of HS during IVF and early embryonic stages of IVC on CTSB activity and developmental competence in bovine embryos. HS (40 °C for 6 h during IVF and 20 h during IVC) caused a significant increase in CTSB activity irrespective of the developmental stage or duration of HS. The developmental rate to the blastocyst stage was also significantly decreased by HS. Additionally, HS during IVC significantly increased the number of apoptotic cells in blastocysts. Notably, these HS-induced changes in blastocyst development and quality were significantly improved by inhibition of CTSB activity, indicating a key role for CTSB. These results showed that CTSB activity plays an essential role in HS-induced dysfunction in bovine embryo development, and that inhibition of this activity could enhance the developmental competence of heat-shocked embryos.

Carotid stenosis with impaired brain flow reserve is associated with an increased risk of stroke in on-pump cardiovascular surgery.

OBJECTIVES: To prevent haemodynamic stroke during cardiovascular surgery in patients with carotid stenosis, we routinely evaluated magnetic resonance angiography and selectively evaluated brain perfusion single-photon emission computed tomography with acetazolamide challenge. Off-pump surgery was preferred when cerebral blood flow reserve was impaired. This strategy's usefulness was investigated. METHODS: Among the 1059 consecutive patients who underwent preoperative carotid screening by magnetic resonance angiography, 84 (7.9%) patients had >50% stenosis; 45 of them underwent brain single-photon emission computed tomography. The severity of cerebral blood flow compromise was estimated by the proportion of Stage 2 area in the affected territory, in which both resting blood flow (<32 ml/min) and flow reserve (<10%) were reduced. RESULTS: Perioperative stroke occurred in 1.7% overall (18/1059), in 6% (5/84) of those with carotid stenosis and in 1.3% (13/975) of those without stenosis (P = 0.010). On subgroup analysis, carotid stenosis was associated with an increased risk of stroke in the on-pump surgery group [n = 949, 5/59 (9%) with stenosis vs 11/890 (1.1%) without stenosis, P = 0.002], while it was not in the off-pump group [n = 110, 0/25 (0%) with stenosis vs 2/85 (2%) without stenosis, P = 0.59]. With respect to the role of acetazolamide single-photon emission computed tomography, 2 of the 4 patients with Stage 2 area >10% undergoing on-pump surgery without preceding carotid revascularization developed stroke, while none of the 21 patients with Stage 2 area <10% undergoing on-pump surgery developed stroke (P = 0.020). CONCLUSIONS: Carotid stenosis is a risk factor for perioperative stroke in on-pump surgery. Patients with large Stage 2 area (>10%) are at increased risk of perioperative stroke when on-pump surgery is performed.

Re(bpy)(CO)3 Cl Immobilized on Bipyridine-Periodic Mesoporous Organosilica for Photocatalytic CO2 Reduction.

This paper describes the physicochemical properties of a rhenium (Re) complex [Re(bpy)(CO)3 Cl] immobilized on a bipyridine-periodic mesoporous organosilica (BPy-PMO) acting as a solid support. The immobilized Re complex generated a metal-to-ligand charge transfer absorption band at 400 nm. This wavelength is longer than that exhibited by Re(bpy)(CO)3 Cl in the polar solvent acetonitrile (371 nm) and is almost equal to that in nonpolar toluene (403 nm). The photocatalytic activity of this heterogeneous Re complex was lower than that of a homogeneous Re complex due to the reduced phosphorescence lifetime resulting from immobilization. However, the catalytic activity was enhanced by the co-immobilization of the ruthenium (Ru) photosensitizer [Ru(bpy)3 ]2+ on the PMO pore surfaces. Quantum chemical calculations suggest that electron transfer between the Ru and Re complexes occurs through interactions between the molecular orbitals in the pore walls. These results should have applications to the design of efficient heterogeneous CO2 reduction photocatalysis systems.

Secondary graft-oesophageal fistula after total arch replacement.

Secondary graft-oesophageal fistula is a fatal complication of aortic arch replacement. We report a successful two-stage surgical management of a graft-oesophageal fistula seen in a 68-year-old woman 3 years after total aortic arch replacement. She presented with a prolonged intractable fever without haematemesis. The fistula occurred between the distal aortic anastomosis and oesophagus; the entire graft was surrounded by air. In the first-stage operation, we performed re-replacement of the entire infected graft, oesophagectomy with cervical oesophagostomy, omental transfer and cervical routing of the stomach roll, through an extended left thoracotomy incision with sternal transection. Intravenous antibiotics were administered for 6 weeks; the second-stage cervico-oesophageal anastomosis was performed 57 days later. She was discharged without complications and is doing well 6 months postoperatively.

Epigenetic analysis of bovine parthenogenetic embryonic fibroblasts.

Although more than 100 imprinted genes have already been identified in the mouse and human genomes, little is known about genomic imprinting in cattle. For a better understanding of these genes in cattle, parthenogenetically activated bovine blastocysts were transferred to recipient cows to obtain parthenotes, and fibroblasts derived from a Day 40 (Day 0 being the day of parthenogenetic activation) parthenogenetic embryo (BpEFs) were successfully obtained. Bovine embryonic fibroblasts (BEFs) were also isolated from a normal fertilized embryo obtained from an artificially inseminated cow. The expression of imprinted genes was analyzed by RT-PCR. Paternally expressed genes (PEGs) in mouse (viz., IGF2, PEG3, ZAC1, NDN, DLK1, SGCE, and PEG10) were expressed in BEFs, but not in BpEFs, suggesting that these genes are also imprinted in cattle. However, other PEGs in mouse (viz., IMPACT, MAGEL2, SNRPN, and PEG1/MEST) were expressed in both BEFs and BpEFs. These genes may not be imprinted in BEFs. The expression of seven maternally expressed genes in mouse was also analyzed, and only CDKN1C was not expressed in BpEFs. The DNA methylation patterns of repetitive elements (Satellite I, Satellite II, alpha-satellite, and Art2) were not different between the BEFs and BpEFs; however, the differentially methylated region (DMR) of paternally methylated H19 was hypomethylated, whereas those of maternally methylated PEG3 and PEG10 were hypermethylated in BpEFs, as expected. The methylation of the SNRPN DMR was not different between the BEFs and BpEFs, in accordance with the SNRPN expression levels in both cell types. The XIST gene, which is essential for X chromosome inactivation in females, was expressed in BpEFs, whereas its DMR was half-methylated, suggesting that X chromosome inactivation is normal in these cells. Microarray analysis was also applied to identify novel PEGs that should be expressed only in BEFs but not in BpEFs. More than 300 PEG candidate genes, including IGF2, PEG3, and PEG10, were obtained. These results illustrate the epigenetic characteristic of bovine parthenogenetic embryos and contribute to the identification of novel imprinted genes in cattle.