Vitrification of porcine immature oocytes and zygotes results in different levels of DNA damage which reflects developmental competence to the blastocyst stage.

The present study investigated the effects of vitrification of porcine oocytes either at the immature Germinal Vesicle (GV) stage before in vitro maturation (GV-stage oocytes) or at the pronuclear stage after in vitro maturation and fertilization (zygotes) on DNA integrity in relevance with their subsequent embryo development. Vitrification at the GV stage but not at the pronuclear stage significantly increased the abundance of double-strand breaks (DSBs) in the DNA measured by the relative fluorescence after γH2AX immunostaining. Treatment of GV-stage oocytes with cryoprotectant agents alone had no effect on DSB levels. When oocytes were vitrified at the GV stage and subjected to in vitro maturation and fertilization (Day 0) and embryo culture, significantly increased DSB levels were detected in subsequent cleavage-stage embryos which were associated with low cell numbers on Day 2, the upregulation of the RAD51 gene at the 4-8 cell stage (measured by RT-qPCR) and reduced developmental ability to the blastocyst stage when compared with the non-vitrified control. However, total cell numbers and percentages of apoptotic cells (measured by TUNEL) in resultant blastocysts were not different from those of the non-vitrified control. On the other hand, vitrification of zygotes had no effect on DSB levels and the expression of DNA-repair genes in resultant embryos, and their development did not differ from that of the non-vitrified control. These results indicate that during vitrification GV-stage oocytes are more susceptible to DNA damages than zygotes, which affects their subsequent development to the blastocyst stage.

Dibutyryl-cAMP and roscovitine differently affect premature meiotic resumption and embryo development of vitrified immature porcine oocytes.

Vitrification and warming can trigger premature meiosis in immature porcine oocytes. Our aim was to compare the efficacies of two meiotic inhibitors, dibutyryl-cAMP and roscovitine for the meiosis synchronization during in vitro maturation (IVM) of porcine oocytes vitrified at the germinal vesicle (GV) stage. We first compared the efficacy of 1 mM dibutyryl-cAMP and 25 µM roscovitine on meiotic arrest during the first 22 h of IVM. Dibutyryl-cAMP could maintain the GV stage in 83.5% of oocytes; however, roscovitine was even more effective (96.6%), whereas only 17.4% of the oocytes remained at the GV stage without these additives. Temporal meiotic arrest for 22 h by roscovitine did not reduce the percentage of oocytes reaching the Metaphase II stage during subsequent IVM. However, after parthenogenetic stimulation or in vitro fertilization, subsequent embryo development to the blastocyst stage was compromised after roscovitine treatment, whereas dibutyryl-cAMP improved the percentage of blastocyst development. In conclusion, dibutyryl-cAMP could derogate but not completely prevent premature meiosis in vitrified oocytes, whereas roscovitine could more efficiently prevent it. However, for embryo production, the use of roscovitine was disadvantageous, whereas the use of dibutyryl-cAMP was beneficial.

Size adjustment occurs during the larval growth of the separated blastomeres of the starfish, Patiria Pectinifera.

Starfish embryos derived from blastomeres separated at the early cleavage stage exhibit morphogenesis to form normally shaped, but smaller-sized dwarf bipinnaria larvae. To further understand this developmental capacity, we primarily characterized the morphogenetic processes of separated 2-cell and 4-cell stage blastomeres during the embryonic and larval periods of the starfish, Patiria Pectinifera. Using non-separated blastomeres as the control, we subjected the separated blastomeres to morphological analyses in conjunction with quantitative measurements of the changes in their body sizes with time post-fertilization. Our results were as follows: (i) Blastomeres separated at 2-cell and 4-cell stages synchronously developed into dwarf-sized bipinnaria larvae. (ii) Upon reaching a body size of 500-700 µm, all the bipinnaria larvae originating from the separated blastomeres and controls began to undergo a series of similar organ formation events in preparation for metamorphosis-recognized as the demarcation between the early and late substages of the bipinnaria larval period. (iii) The separated blastomeres became brachiolaria larvae capable of undergoing metamorphosis at differing rates after reaching approximately 1000-1200 µm body sizes, with adult rudiment and sensory organ forming functionally. (iv) The unfed controls and dwarf bipinnaria larvae derived from blastomeres separated at the 4-cell stage arrested their development synchronously without reaching the threshold size required for the latter half of the bipinnaria stage. These results, taken together, suggested that separated blastomeres possess the developmental capacity to become brachiolaria larvae through a shift in morphogenetic regulation from a synchronous growth to size adjustment during the larval period.

One-Year Outcome of Intravitreal Tissue Plasminogen Activator, Ranibizumab, and Gas Injections for Submacular Hemorrhage in Polypoidal Choroidal Vasculopathy.

This study investigated one-year outcomes of treatment with one session of intravitreal recombinant tissue plasminogen activator, ranibizumab, and gas injections for submacular hemorrhage secondary to polypoidal choroidal vasculopathy (PCV). An extended study of a previous prospective trial of this treatment modality in PCV patients was conducted in 64 patients (64 eyes). Early Treatment Diabetic Retinopathy Study (ETDRS) score, central retinal thickness (CRT), and central pigment epithelial detachment thickness (CPEDT) before and 1, 3, and 12 months after treatment were analyzed. Mean ETDRS score increased from 58 at baseline to 64 letters (p = 0.0122), CRT decreased from 543 to 192 µm (p < 0.0001), and CPEDT decreased from 161 to 103 µm (p = 0.0668) at 3 months and were maintained until 12 months. Complications requiring reoperation occurred within one month in four eyes. Recurrence was observed in 46 eyes (72%), and 1.6 ± 1.5 (0−7) intravitreal aflibercept injections were given pro re nata. Univariate and multivariate analyses identified CPEDT as the pre- and post-treatment factor affecting 12-month ETDRS score (p < 0.0001). Improved visual acuity stabilized 3 months after treatment. Although 72% of patients experienced recurrence, an average of 1.6 aflibercept injections/patient maintained visual acuity up to 12 months. CPEDT was the most important factor associated with visual outcome.

Production of Agu piglets after transfer of embryos produced in vitro.

The present study was conducted to examine the feasibility of in vitro embryo production and transfer technologies for producing piglets of Agu, an Okinawan indigenous pig breed. After collection of oocytes from surgically dissected ovaries, they were subjected to in vitro maturation. After in vitro maturation/fertilization, a total of 616 putative embryos were transferred into four commercial Western pig recipients, one of which became pregnant and farrowed a total of eight Agu piglets. These results demonstrate that in vitro embryo production using ovaries from Agu females is useful for breeding management and conservation of indigenous breeds.

Effect of nucleocytoplasmic ratio on the in vitro porcine embryo development after in vitro fertilization or parthenogenetic activation.

This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization.

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = -0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = -0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.

Laparoscopic approach for surgical treatment of pleuroperitoneal communication interfering with peritoneal dialysis: a case report.

BACKGROUND: Pleuroperitoneal communication is a rare disorder that interferes with peritoneal dialysis. Although favorable results of thoracoscopic fistula closure have been reported, there are some cases in which the fistulas cannot be identified by thoracoscopy and the patients are forced to switch to hemodialysis. CASE PRESENTATION: We present two cases of pleuroperitoneal communication in which diaphragmatic fistulas could not be identified thoracoscopically, but could be identified laparoscopically. Patient 1 had difficulty continuing peritoneal dialysis 9 months after its introduction due to right pleural effusion. Although we could not detect the fistula thoracoscopically, we could laparoscopically identify the fistula in the center of the tendon of the right diaphragm and closed the site from the thoracic side. Patient 2 developed dyspnea due to right pleural effusion 6 months after the introduction of peritoneal dialysis. We could not find the fistulas with a thoracoscopic approach, but could identify multiple diaphragmatic fistulas with a laparoscopic approach and close the sites from the thoracic side. CONCLUSION: In the surgical treatment of pleuroperitoneal communication, diaphragmatic fistulas can be identified laparoscopically even when thoracoscopic observation fails to find any fistulas.

Cell sorting and germ layer formation in reconstructed starfish embryos.

Germ layer formation is driven by embryonic cell sorting during the early developmental stages. Starfish (Patiria pectinifera) embryos have a connected endoderm and ectoderm, albeit with few contact surfaces between the epithelia. To better understand the association between cell sorting and germ layer formation, we reconstructed P. pectinifera embryos and examined their germ layer formation. Initial observations showed that the presumptive endodermal (pEN) and presumptive ectodermal (pEC) portions of the embryonic body at the late-blastula stage were preserved throughout development. Based on this, cells that were dissociated from each dermal fragment were mixed in a reconstruction experiment. Our results showed that the pEN and pEC cells were located inside and outside the reaggregates, respectively, to form an embryonic body containing two epithelial layers, separated by a blastocoel. During this process, the pEN cells were motile and shifted from smaller clumps to form a large clump. In contrast, in reaggregates formed in separate cultures, the pEN cells showed strong adhesion abilities, whereas the pEC cells underwent epithelialization. Unlike that in pEN cells, the reaggregation of pEC cells preceded cadherin expression. Filamentous actin was similarly observed in both reaggregates. These results suggest that during the reconstruction of starfish embryos, germ layer formation occurs via the sorting of pEN and pEC cells, depending on their adhesiveness, motility, and epithelialization. In vivo, these properties might embody the physiological significance of cell adhesion in the germ layers constituting the epithelial monolayer.

Submandibular lymph node metastasis of occult thyroid carcinoma first suspected to be a salivary gland tumor: a case report.

BACKGROUND: When diagnosing and treating neck masses, various diseases need to be considered, including benign or malignant tumors, lymph node-related diseases, and cysts. Thus, there may be cases in which making a definitive diagnosis is difficult on the basis of blood testing and imaging alone. CASE PRESENTATION: The patient was an 80-year-old Japanese female who presented with swelling in the right submandibular area. Magnetic resonance imaging and ultrasonography revealed a solid tumor with inhomogeneous content continuous with the submandibular gland. Therefore, the clinical diagnosis was salivary gland tumor. Surgical treatment was performed, and intraoperative frozen-section examination demonstrated submandibular lymph node metastasis of thyroid carcinoma. After surgical treatment, blood test for thyroid gland function yielded normal results except for increased thyroglobulin levels. Further positron-emission tomography-computed tomography and ultrasonography were performed, in addition to fine-needle aspiration biopsy of the thyroid gland and other tests; however, no other thyroid abnormalities were observed. Fine-needle aspiration biopsy revealed no carcinomatous components. Close observational follow-up has been continued without thyroid gland treatment, and as of approximately 8 years postoperation, no recurrence, metastases, or thyroid carcinoma have developed. CONCLUSION: The mass was lymph node metastasis of occult thyroid carcinoma. In general, occult thyroid carcinoma metastasizes to level II-V. To the best of our knowledge, this is the first report of submandibular lymph node metastasis alone of occult thyroid carcinoma.

Early and Middle-Term Results and Anticoagulation Strategy after Left Atrial Appendage Exclusion Using an Epicardial Clip Device.

OBJECTIVE: The present study aimed to evaluate short- and middle-term results and postoperative anticoagulation of left atrial appendage (LAA) exclusion with an epicardial clip device. MATERIALS AND METHODS: From September 2017 to August 2019, 102 patients at our institution underwent epicardial LAA exclusion using the AtriClip device. Anticoagulation therapy was resumed in the very early postoperative period and continued for at least three months after surgery. The patients' data were obtained by reviewing their medical records retrospectively. RESULTS: The mean and median durations of follow-up was 510 ± 184 days and 482 days (range, 216-938 days), respectively. Successful LAA exclusion was confirmed in all but one patient. No device-related complications occurred during surgery. Postoperative computed tomography (CT) findings revealed no migration or displacement of the clips in any patient; however, small clots were observed at the LAA stump in seven patients. Stroke-free rate during the follow-up period was 98.9%. CONCLUSION: LAA exclusion using the AtriClip device was a feasible treatment method in terms of its early and middle-term safety and efficacy. In addition, our postoperative anticoagulation strategy could be optimal for maximizing the procedure's merits, although further studies, involving a larger number of patients and longer duration of follow-up, are needed.

Embryo production by intracytoplasmic injection of sperm retrieved from neonatal testicular tissue of Agu pigs after cryopreservation and grafting into nude mice.

The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0 = xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1 ± 0.1%, mean ± SEM for four mice), the proportion reached 36.9 ± 16.7% (n = 4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation.

Total debranching thoracic endovascular aortic repair with elephant trunk insertion technique.

We report successful total debranching thoracic endovascular aortic repair using the elephant trunk insertion technique without hypothermic circulatory arrest for a 56-year-old man who developed aortic arch dissection and ascending aortic aneurysm. In the first step, an elephant trunk graft was inserted into the ascending aorta under cardiopulmonary bypass, and a branched prosthetic graft was attached to the ascending aorta. The left common carotid artery and brachiocephalic artery were sequentially anastomosed to the branched graft. The second step was thoracic endovascular aortic repair covering the elephant trunk to the distal arch. Postprocedure digital subtraction angiography showed no endoleaks or false lumen.

Doubling of the cytoplasm volume improves the developmental competence of porcine oocytes injected with freeze-dried somatic cells.

In the present study using pig cells, we examined the effect of the cryoprotectant trehalose on the DNA integrity of freeze-dried cells. We then investigated whether donor cell types and storage duration had impact on DNA integrity in freeze-dried cells or developmental competence of oocytes injected with freeze-dried somatic cells. We also examined whether double cytoplasm nuclear transfer (DCNT) would improve developmental competence of such oocytes. Furthermore, using a PCR-based method for sex identification, we determined whether the blastocysts obtained had actually been generated from the freeze-dried cells. It was found that, for a short storage duration at low temperature, trehalose had no beneficial effect on protection from DNA damage, and that donor cell type had no effect on the DNA integrity of freeze-dried somatic cells or the developmental competence of oocytes injected with them. We also confirmed that all of the blastocysts obtained following nuclear transfer were of freeze-dried somatic cell origin. Storage of freeze-dried somatic cells for up to 1 year at low temperature did not degrade DNA integrity in comparison with storage for 1 month, 1 week or 1 day. Following injection of freeze-dried cells, the proportion of oocytes that developed to blastocysts after storage for up to 1 year was similar to that after storage for 1 month, 1 week or 1 day. Moreover, DCNT significantly improved the developmental competence of oocytes treated in this way. In summary, using DCNT, we have demonstrated that freeze-dried porcine somatic cells subjected to long-term storage at 4 °C have nearly the same potential to develop to blastocysts as non-freeze-dried cells.

Pluripotency-associated genes reposition during early embryonic developmental stages in pigs.

We examined the allelic expression and positioning of two pluripotency-associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4- and 8-cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi-allelically increased from 45% at the 4-cell stage to 60% at the 8-cell stage. Moreover, in 8-cell embryos, SOX2 was expressed bi-allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4- to 8-cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4- or 8-cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency-associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.

Selection based on morphological features of porcine embryos produced by in vitro fertilization: Timing of early cleavages and the effect of polyspermy.

The aim of this study was to examine whether a morphological approach is efficient for selecting high-quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen-thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105  sperms/ml). Under conditions of moderate polyspermy, 4-cell embryos selected at 48 hr after IVF (single selection) and 8-cell embryos selected at 79 hr after IVF from the collected 4-cell embryos (double selection) showed high developmental competence. Likewise, 4- and 8-cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4- and 8-cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4- and 8-cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.

Combined refinements to somatic cell nuclear transfer methods improve porcine embryo development.

The discovery of how to utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats)-Cas (CRISPR-associated) systems for genome modification has accelerated development of the field of genome editing, especially in large animals such as pigs. The low efficiency of somatic cell nuclear transfer (SCNT) is now becoming a major obstacle in the production of genome-edited animals via cell-mediated approaches and improving efficacy of this technique is crucial. In this study, we propose a few simple modifications to a zona-free SCNT protocol that are effective to produce numerous high-quality blastocysts. To refine the SCNT protocol we modified the following steps/factors: 1) culture medium for SCNT embryos, 2) chemical treatment to prevent precocious activation of the manipulated/reconstructed oocytes and 3) donor cell serum starvation treatment. Although changes in each of these steps only resulted in small improvements, the combination of all modifications altogether significantly enhanced developmental competence of SCNT embryos. Our modified method yielded approximately three times greater blastocyst formation rates. Moreover, resulting blastocysts had roughly twice as many cells as compared to blastocysts produced by the conventional SCNT method. With these significant in vitro improvements, our refined SCNT method is potentially suited for use in the production of genome edited pigs.

Vitrification of porcine cumulus-oocyte complexes at the germinal vesicle stage does not trigger apoptosis in oocytes and early embryos, but activates anti-apoptotic Bcl-XL gene expression beyond the 4-cell stage.

The aim of the present study was to clarify whether or not our vitrification procedure at the germinal vesicle (GV)-stage triggers the apoptotic cascade in oocytes and subsequent embryos. Immature porcine cumulus-oocyte complexes were either vitrified and warmed (vitrified group) or subjected to cryoprotectant agents (CPA group) or cultured without any treatment (control). Oocytes of all treatment groups were subjected to in vitro maturation (IVM), fertilization, and embryo culture. Apoptosis was assayed in live oocytes at the end of IVM culture and in cleavage-stage embryos after in vitro fertilization (IVF). We detected similar frequencies of DNA fragmentation, levels of caspase activity, phosphatidylserine externalization, and mRNA levels for pro-apoptotic Bax and CASP3 genes in oocytes at the end of IVM and in early embryos among all groups. However, in the vitrified group, the anti-apoptotic Bcl-XL gene was upregulated in 4-8 cell embryos, which caused an 8-fold significant increase in the Bcl-XL/Bax mRNA ratio compared with the control and CPA groups (P < 0.05). In conclusion, vitrification of porcine oocytes at the GV stage by our method did not trigger the apoptotic cascade in oocytes and subsequent embryos but triggered the upregulation of the anti-apoptotic Bcl-XL gene in embryos.

Suppression of poisoning of photocathode catalysts in photoelectrochemical cells for highly stable sunlight-driven overall water splitting.

A photoelectrochemical (PEC) cell composed of two semiconductor electrodes, a photocathode, and a photoanode is a potentially effective means of obtaining hydrogen through spontaneous overall water splitting under light irradiation. However, the long-term stability (that is, operation for more than one day) of a PEC cell has not yet been demonstrated. In addition to the corrosion of both photoelectrodes, the gradual migration of heavy metal cations from the photoanode into the electrolyte can also result in degradation of the cell by contamination of the photocathode surface. In the present work, BiVO4-based photoanodes were used in conjunction with two different modifications: dispersion of a chelating resin in the electrolyte and coating of the photoanode surface with an anion-conducting ionomer. The chelating resin was found to capture Bi3+ cations in the electrolyte before they became deposited on the cathode surface. Consequently, a PEC cell incorporating a BiVO4-based photoanode and a (ZnSe)0.85(CuIn0.7Ga0.3Se2)0.15-based photocathode showed stable overall water splitting over a span of two days under simulated sunlight. To the best of our knowledge, this represents the longest period over which stable PEC cell performance has been established. A considerable decrease in the performance of the BiVO4-based photoanode was still observed due to the continuous dissolution of Bi species, but surface coating of the photoanode with an anion-conducting ionomer prevented the movement of Bi3+ ions into the electrolyte because of the selective conduction of ions. The coating also served as a protective layer that improved the durability of the photoanode. This study therefore suggests a simple yet effective method for the construction of stable PEC cells using semiconductor photoelectrodes.