Exploration of the Cytoplasmic Function of Abnormally Fertilized Embryos via Novel Pronuclear-Stage Cytoplasmic Transfer.

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory "aged" oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.

A Preclinical Evaluation towards the Clinical Application of Oxygen Consumption Measurement by CERMs by a Mouse Chimera Model.

We have developed an automated device for the measurement of oxygen consumption rate (OCR) called Chip-sensing Embryo Respiratory Measurement system (CERMs). To verify the safety and the significance of the OCR measurement by CERMs, we conducted comprehensive tests using a mouse model prior to clinical trials in a human in vitro fertilization (IVF) program. Embryo transfer revealed that the OCR measured by CERMs did not compromise the full-term development of mice or their future fertility, and was positively correlated with adenosine triphosphate (ATP) production and the mitochondrial membrane potential (ΔΨm), thereby indirectly reflecting mitochondrial oxidative phosphorylation (OXPHOS) activity. We demonstrated that the OCR is independent of embryo morphology (the size) and number of mitochondria (mitochondrial DNA copy number). The OCR correlated with the total cell numbers, whereas the inner cell mass (ICM) cell numbers and the fetal developmental rate were not. Thus, the OCR may serve as an indicator of the numbers of trophectoderm (TE) cells, rather than number or quality of ICM cells. However, implantation ability was neither correlated with the OCR, nor the embryo size in this model. This can probably be attributed to the limitation that chimeric embryos contain non-physiological high TE cells counts that are beneficial for implantation. CERMs can be safely employed in clinical IVF owing to it being a safe, highly effective, non-invasive, accurate, and quantitative tool for OCR measurement. Utilization of CERMs for clinical testing of human embryos would provide further insights into the nature of oxidative metabolism and embryonic viability.

Triptolide suppresses ultraviolet B-enhanced sebum production by inhibiting the biosynthesis of triacylglycerol in hamster sebaceous glands in vivo and in vitro.

Ultraviolet B (UVB) irradiation causes alterations in cutaneous barrier function, including excessive production of sebum in sebaceous glands, which is associated with the aggravation of acne. This study aimed to evaluate the inhibitory effects of triptolide, a diterpenoid triepoxide from Tripterygium wilfordii Hook F, on sebocytic lipogenesis in UVB-irradiated hamster skin in vivo and in vitro. Topical application of triptolide decreased the UVB-enhanced sebum accumulation in the sebaceous glands of hamster skin. The level of triacylglycerol (TG), a major sebum component, on the skin surface was reduced by triptolide treatment in UVB-irradiated hamsters, whereas there was no change in that of free-fatty acids and cholesterol, which are minor sebum components. UVB irradiation significantly enhanced TG production (P<0.01 in extracellular lipids, P<0.05 in intracellular lipids), and the activity of acyl coenzyme A/diacylglycerol acyltransferase (DGAT), a rate-limiting enzyme of TG synthesis, in differentiated hamster sebocytes (P<0.05 at 6 h and UVB of 0.62 kJ/m2, P<0.001 at 24 h and UVB 0.37 or 0.62 kJ/m2). Furthermore, triptolide significantly inhibited UVB-enhanced TG production (P<0.05 at 28 nM and P<0.01 at 56 and 112 nM triptolide) and DGAT activity (P<0.01 at 28 nM and P<0.001 at 56 and 112 nM triptolide) in differentiated hamster sebocytes. These results provide novel evidence that triptolide decreases UVB-enhanced sebum production by inhibiting DGAT-dependent TG biosynthesis in differentiated hamster sebocytes. These findings may be applicable to the prevention of acne aggravation.

Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

STUDY QUESTION: Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? SUMMARY ANSWER: The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. WHAT IS ALREADY KNOWN: To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. STUDY DESIGN, SIZE, DURATION: This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. PARTICIPANTS/MATERIALS, SETTING, METHODS: We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. MAIN RESULTS AND THE ROLE OF CHANCE: The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. LIMITATIONS, REASONS FOR CAUTION: Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. WIDER IMPLICATIONS OF THE FINDINGS: Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality embryos by CERMs may enable the omission of long-term in vitro embryo culture to the blastocyst stage. CERMs is scalable technology that can be integrated into incubators and/or other embryo evaluation systems, such as the time-lapse systems, due to its chip-based architecture. Thus, CERMS would enable automatic measurement of oxygen consumption, under 5% CO2, in the near future, in order to reduce oxidative stress from exposure to atmospheric air. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Health and Labor Sciences Research Grant (H24-Hisaichiiki-Shitei-016). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Foster placement disruptions associated with problem behavior: mitigating a threshold effect.

OBJECTIVE: Placement disruptions have adverse effects on foster children. Identifying reliable predictors of placement disruptions might assist in the allocation of services to prevent disruptions. There were two objectives in this study: (a) to replicate a prior finding that the number of daily child problem behaviors at entry into a new foster home predicts subsequent placement disruptions in foster preschoolers and (b) to determine whether this association is mitigated by a treatment foster care intervention. METHOD: Problem behavior and placement disruptions were examined in 60 children in regular foster care (age range = 3.10-5.91 years [M = 4.34, SD = 0.83], 58.3% male, 93.4% Caucasian) and 57 children in a treatment foster care program (age range = 3.01-6.78 years [M = 4.54, SD = 0.86], 49.1% male, 82.5% Caucasian). Using the Parent Daily Report Checklist (Chamberlain & Reid, 1987), a brief telephone interview, foster caregivers reported problem behavior 6 times over 3 months. Placement disruptions were tracked over 12 months. RESULTS: The regular foster care children with 5 or fewer problem behaviors were at low risk for disruption, but their risk increased 10% for each additional behavior (p = .013). The intervention appeared to mitigate this "threshold effect"; number of problem behaviors did not predict risk of placement disruption in the treatment foster care group (p = .63). CONCLUSIONS: These findings replicate previous evidence linking child problem behavior to placement disruptions and further highlight the need for early preventative interventions.

Successful pregnancy after oocyte activation by a calcium ionophore for a patient with recurrent intracytoplasmic sperm injection failure, with an assessment of oocyte activation and sperm centrosomal function using bovine eggs.

OBJECTIVE: To report a successful pregnancy after intracytoplasmic sperm injection (ICSI) with artificial oocyte activation (AOA) on a patient whose fertilization rate after ICSI was extremely low; and to report on cytologic analyses of the fertilization failure (FF) eggs after ICSI and a biologic assessment of the sperm of this patient. DESIGN: Case report with an assessment of gamete function. SETTING: University hospital and an experimental laboratory. PATIENT(S): A couple with severe oligoasthenozoospermia, whose seventh attempt at ICSI ended in the failure. INTERVENTION(S): Cytologic analyses of FF eggs after ICSI, AOA after ICSI, and analyses of human sperm oocyte activation ability and centrosomal function. RESULT(S): Fertilization arrest after ICSI was observed in FF eggs at various stages of fertilization. After artificial oocyte activation by exposure to ionomycin, clinical pregnancy was confirmed, and a healthy baby was born. As assessed by heterologous ICSI of human sperm into bovine oocytes, there was no significant difference in the oocyte activation rates between the patient's and control sperm, but the sperm centrosomal function was low in the patient's sperm (48.5% vs. 69.6%). CONCLUSION(S): We report a successful pregnancy after ICSI with AOA using a calcium ionophore, after critical cytologic analyses of the FF eggs. Furthermore, sperm centrosomal function was low, indicating that sperm's ability to process the events of fertilization after the oocyte activation was poor in this patient.

The H-Invitational Database (H-InvDB), a comprehensive annotation resource for human genes and transcripts.

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts. H-InvDB, originally developed as an integrated database of the human transcriptome based on extensive annotation of large sets of full-length cDNA (FLcDNA) clones, now provides annotation for 120 558 human mRNAs extracted from the International Nucleotide Sequence Databases (INSD), in addition to 54 978 human FLcDNAs, in the latest release H-InvDB_4.6. We mapped those human transcripts onto the human genome sequences (NCBI build 36.1) and determined 34 699 human gene clusters, which could define 34 057 (98.1%) protein-coding and 642 (1.9%) non-protein-coding loci; 858 (2.5%) transcribed loci overlapped with predicted pseudogenes. For all these transcripts and genes, we provide comprehensive annotation including gene structures, gene functions, alternative splicing variants, functional non-protein-coding RNAs, functional domains, predicted sub cellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs, co-localization with orphan diseases, gene expression profiles, orthologous genes, protein-protein interactions (PPI) and annotation for gene families. The current H-InvDB annotation resources consist of two main views: Transcript view and Locus view and eight sub-databases: the DiseaseInfo Viewer, H-ANGEL, the Clustering Viewer, G-integra, the TOPO Viewer, Evola, the PPI view and the Gene family/group.

A citrus polymethoxy flavonoid, nobiletin inhibits sebum production and sebocyte proliferation, and augments sebum excretion in hamsters.

Acne vulgaris is characterized by excess sebum production, and apart from all-trans retinoic acid (atRA) or 13-cis retinoic acid (13-cisRA), there are few effective agents for acne therapy that directly suppresses sebaceous lipogenesis. In this study, we demonstrated that topical application of a citrus polymethoxy flavonoid, nobiletin, to hamster auricles decreased skin surface triacylglycerols (TG) level and the size of sebaceous glands along with inhibition of diacylglycerol acyltransferase (DGAT)-dependent TG synthesis and sebocyte proliferation. The inhibitory actions were similar to that observed with atRA and 13-cisRA in hamster sebocytes. The antilipogenic and antiproliferative actions of nobiletin were also reproduced in UVB (5.4 kJ/m2)-irradiated hamsters, which showed aberrant enhancement of sebum accumulation and sebaceous enlargement. Furthermore, nobiletin, but not 13-cisRA, augmented sebum excretion along with increases in intracellular cAMP level, protein kinase A (PKA) activation, and apoptosis-independent phosphatidylserine (PS) externalization in cell membrane. These phenomena were reproduced by forskolin and inhibited by a PKA inhibitor, H-89. These results provide early evidence that nobiletin is an effective candidate for acne therapy through mechanisms that include the inhibition of DGAT-dependent TG synthesis and sebocyte proliferation, and the progression of apoptosis-independent and PS-externalization-dependent sebum excretion by PKA activation.

A systematic evaluation of the function of the protein-remodeling factor Hsp104 in [PSI+] prion propagation in S. cerevisiae by comprehensive chromosomal mutations.

The yeast prion [PSI(+)] represents an aggregated state of the translational release factor Sup35 (eRF3) and deprives termination complexes of functional Sup35, resulting in nonsense codon suppression. Protein-remodeling factor Hsp104 is involved in thermotolerance and [PSI(+)] propagation, however the structure-and-function relationship of Hsp104 for [PSI(+)] remains unclear. In this study, we engineered 58 chromosomal hsp104 mutants that affect residues considered structurally or functionally relevant to Hsp104 remodeling activity, yet most remain to be examined for their significance to [PSI(+)] in the same genetic background. Many of these hsp104 mutants were affected both in thermotolerance and [PSI(+)] propagation. However, nine mutants were impaired exclusively for [PSI(+)], while two mutants were impaired exclusively for thermotolerance. Mutations exclusively affecting [PSI(+)] are clustered around the lateral channel of the Hsp104 hexamer. These findings suggest that Hsp104 possesses shared as well as distinct remodeling activities for stress-induced protein aggregates and [PSI(+)] prion aggregates and that the lateral channel plays a role specific to [PSI(+)] prion propagation.