Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Lipid content, ATP content, and histone acetylation are thought to reflect the energy state of cells. In addition, the energy state closely associates with growth and developmental ability of oocytes. Oocyte growth is accompanied by active proliferation of the surrounding granulosa cells (GCs), and GCs play a key role in the provision of energy substrates to the oocytes. In the present study, we first examined the relationship among the average number of GCs per follicle, the average number of cumulus cells (CCs) per oocyte, and the average lipid content in oocytes that developed in vivo within individual donor gilts. Second, we validated the relationship between the number of cells surrounding oocytes and the energy states of oocytes by using an IVC system of oocyte granulosa cell complexes (OGCs) derived from early antral follicles. We collected cumulus cells and oocyte complexes (COCs) from antral follicles (3-5 mm in diameter) and found that average lipid content in oocytes significantly correlated with the average number of both GCs/follicle and CCs/oocyte (P < 0.05). In the next series of experiments, we collected OGCs from early antral follicles (0.5-0.7 mm in diameter), and cultured them for 14 days, and then determined the cell number of OGCs, as well as the lipid content, ATP content, and acetylation level of H4K12 in oocytes grown in vitro. In addition, glucose consumption by OGCs was calculated from the sample media collected at Days 13 and 14. The lipid content of oocytes grown in vitro, significantly correlated with the number of cells surrounding the oocytes (P < 0.01) and with the level of glucose consumption by OGCs (P < 0.01). In addition, both ATP content and H4K12 acetylation levels of oocytes grown in vitro significantly correlated with the number of cells surrounding the oocytes (P < 0.05) and glucose consumption by OGCs (P < 0.05). In conclusion, the lipid content of oocytes depends on the number of cells surrounding the oocytes, and glucose uptake by OGCs is crucial for lipid content and ATP content, and H4K12 acetylation in oocytes. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2016.05.036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

Follicle development is accompanied by proliferation of granulosa cells and increasing oocyte size. To obtain high-quality oocytes in vitro, it is important to understand the processes that occur in oocytes and granulosa cells during follicle development and the differences between in vivo and in vitro follicle development. In the present study, oocytes and granulosa cells were collected from early antral follicles (EAFs, 0.5-0.7 mm in diameter), small antral follicles (SAFs, 1-3 mn in diameter), large antral follicles (LAFs, 3-7 mm in diameter), and in vitro grown oocyte-and-granulosa cell complexes (OGCs), which were cultured for 14 days after collection from EAFs. Gene expression was analyzed comprehensively using the next-generation sequencing technology. We found top upstream regulators during the in vivo follicle development and compared them with those in in vitro developed OGCs. The comparison revealed that HIFI is among the top regulators during both in vivo and in vitro development of OGCs. In addition, we found that HIF1-mediated upregulation of glycolysis in granulosa cells is important for the growth of OGCs, but the cellular metabolism differs between in vitro and in vivo grown OGCs. Furthermore, on the basis of comparison of upstream regulators between in vivo and in vitro development of OGCs, we believe that low expression levels of FLT1 (VEGFA receptor), SPP1, and PCSK6 can be considered causal factors of the suboptimal development under in vitro culture conditions.

DOI： 10.1262/jrd.2016-022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

A plasmid-based reverse genetics system for human astrovirus type 1 (HAstV1) is examined. Upon transfection into 293T cells, the plasmid vector, which harbors a HAstV1 expression cassette, expressed astroviral RNA that appeared to be capable of viral RNA replication, as indicated by the production of subgenomic RNA and capsid protein expression irrespective of the heterologous 5 ends of the transcribed RNA. Particles infectious to Caco-2 cells were made in this system; however, their infectivity was much lower than would be expected from the amount of particles apparently produced. Using Huh-7 cells as the transfection host with the aim of improving viral capsid processing for virion maturation partially restored the efficiency of infectious particle formation. Our results support the possibility that the DNA transfection process induces a cellular response that targets late, but not early, stages of HAstV1 infection.

DOI： 10.1111/1348-0421.12317

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

In this study, we examined the effects of reconstructed oocyte granulosa cell complexes (OGCs) on the development of porcine oocytes derived from early antral follicles (EAFs; 0.5-0.7 mm in diameter). When denuded oocytes were cocultured with granulosa cells derived from other EAFs, the oocytes and granulosa cells aggregated to form OGCs after 2 days of culture. After 14 days of culture, we compared cell number, oocyte diameter, and oocyte chromatin configuration in unmanipulated (natural) OGCs, reconstructed OGCs, and OGCs collected from antral follicles (AFs, 3.0-6.0 mm in diameter). The diameters of oocytes from reconstructed OGCs grown in vitro were not different from those of oocytes from natural OGCs, although they were significantly smaller than those of oocytes from antral follicle (AF) OGCs. Oocyte chromatin configuration did not differ among the 3 OGC groups, but the oocyte nuclear maturation rate was lower in the reconstructed OGCs and higher in the AF OGCs. However, when the in vitro culture period for the reconstructed OGCs was extended by 2 days, the nuclear maturation rate of oocytes from reconstructed OGCs was similar to that of oocytes from natural OGCs. In addition, blastocysts were successfully obtained from oocytes from reconstructed OGCs. In conclusion, we established an innovative culture method that allows oocytes and granulosa cells from EAFs to reaggregate as reconstructed OGCs, which yield oocytes with the ability to develop to the blastocyst stage.

DOI： 10.1262/jrd.2014-123

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Language：English   Publishing type：Research paper (scientific journal)  

This study examined the effect of hyperglycemic culture conditions on the development of oocytes derived from early antral follicles (EAFs). Oocyte - granulosa cell complexes (OGCs) derived from EAFs were cultured for 12 days in medium containing 5.56 mM or 11 mM glucose, and the rate of antrum formation and glucose consumption by the OGCs, and the characteristics of the oocytes grown in vitro were studied. The results were compared with those obtained from in vivo grown oocytes derived from antral follicles (AFs; 3-5 mm in diameter). In addition, the effect of a high glucose concentration in the maturation medium on the quality of oocytes derived from AFs was examined. The high glucose condition increased the glucose consumption of the OGCs but did not affect antrum formation, oocyte diameter, chromatin configuration, levels of histone 4 K12 acetylation, nuclear maturation, and the developmental ability of oocytes grown in vitro. In contrast, high glucose-maturation medium increased the amount of reactive oxygen species and adversely affected the developmental ability of the oocytes. In conclusion, the results of this study suggest that culture under hyperglycemic conditions is detrimental to oocyte maturation but not for oocyte growth from the EAF stage to the AF stage.

DOI： 10.1274/jmor.32.41

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The present study assessed the effect of resveratrol on the expression of SIRT1 and mitochondrial quality and quantity in porcine oocytes. Supplementing the maturation medium with 20 mu M resveratrol increased the expression of SIRT1, and enhanced mitochondrial functions, as observed from the increased ATP content and mitochondrial membrane potential. Addition of resveratrol also improved the ability of oocytes to develop into the blastocyst stage following activation. The effects of resveratrol on mitochondrial number were examined by comparing the mitochondrial DNA copy number (Mt number) between group of oocytes collected from the same donor gilt ovaries. Supplementing the maturation medium with only resveratrol did not affect the Mt number in the oocytes. However, supplementing the maturation medium with 10 mu M MG132, a proteasome inhibitor, significantly increased the amount of ubiquitinated proteins and Mt number by 12 and 14%, respectively. In addition, when resveratrol was added to the medium containing MG132, the Mt number increased significantly by 39%, this effect was diminished by the addition of the SIRT1 inhibitor EX527. Furthermore, supplementing the medium with MG132 and EX527 did not affect Mt number. The mean SIRT1 expression in 20 oocytes was significantly and positively correlated with the Mt number in oocytes collected from the same donor. This study suggests that the expression of SIRT1 is associated with the Mt number in oocytes. In addition, activation of SIRT1 by resveratrol enhances the biosynthesis and degradation of mitochondria in oocytes, thereby replenishing and improving mitochondrial function and the developmental ability of oocytes.

DOI： 10.1371/journal.pone.0094488

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Antrum formation and estradiol (E2) secretion occur during early folliculogenesis. The objective was to determine the role of E2 in antrum formation of oocyte-granulosa cell complexes (OGCs) derived from porcine preantral follicles (PAFs). Supplementation of the culture medium with E2 (1 mu g/mL) improved antrum formation of OGCs during 14 days of in vitro culture. Furthermore, adding 0.1 mu g/mL androstenedione (a precursor of E2) to the medium also improved antrum formation. Concentration of E2 was higher in the medium of developmentally competent OGCs versus incompetent OGCs (8.5 vs. 3.5 ng/mL, P < 0.05). Fulvestrant (1 mu g/mL), a competitive inhibitor of E2, completely inhibited antrum formation of OGCs that were cultured in medium containing either E2 (0.1 mu g/mL) or androstenedione (0.1 mu g/mL); however, increasing E2 to 1 mu g/mL ameliorated the inhibitory effect. Conversely, in the case of early antral follicles, OGCs formed antrums without E2 supplementation. After E2 pretreatment, OGCs derived from PAFs formed antrums even when the OGCs were subsequently cultured in medium without E2. Furthermore, when OGCs derived from PAFs were cultured without E2 followed by an additional in vitro culture with E2, antrums were formed, albeit with the same period delay by the same pretreatment periods. In conclusion, E2 in the culture medium was indispensable for in vitro antrum formation of OGCs derived from PAFs; therefore, one of the roles of E2 is in the initiation of antrum formation. (c) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2012.12.009

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Language：Japanese   Presentation type：Oral presentation (general)  

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