Takehito Kuwayama

Effect of maternal age on mitochondrial DNA copy number, ATP content and IVF outcome of bovine oocytes

The primary aim of the present study was to examine the effect of maternal age (in months) on mitochondrial DNA copy number (Mt number), ATP content and IVF outcome of bovine oocytes. We also compared the Mt number of oocytes with fertilisation outcome and ATP content. Oocytes were collected from cows aged 20-204 months and the Mt number was determined by real-time polymerase chain reaction. The Mt number in immature and mature oocytes was determined to be 368,118 and 807,794, respectively; the ATP content in these oocytes was 1.2 and 2.0 pM, respectively. Both Mt number and ATP content increased during oocyte maturation. However, after 90 months of age, the Mt number of mature oocytes decreased with increasing maternal age, whereas the ATP content of mature oocytes was positively correlated with maternal age (P<0.01); there was no obvious relationship observed between Mt number and ATP content. Furthermore, maternal age was positively correlated with the abnormal fertilisation rate (P<0.01). Mt number and fertilisation outcome were unrelated, but the nature of this relationship differed between young (21-89 months) and old (>89 months) cows. Thus, we conclude that Mt number, the ATP content and fertilisation outcome of bovine oocytes are affected by maternal age.

Relationship between mitochondrial DNA Copy Number and SIRT1 Expression in Porcine Oocytes

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 µM 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 µM 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.

Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes

The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization.

Next-generation sequencing reveals genomic features in the Japanese quail.

The Japanese quail has several advantages as a laboratory animal for biological and biomedical investigations. In this study, the draft genome of the Japanese quail was sequenced and assembled using next-generation sequencing technology. To improve the quality of the assembly, the sequence reads from the Japanese quail were aligned against the reference genome of the chicken. The final draft assembly consisted of 1.75 Gbp with an N50 contig length of 11,409 bp. On the basis of the draft genome sequence obtained, we developed 100 microsatellite markers and used these markers to evaluate the genetic variability and diversity of 11 lines of Japanese quail. Furthermore, we identified Japanese quail orthologs of spermatogenesis markers and analyzed their expression using in situ hybridization. The Japanese quail genome sequence obtained in the present study could enhance the value of this species as a model animal.

Age-associated changes in gene expression and developmental competence of bovine oocytes, and a possible countermeasure against age-associated events.

In general, maternal age affects the quality of oocytes and embryos. The present study aimed to examine the features and age‐associated gene expression profiles of bovine oocytes and embryos as well as to discover possible countermeasures against age‐associated events. Comprehensive gene expression assays of germinal vesicle and metaphase II (MII)‐stage oocytes and 8‐ to 16‐cell‐stage embryos were conducted using next‐generation sequencing technology. The gene expression profiles of aged cows showed high expression of genes related to oxidative phosphorylation, eIF4 and p70S6K signaling, and mitochondrial dysfunction in MII‐stage oocytes. Oocytes derived from aged cows, compared with those derived from their younger counterparts, exhibited high levels of abnormal fertilization and blastocysts with low total cell numbers. Levels of reactive oxygen species (ROS) and SIRT1 were higher in in vitro‐matured oocytes derived from aged cows than in those derived from their younger counterparts. Supplementation of maturation medium with N‐acetyl‐cysteine (NAC), but not resveratrol, reduced the levels of ROS in the oocytes derived from cows of both age groups; however, resveratrol, but not NAC, improved the fertilization ratio. Conversely, EX 527, an inhibitor of SIRT1, increased the ratio of abnormal fertilization. In conclusion, gene expression profiles of oocytes and embryos derived from aged cows differ from those of oocytes and embryos derived from young cows; in particular, oocytes derived from aged cows show protein and mitochondrial dysfunction. In addition, activation of SIRT1 in oocytes may be a potential countermeasure against age‐associated events in oocytes derived from aged cows. Mol. Reprod. Dev. 80: 508–521, 2013.

Effect of Maternal Age on the Ratio of Cleavage and Mitochondrial DNA Copy Number in Early Developmental Stage Bovine Embryos

Abstract Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease.

Effect of maternal age on the developmental competence and progression of nuclear maturation in bovine oocytes.

Progression of meiotic division in oocytes and early embryonic development are affected by oocytes quality. In most mammals, oocyte quality declines with increase in maternal age. The main aim of the present study is to investigate the effect of maternal age on developmental competence, progression of meiotic division, and associated kinetics of maturation promoting factor (MPF) activity in bovine oocytes. Oocytes were collected from the ovaries of young and old cows (here after referred to as young cow oocytes and old cow oocytes, respectively). When old cow oocytes were matured and fertilized in vitro, the rate of abnormal fertilization was greater than that in young cow oocytes. Moreover, progression of nuclear maturation and activation of MPF during oocyte maturation (or inactivation of MPF and formation of pronucleus after insemination) were faster in old cow oocytes than in young cow oocytes. Relative expression of cyclin B, cyclin‐dependent kinase 1 and MAD2 transcripts in either immature or mature oocytes did not differ between the two groups. When cumulus cells (CC) were removed and denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two age groups. Moreover gap junctions between oocytes and CC disappeared more rapidly during maturation of old cow oocytes than of young cow oocytes. These results suggest that the fertilization ability of old cow oocytes is low and that premature progression of meiotic division in these oocytes is partly due to impaired oocyte–CC gap junctions communication. Mol. Reprod. Dev. 77: 595–604, 2010.

Estradiol has a major role in antrum formation of porcine preantral follicles cultured in vitro

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 μg/mL) improved antrum formation of OGCs during 14 days of in vitro culture. Furthermore, adding 0.1 μg/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 μg/mL), a competitive inhibitor of E2, completely inhibited antrum formation of OGCs that were cultured in medium containing either E2 (0.1 μg/mL) or androstenedione (0.1 μg/mL); however, increasing E2 to 1 μg/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.

Estradiol supports in vitro development of bovine early antral follicles

Antrum formation and estradiol (E(2)) secretion are specific features of oocyte and granulosa cell complexes (OGCs). This study investigates the effect of E(2) on the in vitro development of bovine OGCs derived from early antral follicles as well as on the expression of genes in granulosa cells (GCs). The supplementation of culture medium with either E(2) or androstenedione (A(4)) improved the in vitro development of OGCs and the nuclear maturation of enclosed oocytes. When OGCs were cultured in medium containing A(4), developmentally competent OGCs secreted more E(2) than OGCs that were not competent. In addition, fulvestrant inhibited the effect of both E(2) and A(4) on OGCs development. Comprehensive gene expression analysis using next-generation sequence technology was conducted for the following three types of GCs: i) GCs of OGCs cultured for 4 days with E(2) (1 μg/ml; E(2)(+)), ii) GCs of OGCs cultured for 4 days without E(2) (E(2)(-)) or iii) OGCs that formed clear antrum after 8 days of in vitro culture in medium containing E(2) (1 μg/ml; AF group). GCs of the E(2)(+) group had a similar gene expression profile to the profile reported previously for the in vivo development of large follicles. This genetic profile included factors implicated in the up-regulation of E(2) biosynthesis and down-regulation of cytoskeleton and extracellular matrices. In addition, a novel gene expression profile was found in the AF group. In conclusion, E(2) impacts the gene expression profile of GCs to support the in vitro development of OGCs.

Resveratrol-induced mitochondrial synthesis and autophagy in oocytes derived from early antral follicles of aged cows

Mitochondrial numbers increase during oocyte growth. In this study, we collected oocytes and granulosa cell complexes (OGCs) from early antral follicles (EAFs) of aged cows (> 120 months of age) and examined the effects of resveratrol on mitochondrial generation, degradation, and quality in oocytes grown in vitro. We also examined the effects of resveratrol on gene expression of the granulosa cells. Resveratrol (20 µM) enhanced the expression of SIRT1 and induced autophagy in both granulosa cells and oocytes derived from aged cows. Culturing the OGCs with resveratrol increased mitochondrial DNA copy numbers in oocytes grown in vitro. Furthermore, resveratrol increased the ATP content in oocytes and improved the developmental ability of the oocytes to the blastocyst stage. Gene expression profiles in granulosa cells, as evaluated by next-generation sequencing technology, revealed that resveratrol enhanced the expression of EIF2-related genes but downregulated the expression of mammalian target of rapamycin (mTOR)-, inflammation-, and cholesterol homeostasis-related genes in granulosa cells. In conclusion, resveratrol affected both oocytes and granulosa cells derived from aged cows and improved the quality of oocytes grown in vitro through upregulation of mitochondrial biogenesis and degradation in growing oocytes and conditioning of granulosa cells.