Naoki Takenouchi

In Vitro Growth and Development of Bovine Oocyte-Granulosa Cell Complexes on the Flat Substratum: Effects of High Polyvinylpyrrolidone Concentration in Culture Medium

Abstract The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 μm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 μg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360 000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.

Effects of Sodium Pyruvate in Nonserum Maturation Medium on Maturation, Fertilization, and Subsequent Development of Bovine Oocytes With or Without Cumulus Cells

Abstract The present study was conducted to determine the effects of cumulus cells and sodium pyruvate during in vitro maturation of bovine oocytes on maturation, fertilization, and subsequent development. Cumulus-enclosed oocytes (CEOs) and cumulus-denuded oocytes (CDOs) were cultured for 24 h in polyvinylpyrrolidone-Hepes-tissue culture medium 199 with or without sodium pyruvate. Oocytes were fertilized in vitro and then cultured in CR1aa for 10 days. Before in vitro fertilization, the glutathione (GSH) content of some oocytes was measured. Maturation and normal fertilization rates of CDOs cultured with sodium pyruvate and CEOs were higher than that of CDOs cultured without sodium pyruvate. The CEOs showed significantly higher rates of development to the blastocyst stage than CDOs. The GSH contents of oocytes significantly decreased in CDOs after maturation culture, but the GSH contents of oocytes in CEOs remained at the same level as oocytes before culture. These results indicate that sodium pyruvate promotes nuclear maturation of bovine CDOs and that a continuing presence of cumulus cells during maturation is important for subsequent development of zygotes to the blastocyst stage. However, blastocysts produced from CDOs in the presence of sodium pyruvate showed a developmental competence to be normal calves, but it is not known if CDOs cultured without sodium pyruvate also were capable of developing into calves.

In vitro maturation and glutathione synthesis of porcine oocytes in the presence or absence of cysteamine under different oxygen tensions: role of cumulus cells

The present study was conducted to evaluate the effect of cumulus cells on the in vitro maturation (IVM) and glutathione (GSH) synthesis of porcine oocytes cultured in the presence or absence of cysteamine under different oxygen tensions, and on their subsequent male pronucleus formation after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 45 h in modified TCM-199 supplemented with or without 150 microM cysteamine under a humidified atmosphere of 5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2. When cultured in medium supplemented with cysteamine under 20% O2 tension, the rates of COC maturation to the metaphase II (MII) stage were significantly higher than those of DOs (P<0.05). Regardless of the addition of cysteamine and oxygen tension, the rates of male pronucleus formation in COCs after IVM and IVF were significantly higher than in DOs (P<0.05). The GSH content of oocytes was significantly increased by the addition of cysteamine to the maturation medium (P<0.05), with significantly higher GSH content in COCs than in DOs (P<0.05). However, the GSH content of COCs and DOs was not significantly different when cultured in medium without cysteamine. These results indicate that cumulus cells play an important role in nuclear maturation to MII, GSH synthesis in porcine oocytes cultured in the presence of cysteamine, and subsequent male pronucleus formation after IVF.

Effects of thioredoxin on the preimplantation development of bovine embryos

Thioredoxin (TRX) is an ubiquitous protein disulfide reductase, which is known to be involved in the implantation development of mouse embryos. In the present study, recombinant human TRX was used to evaluate its effect on the promotion of preimplantation development of bovine embryos derived from in vitro maturation and fertilization. Supplementation of the medium 24h post insemination with TRX significantly (P<0.05) enhanced the frequency of development to the blastocyst stage in 5% O(2) concentration. The optimal concentration was 0.5 microg/ml (P<0.05, compared with 0, 0.1 and 1.0 microg/ml). This effect of TRX was evident only when added around the time of the first cleavage stage (24 h post insemination); no promotion was found with treatment at 6h (one-cell) or 44 h (six- to eight-cell) after insemination. Moreover, it is of interest that even with the best combination of the dose and timing of TRX treatment (0.5 microg/ml, at 24 h post insemination), no promotion of development was observed when embryos were cultured under 20% O(2). However, a preincubation of TRX in the culture medium under 20% oxygen for 24h did not diminish the promoting effect in the subsequent TRX treatment under optimal conditions, thus suggesting that the possible oxidation of TRX alone may not be the reason for the disappearance of the effect under a high oxygen concentration. These results indicate that TRX does improve the development of bovine embryos in vitro, though unlike the general reducing reagents such as beta-mercaptoethanol or cysteamine, TRX may have to exert its effect at specific times and in more physiologic oxygen environments.

Observation by ultrasonography of embryonic loss following the transfer of two or three embryos in beef cows

Abstract Ultrasonographic observations were carried out at 10-day intervals from 27 to 107 days of gestation, to monitor embryonic losses in Japanese Black cows which received two or three embryos that had been transferred nonsurgically on Day 7 (Day 0 = estrus). Group I cows (n=60) received two embryos, one in each uterine horn. Group II cows (n=31) received three embryos, two embryos in the uterine horn ipsilateral to the corpus luteum and one embryo in the uterine horn contralateral to the corpus luteum. The Group II pregnancy rate was maintained above 83.9% until Day 37, but it decreased significantly (P

Influence of meiotic inhibition by butyrolactone-I during germinal vesicle stage on the ability of porcine oocytes to be activated by electric stimulation after nuclear maturation.

Butyrolactone-I (BL-I) is a specific inhibitor of cyclin-dependent kinases and prevents germinal vesicle breakdown (GVBD) in porcine oocytes. This study first focused on the effect of BL-I on the time course of GVBD and progression to metaphase II (MII) in oocytes after the removal of BL-I. When porcine oocytes were treated with 20 microM BL-I for 28 h, the intervals taken to undergo GVBD and progress to MII were 4-8 h and 16-20 h after washing out BL-I, respectively. These intervals were both approximately 8 h behind those of the control oocytes. When the BL-I treatment was performed for 20 h, instead of 28 h, there were no differences in the timing or frequency of progression to MII between the BL-I treatment and control groups. To determine whether the cytoplasmic maturation was the same, the ability of oocytes to form a female pronucleus in response to an electric stimulus was examined. When oocytes were stimulated at 28 h after the removal of BL-I, the rate of pronucleus formation was significantly lower in oocytes treated with BL-I than in untreated oocytes, despite the fact that both groups reached MII at a similar time. When the electric stimulus was given at 36 h after the removal of BL-I, oocytes had a pronucleus formation rate comparable to that of control oocytes. Therefore, the findings suggest that BL-I treatment of porcine oocytes at the germinal vesicle stage affects not only the progression of meiosis, but also the events involved in cytoplasmic maturation.

Production of Fertile Offspring from Oocytes Grown In Vitro by Nuclear Transfer in Cattle

ABSTRACT Because of recent advancements in reproductive technology, oocytes have attained an increasingly enriched value as a unique cell population in the production of offspring. The growing oocytes in the ovary are an immediate potential source that serve this need; however, complete oocyte growth before use is crucial. Our research objective was to create in vitro-grown (IVG) oocytes that would have the ability to perform specialized activities, including nuclear reprogramming, as an alternative to in vivo-grown oocytes. Bovine oocyte–granulosa cell complexes with a mean oocyte diameter of approximately 100 μm were cultured on Millicell membrane inserts, with culture medium supplemented with 4% polyvinylpyrrolidone (molecular weight, 360 000), 20 ng/ml androstenedione, 2 mM hypoxanthine, and 5 ng/ml bone morphogenetic protein 7. Oocyte viability after the 14-day culture period was 95%, and there was a 71% increase in oocyte volume. Upon induction of oocyte maturation, 61% of the IVG oocytes extruded a polar body. Eighty-four percent of the reconstructed IVG oocytes that used cumulus cells as donor cells underwent cleavage, and half of them became blastocysts. DNA methylation analyses of the satellite I and II regions of the blastocysts revealed a similar highly methylated status in the cloned embryos derived from in vivo-grown and IVG oocytes. Finally, one of the nine embryos reconstructed from the IVG oocytes developed into a living calf following embryo transfer. Fertility of the offspring was confirmed. In conclusion, the potential of a proportion of the IVG oocytes was comparable to that of in vivo-grown oocytes.

Effect of androstenedione on the growth and meiotic competence of bovine oocytes from early antral follicles.

Medium that contains 17β-estradiol has been reported to support in vitro growth of bovine oocytes, isolated from early antral follicles, until the final stage. The aim of this study was to determine the effects of androstenedione in medium on such growing bovine oocytes. Oocyte-granulosa cell complexes were collected from early antral follicles and cultured for 14 days in medium supplemented with 17β-estradiol (0, 10 and 100 ng/ml) or androstenedione (0, 10 and 100 ng/ml). The mean diameter of oocytes measured after seeding on the culture substrate was 96.9 μm (n = 191). Either steroid was necessary for maintainance of the organization of oocyte-granulosa cell complexes over the 14-day culture period. In the 17β-estradiol- or the androstenedione-supplemented medium about 80% or 65%, respectively, of viable oocytes were recovered. In both groups the increase in oocyte size was significant after 14 days. The in vitro grown oocytes were cultured for a further 22-24 h for oocyte maturation; 13% and 30% of oocytes grown in the 10 and 100 ng/ml 17β-estradiol-supplemented medium reached metaphase II, respectively; more than 64% of oocytes grown in the androstenedione-supplemented medium matured to metaphase II. These results show that androstenedione, as 17β-estradiol, can maintain the viability of bovine oocyte-granulosa cell complexes and support the growth of oocytes, and that androstenedione promotes the acquisition of oocyte meiotic competence efficiently at a low dose.

Effects of selenium supplementation on plasma progesterone concentrations in pregnant heifers

It is known that selenium (Se) has various functions in animals. Many investigations on the biochemical and physiological effects of Se have been previously reported; however, the detailed function of Se in reproduction is not yet clear. We proposed the possibility that Se plays a notable role in progesterone production. The aim of this study was to clarify the effects of Se supplementation on progesterone levels of pregnant Holstein heifers. Eight Holstein heifers (-Se) were fed basal diet (containing 0.022 ppm of Se) throughout the experiment. While a 0.3 ppm diet of Se (sodium selenite) was fed to another seven animals (+Se) with basal diet. Blood sampling was carried out every week. Plasma Se concentrations were higher in Se-supplemented cows compared with controls (-Se) (P < 0.01) throughout the experiment. Se supplementation increased plasma progesterone in the 29-39 weeks of pregnancy from 4.98 ± 0.64 to 6.86 ± 0.49 ng/mL on average (P < 0.05). The present findings suggest that Se contributes to maintaining the function of the corpus luteum and/or placenta in the latter period of pregnancy.

Vaginal temperature measurement by a wireless sensor for predicting the onset of calving in Japanese Black cows

We evaluated the utility of the continuous measurement of vaginal temperature by a wireless sensor and wireless connection for predicting the onset of calving and for clarifying the relationships among dystocia, calf conditions, and temperature changes at a commercial beef cattle farm in Japan. A total of 625 effective delivery data was collected. The temperature sensor inserted to the vagina on 7 days before the expected due date and collected the vaginal temperature every 5 min. The sensor detected two alerts according to the temperature change, one was the vaginal temperature of 4 h moving average compared to the same time temperature of last two days decreased more than 0.4 °C (Alert 1) and the other was the rupture of the allantoic sac and the dropped sensor temperature reached to the ambient temperature (Alert 2). The detection rates of Alert 1 and Alert 2 were 88.3% and 99.4%, respectively. The average time between Alert 1 and Alert 2 (Time 1) was 22 h, and that between Alert 2 and delivery (Time 2) was 2 h. These results indicated that the continuous measurement of vaginal temperature is effective for predicting the calving time. The necessity of assistance was correlated with dystocia, calf birth weight (BW), sex, and gestation periods. Interestingly, the durations of Times 1 and 2 were also associated with dystocia. The calf BW, sex, and gestation periods affected the length of Time 2. Our findings indicate that the BW of the calf is the most important factor for dystocia risk, and that the continuous measurement of vaginal temperature could become a good indicator for predicting not only the onset of calving, but also the necessity of assistance.