-
[show abstract]
[hide abstract]
ABSTRACT: A previous study revealed that follicle superstimulation significantly improved the developmental competence of immature bovine oocytes collected by ovum pickup (OPU; Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The aim of the present study was to investigate the effect of follicle superstimulation on the expression of developmentally important genes in bovine oocytes collected by OPU. Follicular oocytes were collected by OPU without (OPU group) or after follicle superstimulation by FSH (FSH/OPU group) by using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner according to Imai et al. (2008). In the FSH/OPU group, after dominant follicle removal from Holstein dry cows by OPU, a CIDR was inserted on Day 5 (dominant follicle removal=Day 0). Cows then received 30mg of FSH twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2mg) by IM injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75mg) was administered in the morning of Day 9 (third day of superstimulation). Oocyte collection by OPU was performed 48h after PGF administration (Day 11) by the aspiration of follicles larger than 5mm in diameter. In the OPU group, 3-to-6-mm follicles were aspirated without any previous hormone treatment. In vitro oocyte maturation (IVM) was performed according to Imai et al. (2006 J. Reprod. Dev. 52(Suppl), 19-29). Gene expression was assessed before (0h IVM) and after IVM (22h IVM) by RT quantitative PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1 and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using Qiagen RNeasy Micro kit (Qiagen, Valencia, CA, USA). Gene expression was analysed by a Roche Light Cycler 480 device. The relative expression of each gene was normalized to ACTB. Three replications were performed. Data were analysed by ANOVA. At 0h IVM, PAP and DYNC 1/1 were found to be down-regulated (P<0.05) in the FSH/OPU group compared with the OPU group, whereas the rest of the studied genes showed similar expression in the FSH/OPU and OPU groups. At 22h IVM, PAP and DYNC 1/1 remained down-regulated in FSH/OPU oocytes. However, at this time the expression of GDF9 appeared significantly higher (P<0.05) in FSH/OPU oocytes than in OPU oocytes. The expression of GDF9 was found to decrease during IVM in both groups; however, this decrease was less drastic in FSH/OPU oocytes. The results suggest that follicle superstimulation caused reduced expression of mRNA levels of PAP and DYNC 1/1 irrespective of maturation status and it also moderated the reduction of mRNA levels of GDF9 during IVM.
Reproduction Fertility and Development 12/2011; 24(1):181-2. · 2.11 Impact Factor
-
T Somfai,
K Kikuchi,
M Kaneda,
S Akagi,
S Watanabe,
E Mizutani,
S Haraguchi,
T Q Dang-Nguyen, Y Inaba,
M Geshi,
T Nagai
[show abstract]
[hide abstract]
ABSTRACT: We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.
Anantomia Histologia Embryologia 05/2011; 40(5):335-44. · 0.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The efficiency of somatic cell cloning is very low, probably because of incomplete reprogramming of the somatic cell nucleus. In recent studies, it is suggested that transient exposure of donor somatic cells to mouse embryonic stem cell (ESC) extract enhances pluripotency of the cells in vitro (Bru et al. 2008 Exp. Cell Res. 314, 1634-1642; Xu et al. 2009 Anat. Rec. 292, 1229-1234). In the present study, we examined the effect of treatment of donor cells with mouse ESC extract on the in vitro development of bovine NT embryos. First, in order to examine effect of treatment of donor cells with streptolysin O (SLO), which reversibly permeabilizes the plasma membrane, we compared the in vitro development of NT embryos using donor cells treated with 5μgmL(-1) SLO (SLO group) and untreated donor cells (control group). As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used. Fibroblasts were treated with 5μgmL(-1) SLO for 45min, and then incubated for resealing in DMEM including 2mM CaCl(2) for 60min. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264-272). After in vitro culture for 8 days, blastocyst formation and cell number of blastocysts were examined. There were no significant differences between SLO and control groups in the fusion rate (80% and 72%, respectively), cleavage rate (60% and 65%, respectively), developmental rate to the blastocyst stage of NT embryos (31% and 28%, respectively), and blastocyst cell number (127±6 and 112±14, respectively). These results suggest that SLO treatment of donor cells has no negative effect on the in vitro development of NT embryos. Next, we examined the in vitro developmental ability of NT embryos using donor cells treated with mouse ESC extract (ES extract group). After SLO treatment for 45min, permeabilized fibroblast cells were treated with mouse ESC extract for 45min, and then incubated in DMEM including 2mM CaCl(2) for 60min, and used for producing NT embryos. There were no differences between ES extract and control groups in the fusion rate (68% and 69%, respectively), cleavage rate (86.7% and 80.6%, respectively), and developmental rate to the blastocyst stage of NT embryos (39.8% and 43.5%, respectively). The cell number of NT embryos at the blastocyst stage in ES extract group (201±30) was significantly (t-test; P<0.05) higher than that in control group (140±14). In conclusion, treatment of bovine donor cell with mouse ESC extract did not affect the in vitro developmental ability of NT embryos, but improved the quality of blastocysts.
Reproduction Fertility and Development 01/2011; 23(1):119. · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bovine interferon (bIFN) τ has been implicated as a mediator of maternal recognition of pregnancy in cattle. (Geshi et al. 2001 Theriogenology 55, 325) reported that daily intrauterine infusion of recombinant (r) bIFNτ from Day 13 to 24 extended interestrous intervals in heifers. The objective of this study was to determine whether a single infusion of sustained release bIFNτ into the uterine horn would extend corpus luteum lifespan in cyclic cows. RbIFNτ was prepared from the Silkwarm-Baculovirus gene expression system (1mgmL(-1), 1×10(8)IUmg(-1), Nagaya et al. 2004 J. Vet. Med. Sci. 66, 1395-1401). Two sustained release carriers, liposome and aluminum hydroxide (Al) gel were tested. Liposome encapsulated (lipo) BSA (control) and rbIFNτ were prepared from a lipid mixture solution contained 2mg of each protein by the Bangham method including lyophilization and rehydration. The same amount of BSA or rbIFNτ was adsorbed to Al-gel containing 2.5mg Al. Adsorption was allowed to proceed for 5min at room temperature and unbound proteins were removed by centrifugation. Subsequently, lipo- or Al-gel adsorbed BSA and rbIFNτ were adjusted to 0.5mL with saline and loaded into 0.5-mL AI straws. BIFNτ free from liposome or unbound to Al-gel was measured by RIA. Eight Japanese Black cows were used in this study. Their signs of oestrus were monitored twice a day. Cows were randomly assigned to four groups, receiving a single infusion of 1) lipo-BSA (n=3); 2) lipo-rbIFN τ (n=3); 3) BSA with Al-gel (n=4); or 4) rbIFNτ with Al-gel (n=4) on Day 13 (oestrus=Day 0). The BSA or rbIFNτ solution was introduced into the uterine horn ipsilateral to the corpus luteum by the cervical route. Blood samples were collected and rectal temperatures were recorded immediately preceding the infusion (0h), thereafter every 3h until 12h, and 24h after the infusion. Total white blood cells (WBC) were counted. Data were analysed by ANOVA using the STATVIEW program. Unbound bIFNτ in liposomal encapsulation and adsorption to Al-gel were estimated to be ~90% and 2%, respectively. Corpus luteum lifespan was normal in controls (lipo-BSA; 21.6±0.33 and BSA in Al-gel; 21.2±0.25 days) but was extended in cows receiving rbIFNτ with Al-gel (27.0±0.40 days; P<0.01), whereas lipo-rbIFNτ was less effective. Rectal temperatures increased following rbIFNτ treatment with a peak at 6h after infusion (P<0.05). Correspondingly, total WBC was decreased following rbIFNτ treatment with a minimum at 9h. Those changes were larger in liposomal encapsulation than in adsorbed to Al-gel. In conclusion, a single infusion of bIFNτ adsorbed to Al-gel can extend corpus luteum lifespan in cyclic cows.
Reproduction Fertility and Development 01/2011; 23(1):164. · 2.11 Impact Factor
-
T Somfai,
K Imai,
M Kaneda,
S Akagi,
S Haraguchi,
S Watanabe,
E Mizutani,
T Q Dang-Nguyen, Y Inaba,
M Geshi,
T Nagai
[show abstract]
[hide abstract]
ABSTRACT: The aim of the present study was to investigate the effect of oocyte source and in vitro maturation (IVM) on the expression of selected genes in bovine oocytes and their contribution to in vitro embryo development. Follicular oocytes were collected either by ovum pick-up from live cows or by the aspiration of ovaries of slaughtered cows following storage in Dulbecco's PBS at 15°C for overnight. In vitro maturation was performed according to the method of (Imai et al. 2006 J. Reprod. Dev. 52, 19-29 suppl.). Gene expression was assessed before and after IVM by real-time PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1, and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using a Qiagen RNeasy Micro Kit (Qiagen, Valencia, CA). Gene expression was analysed by a Roche Light Cycler 480 device and software (Roche, Indianapolis, IN). Relative expression of each gene was normalized to CCNB1, which in preliminary experiments appeared the most stably expressed irrespective of oocyte source and meiotic stage. Three replications were performed. Data were analysed by paired t-test. In immature ovum pick-up oocytes, genes related to metabolism (GAPDH, G6PDH, GLUT8) and stress (MnSOD, HSP70), and also OCT4, ATP1A1, and DYNC1/1 showed significantly (P<0.05) higher expression compared with immature oocytes collected from slaughtered-stored ovaries. The expression of GDF9, GLUT8, CTNNB1, and PMSB1 was significantly (P<0.05) reduced during IVM irrespective of the oocyte source. In a second experiment, IVF IVM oocytes showing an early (at 22 to 25h after IVF) or late (at 27 to 30h after IVF) first cleavage were either cultured in vitro or analysed for gene expression at the 2-cell stage. A higher (P<0.05) rate of early-cleaving oocytes developed to the blastocyst stage compared with the rate of late-cleaving ones (46.2% v. 15.6%, respectively). Nevertheless, only ATP1A1 showed significantly reduced (P<0.05) expression in late-cleaving embryos compared with early-cleaving ones. Our results suggest that although removal and storage of ovaries and IVM caused a reduction in the relative abundance of several genes in oocytes, in most cases, this did not affect embryo development. Among the genes studied, only ATP1A1 was correlated with in vitro development.
Reproduction Fertility and Development 01/2011; 23(1):199. · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The objective of this study was to investigate the feasibility of the silk protein sericin as an alternative protein supplement for bovine embryo cultures. The effects of sericin supplementation in in vitro culture (IVC) medium on the development and cryosurvival of bovine IVM-IVF embryos were investigated. Cumulus-oocyte complexes were collected from 2- to 8-mm follicles of ovaries obtained from a local abattoir. They were matured for 20 to 22h in TCM-199 supplemented with 5% fetal bovine serum (FBS), 0.002AUmL(-1) of FSH, and 1μgmL(-1) of oestradiol-17β at 38.5°C under an atmosphere of 5% CO(2) in air. After IVF (Day 0), presumptive zygotes were cultured in SOF medium (IVC medium) containing amino acids and supplemented with either 5% FBS (FBS group: n=400) or sericin at 3 different concentrations (wt/vol; 0.05%: n=493; 0.1%: n=419; or 0.15%: n=520; sericin groups) at 38.5°C under an atmosphere of 5% CO(2), 5% O(2), and 90% N(2) for 5 days. They were then transferred into each IVC medium supplemented with 0.1mM β-mercaptoethanol and cultured for an additional 4 days (9 days in total). Cleavage rates were recorded on Day 2 of IVC. The excellent expanded blastocysts harvested on Days 7 and 8 were used for freezing (FBS group: n=51, 0.05% sericin group: n=56, 0.1% sericin group: n=44, 0.15% sericin group: n=44). They were frozen in m-PBS supplemented with 1.5M ethylene glycol, 0.1M sucrose, 20% calf serum, and 4mgmL(-1) of BSA. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1mM β-mercaptoethanol under the same atmosphere used for IVC for 72h. Rates of the embryos that reexpanded and developed to the hatching and hatched blastocyst stages were determined at, respectively, 24, 48, and 72h after thawing. Rates of cleavage and blastocyst formation were expressed as mean±SD and were analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by Fisher's exact test and chi-square test. Four replications were performed. Cleavage and blastocyst formation rates did not differ among the groups (FBS: 61.6±15.1 and 22.1±3.5%; 0.05% sericin: 71.6±12.0 and 19.4±6.7%; 0.1% sericin: 70.3±4.7 and 18.4±5.6%; 0.15% sericin: 66.9±10.3 and 17.9±6.9%, respectively). There were no significant differences in post-thaw survival rates after 24h among the FBS (88.2%), 0.05% sericin (92.9%), 0.1% sericin (84.1%), and 0.15% sericin (93.2%) groups. However, post-thaw survival rates after 72h in the 0.05% sericin (83.9%) and FBS (82.4%) groups were significantly higher than those in the 0.1% sericin (56.8%) and 0.15% sericin (61.4%) groups (P<0.05). These results indicate the feasibility of sericin as an alternative protein supplement for bovine embryo culture. Additionally, in this study, 0.05% sericin was shown to be the best concentration for survival of the resultant embryos after freezing and thawing.
Reproduction Fertility and Development 01/2011; 23(1):205-206. · 2.11 Impact Factor
