228 microRNA REGULATION OF GENES IN BOVINE OOCYTES AND EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 272
Author(s):  
J. P. Barfield ◽  
G. J. Bouma ◽  
G. E. Seidel Jr
Keyword(s):  
Mirna Expression ◽  
Polar Body ◽  
Prostaglandin F2α ◽  
Defined Medium ◽  
Tight Junction Formation ◽  
First Polar Body ◽  
Lysis Buffer ◽  
Bovine Oocytes

Little is known about expression of microRNA (miRNA) in bovine oocytes and pre-implantation embryos. These molecules likely have an important role in regulating development. For example, differences in quality of oocytes matured in vivo v. in vitro might be due, in part, to altered miRNA expression. In Experiment 1, in vivo-matured COC were collected by transvaginal aspiration of 7 superstimulated cows 21 to 23 h after GnRH injection, given 48 h after prostaglandin F2α and the last of 6 FSH injections given b.i.d. Oocytes aspirated from abattoir ovaries were matured in vitro for 23 h in a chemically defined medium. After vortexing, maturation of both groups of oocytes was confirmed by visualization of the first polar body, and oocytes were snap frozen in mirVana lysis buffer (Applied Biosciences, Foster City, CA, USA). In Experiment 2, in vitro-matured oocytes were generated as described. Subsets were fertilized in vitro or activated parthenogenetically by incubation in 5-μM ionomycin for 5 min followed by 10 μg mL-1 cycloheximide plus 5 μg mL-1 cytochalasin B for 5 h. After 18 h and 12 h, respectively, fertilized and activated oocytes were centrifuged at 10 000 × g for 10 min to enable visualization of pronuclei. Zygotes with 2 polar bodies and 2 pronuclei and parthenotes with 2 pronuclei were snap frozen in mirVana lysis buffer. Total RNA was extracted from 30 pooled oocytes for each replicate using the mirVana MiRNA Isolation Kit (Ambion, Inc., Austin, TX, USA). Reverse transcription of RNA was performed using the QuantiMir RT kit (System Biosciences, Mountain View, CA, USA), and miRNA expression was evaluated by real-time PCR using the Mouse miRNome Profiler plate, which contains primers for 384 miRNA (System Biosciences). Three plates were analyzed for each group (30 oocytes per plate). Changes in relative expression levels were analyzed with a t-test of values normalized to miR-181a, which was consistently expressed in all samples. In Experiment 1, compared with in vitro-matured oocytes, in vivo-matured oocytes had 11-fold higher (P = 0.02) expression of miR-375, which targets numerous genes involved in electron transport chain and oxidative phosphorylation pathways according to the bioinformatic database mirGator. MiR-291a-5p, miR-494, miR-539, and miR-547 were expressed in in vivo-matured oocytes only; the converse was found for miR-575-5p. Results from Experiment 2 are in the table. Major pathways associated with potential targets of the detected miRNA include TGF-beta signaling, Wnt signaling, tight junction formation, DNA replication reactome, steroid biosynthesis, mRNA processing binding reactome, and glutamate metabolism. Several of these candidate miRNA might be important for regulation of bovine oocyte maturation and embryo development. Table 1.Experiment 2: Fold change expression of miRNA

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

372 SPERM ASTER FORMATION AND BLASTOCYST DEVELOPMENT OF IN VIVO-MATURED BOVINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION

2007 ◽  
Vol 19 (1) ◽  
pp. 301 ◽  
Author(s):  
T. Horiuchi ◽  
M. Takenaka ◽  
C. Kani ◽  
C. Emuta ◽  
Y. Ogata ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Chi Square ◽  
First Polar Body ◽  
Bovine Oocytes ◽  
Sperm Aster

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P &lt; 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.

Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

2014 ◽  
Vol 26 (8) ◽  
pp. 1084 ◽  
Author(s):  
Yu-Ting Shen ◽  
Yue-Qiang Song ◽  
Xiao-Qin He ◽  
Fei Zhang ◽  
Xin Huang ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Maturation ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body ◽  
Triphenyltin Chloride ◽  
Dose Dependent

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Linker Histone ◽  
Meiotic Maturation ◽  
Control Group ◽  
Bovine Oocyte ◽  
First Polar Body ◽  
Effective Sirnas ◽  
Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

scholarly journals Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 861-874 ◽  
Author(s):  
Korakot Nganvongpanit ◽  
Heike Müller ◽  
Franca Rings ◽  
Michael Hoelker ◽  
Danyel Jennen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Free Water ◽  
Double Stranded Rna ◽  
Immature Oocytes ◽  
Selective Degradation ◽  
First Polar Body ◽  
Bovine Oocytes

RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2,in vitroproduced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

74 IN VIVO SURVIVAL OF DOMESTIC CAT OOCYTES AFTER VITRIFICATION, INTRACYTOPLASMIC SPERM INJECTION, AND TRANSFER TO RECIPIENTS

2008 ◽  
Vol 20 (1) ◽  
pp. 118 ◽  
Author(s):  
M. C. Gómez ◽  
N. Kagawa ◽  
C. E. Pope ◽  
M. Kuwayama ◽  
S. P. Leibo ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Oocyte Retrieval ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
Minimal Amount ◽  
Vitro Fertilization ◽  
First Polar Body

The ability to cryopreserve female gametes efficiently holds immense economic and genetic implications. The purpose of the present project was to determine if domestic cat oocytes could be cryopreserved successfully by use of the Cryotop method. We evaluated (a) cleavage frequency after in vitro fertilization (IVF) v. intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification, and (b) fetal development after transfer of resultant embryos into recipients. In vivo-matured cumulus–oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment, denuded of cumulus cells, and examined for the presence of the first polar body (PB). In vitro-matured COCs were obtained from ovaries donated by local clinics and placed into maturation medium for 24 h before cumulus cells were removed and PB status was determined. Oocytes were cryopreserved by the Cryotop method (Kuwayama et al. 2005 Reprod. Biomed. Online 11, 608–614) in a vitrification solution consisting of 15% DMSO, 15% ethylene glycol, and 18% sucrose. For IVF, oocytes were co-incubated with 1 � 106 motile spermatozoa mL–1 in droplets of modified Tyrode's medium in 5% CO2/air at 38�C (Pope et al. 2006 Theriogenology 66, 59–71). For ICSI, an immobilized spermatozoon was loaded into the injection pipette, which was then pushed through the zona pellucida into the ooplasm. After a minimal amount of ooplasm was aspirated into the pipette, the spermatozoon was carefully expelled, along with the aspirated ooplasm. After ICSI, or at 5 or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006). As assessed by normal morphological appearance after liquefaction, the survival rate of both in vivo- and in vitro-matured oocytes was >90% (93–97%). For in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 73% (16/22) and 53% (30/57), respectively, as compared to 68% (19/28) after ICSI of vitrified oocytes (P > 0.05). For in vivo-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 55% (18/33) and 35% (6/17), respectively, compared to 50% (10/20) after ICSI of vitrified oocytes (P > 0.05). At 18–20 h after ICSI, 18 presumptive zygotes and four 2-cell embryos derived from vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo-matured and 12 in vitro-matured vitrified oocytes were transferred by laparoscopy into the oviducts of two recipients at 24–26 h after oocyte retrieval. The two recipients were 9-month-old IVF/ET-derived females produced with X-sperm sorted by flow cytometry. At ultrasonography on Day 22, both recipients were pregnant, with three live fetuses observed in one recipient and one live fetus seen in the second recipient. On Day 63 and Day 66 of gestation, four live kittens were born, without assistance, to the two recipients. The one male and three female kittens weighed an average of 131 g. In summary, in vivo viability of zygotes/embryos produced by ICSI of cat oocytes vitrified by the Cryotop method was demonstrated by the birth of live kittens following transfer to recipients.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

252 EFFECT OF SUPERSTIMULATORY TREATMENT TO DONOR COWS IN OXYGEN CONSUMPTION OF MATURED OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 273
Author(s):  
K. Imai ◽  
S. Sugimura ◽  
M. Ohtake ◽  
Y. Aikawa ◽  
Y. Inaba ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Prostaglandin F2α ◽  
Gonadotropin Releasing Hormone ◽  
Oestrous Cycle ◽  
Control Group ◽  
Releasing Hormone ◽  
And Control ◽  
Bovine Oocytes

We previously reported that follicular wave synchronization and follicular growth treatment (FGT) before ovum pick-up (OPU) were effective in improving oocyte competence, which was associated with an increase in related embryos obtained by somatic cell nuclear transfer (Sugimura et al. 2012 Cell. Reprogram. 14, 29–37). However, oxygen consumption in oocytes remained unknown. The present study was designed to examine the differences in oxygen consumption between bovine oocytes obtained by OPU with or without FGT after in vitro maturation. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two OPU sessions were conducted in each cow to collect immature oocytes, as described by Sugimura et al. (2012). The first OPU session (OPU group) was performed in cows on arbitrary days of the oestrous cycle, using a 7.5-MHz linear transducer with the needle connected to an ultrasound scanner. Follicles larger than 8 mm in diameter were then aspirated and a controlled internal drug release device (CIDR) was inserted on Day 5 (the day of the first OPU session = Day 0). Then 30 Armour units (AU) of FSH (Antrin, Kyoritsu Seiyaku, Tokyo, Japan) was administrated to cows twice a day from Day 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 AU day–1). Cloprostenol (prostaglandin F2α; 0.75 mg) was administered in the morning of Day 9. The second OPU session (FGT-OPU group) was performed 48 h after prostaglandin F2α administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected cumulus–oocyte complexes in the OPU and FGT-OPU groups were matured in vitro as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. To collect in vivo-matured oocytes (control group), the CIDR was inserted into the cows on arbitrary days of the oestrous cycle (= Day 0), and oestradiol benzoate (0.8 mg) was administered on Day 1. The cows received the FGT treatment (as described above) from Day 6 to 10; however, the CIDR was removed in the evening of Day 8. Buserelin (gonadotropin-releasing hormone; 200 µg) was then administrated in the morning of Day 10, and OPU was performed at 24 h after gonadotropin-releasing hormone administration (Day 11). Oxygen consumption of matured oocytes was measured noninvasively with a scanning electron microscopy system (HV-405SP; Hokuto Denko Co., Tokyo, Japan). Data were analysed by ANOVA followed by a Tukey-Kramer test. There was no difference in the mean oxygen consumption between the FGT-OPU group (0.34 ± 0.02 × 10–14 mol–1, mean ± SEM) and control group (0.40 ± 0.01 × 10–14 mol–1). However, oxygen consumption in the FGT-OPU and control groups was significantly lower (P < 0.01) than that in the OPU group (0.50 ± 0.02 × 10–14 mol–1). These results revealed significantly lower oxygen consumption in OPU-derived in vitro-matured bovine oocytes after FGT treatment compared with those obtained without FGT treatment. Oxygen consumption of oocytes obtained from FGT-OPU was similar to that of in vivo-matured oocytes, which may reflect their cytoplasmic maturation status with high developmental competence.

40 TRICHOSTATIN A TREATMENT EFFECTS ON IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER CAT EMBRYOS DEPEND ON RECIPIENT CYTOPLASM SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 113
Author(s):  
L. T. K. Do ◽  
Y. Sato ◽  
M. Taniguchi ◽  
T. Otoi
Keyword(s):  
Nuclear Transfer ◽  
Treatment Effects ◽  
Trichostatin A ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Control Group ◽  
Fluid Medium ◽  
First Polar Body ◽  
Bovine Oocytes

The developmental ability of interspecies somatic cell nuclear transfer (iSCNT) embryos decreases as the taxonomic distance between the donor and recipient species increases. Treatment of cat iSCNT embryos using bovine oocytes with 50 nM of trichostatin A (TSA) improves in vitro embryonic development (Wittayarat et al. 2013 Cell. Reprogram. 15, 301–308). This study investigated whether the TSA treatment effects differ between the development of cat iSCNT embryos reconstructed with porcine and bovine oocytes. Porcine and bovine cumulus-oocyte complexes were in vitro matured for 44 h and 24 h, respectively. After cumulus cell removal, enucleation was performed by aspiration of the metaphase II plate and the first polar body using a piezo-driven pipette. A cat fibroblast cell was then injected into cytoplasm of successfully enucleated oocyte. Reconstructed cybrids were electrically activated by a single 1.5 kV cm–1 pulse for 100 µs (pig-cat embryos), or a 2.3 kV cm–1 pulse for 30 µs (cow-cat embryos). Pig-cat and cow-cat embryos were cultured in porcine zygote medium (PZM)-5 and modified synthetic oviducal fluid medium (mSOF), respectively. After electrical activation, pig-cat and cow-cat embryos were cultured in medium supplemented with 5 µg mL–1 cytochalasin B + 50 nM TSA (TSA group) or without TSA (control group), and the cow-cat embryo medium was also supplemented with 10 µg mL–1 cycloheximide. After 2 h, TSA-treated pig-cat and cow-cat embryos were incubated in medium supplemented with TSA for 22 h, followed by 48 h incubation without TSA. Pig-cat and cow-cat control embryos were cultured in medium without TSA for 70 h after activation. Then, all pig-cat and cow-cat embryos were cultured in porcine blastocyst medium (PBM) or mSOF medium supplemented with 5% fetal bovine serum, respectively, for 5 additional days. Four to seven replicates were performed for each experiment. Data were analysed using Student's t-test. For pig-cat embryos, no difference was observed in cleavage rates between both groups, but development to the blastocyst stage was higher in the pig control group (n = 147, 8.0%) than that of pig TSA group (n = 131, 0.7%; P < 0.05). In contrast, development to the blastocyst stage in cow-cat embryos was not observed in the cow control group (n = 125, 0%), but it was observed in cow TSA group (n = 136, 3.7%). These results indicate that TSA treatment effects are species-specific, but those effects remain to be clarified.

032. Cdh1: A CELL CYCLE PROTEIN INVOLVED IN FEMALE MEIOSIS AND PREVENTION OF ANEUPLOIDY

2010 ◽  
Vol 22 (9) ◽  
pp. 10
Author(s):  
K. T. Jones
Keyword(s):  
Meiotic Division ◽  
Polar Body ◽  
Single Copy ◽  
Cyclin B1 ◽  
Cell Cycle Protein ◽  
Prophase I ◽  
First Polar Body ◽  
A Cell

Mammalian oocytes are arrested at the dictyate stage of prophase I in the ovary. In growing follicles, oocytes can become responsive to Luteinising Hormone and will undergo meiotic resumption just before ovulation. During the first meiotic division, homologous chromosomes are segregated, a process that is very error prone in human oocytes. By ovulation the oocyte has extruded its first polar body and has re-arrested at metaphase of the first meiotic division. Recent work from our lab has established that the protein Cdh1 is involved uniquely in both in the process of prophase I arrest and the correct segregation of homologs in meiosis I. Thus in cultured oocytes, in vitro antisense knockdown of Cdh1 induces both meiotic resumption and high rates of aneuploidy as a result of non-disjunction during first meiosis. Cdh1 causes prophase I arrest by inducing cyclin B1 degradation and maintaining low levels of the kinase CDK1, whose activity induces meiotic resumption. Cdh1 is an activator of the Anaphase-Promoting Complex (APC), a ubiquitin ligase that earmarks proteins such as cyclin B1 for proteolysis. Cdh1 prevents aneuploidy by causing the degradation of Cdc20, a protein that is responsible for activating the APC once all homologs are correctly aligned at metaphase. Thus loss of Cdh1 seems to prematurely activate APC(Cdc20) activity. It is interesting that a single protein can affect two important meiotic transitions in oocytes. However to explore its functions more fully, and confirm that an in vitro knockdown is faithfully replicated by in vivo loss, a targeted knockout of Cdh1 is needed. Therefore we have generated an oocyte specific Cdh1 knockout by ZP3 promoter driven Cre- recombinase activity in oocytes carrying loxP insertions in the single copy Cdh1 gene. This talk will therefore focus on the effects of an in vivo Cdh1 knockout.

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