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.

The localization of LAP2β in bovine oocytes after in vitro activation and fertilization

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 81-93 ◽  
Author(s):  
Mamiko Isaji ◽  
Hisataka Iwata ◽  
Hiroshi Harayama ◽  
Masashi Miyake
Keyword(s):  
Dna Synthesis ◽  
Nuclear Membrane ◽  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Somatic Cells ◽  
Condensed Chromatin ◽  
Lamin A ◽  
Bovine Oocytes

The present study was designed to clarify the localization of LAP2β and to compare it with those of lamins A/C and B in bovine oocytes after activation and in vitro fertilization (IVF). After fertilization, LAP2β was not found until telophase II, and was observed around condensed chromatin after the extrusion of the second polar body, but not in activated oocytes. Although the reaction of LAP2β was temporally negative or weak on the membrane of the growing small pronuclei, it became strong on the fully grown pronuclei of both activated and fertilized oocytes. Examination of the timing of DNA synthesis using bromodeoxyuridine revealed that the expression of LAP2β on the pronuclear membrane became strong around the end of the DNA synthesis in both activated and fertilized oocytes. Both male and female pronuclei exhibited the same reactivity to all nuclear proteins examined. It was also shown that LAP2β first assembled around condensed chromatin, followed by the integration of lamins B and A/C as in somatic cells. LAP2β staining was maintained on the nuclear membrane of the embryonic cells at interphase until the later stage of preimplantational development. There were no differences between parthenogenetic and fertilized embryos in the expression and localization of LAP2β from the PN-stage oocyte to the blastocyst. The assembly of LAP2β was observed around the telophase chromatin of both blastocyst and cumulus cells. Thus, it was shown that the timing of the aggregation of LAP2β at the second meiosis was different from that in the mitosis of blastocyst and somatic cells. LAP2β was constantly expressed in the nuclear membrane in in vitro fertilized and parthenogenetic embryos as was lamin B, and lamin A/C was expressed stage-dependently in both types of embryos. Lamin A/C was positive in some inner cell mass cells of parthenogenetic blastocysts, but not those of in vitro fertilized embryos.

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.

165 EXPRESSION OF Oct-4 IN BUFFALO (BUBALUS BUBALIS) EMBRYOS GENERATED THROUGH IN VITRO FERTILIZATION OR PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 162
Author(s):  
D. Kumar ◽  
T. Anand ◽  
K. P. Singh ◽  
M. S. Chauhan ◽  
P. Palta ◽  
...  
Keyword(s):  
Relative Humidity ◽  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bubalus Bubalis ◽  
Dnase I ◽  
Parthenogenetic Activation ◽  
Cell Lysate

Octamer-4 (Oct-4) is a member of Class V of the POU transcription factors family, which is involved in transcriptional regulation during early embryonic development and cell differentiation. It is expressed in the inner cell mass of blastocysts and in embryonic stem cells (ESCs), and its expression is widely used as a marker of pluripotency in ESCs in many species. This study was, therefore, carried out to examine the expression of Oct-4 in embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages generated through IVF or parthenogenetic activation. A total of 100 embryos were used in the study, 10 for each embryonic stage from both methods of embryo production. Immature oocytes obtained from slaughterhouse buffalo (Bubalus bubalis) ovaries were subjected to IVM in TCM-199 + 10% FBS + 5 µg mL–1 pFSH + 0.81 mm sodium pyruvate for 24 h in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5�C. IVF was carried out immediately after IVM; the cleaved embryos were cultured for 8 days in CR2 medium containing 0.6% BSA and 10% FBS for production of embryos at different stages. For production of embryos through parthenogenesis, after 24 h of IVM, oocytes were denuded of cumulus cells by incubation in 0.2% hyaluronidase in Dulbecco's phosphate-buffered saline for 2 min. The denuded oocytes with a prominent polar body were parthenogenetically activated by exposure to 7% ethanol for 7 min, followed by incubation with 2 mm 6-dimethyl aminopurine in CR2 medium for 3.5 h in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5�C, and then subjected to IVC as described above. A two-step RT-PCR was carried out using Cells-to-cDNA Kit-II (Ambion, Austin, TX, USA), using bovine primers 52-GTT CTC TTT GGA AAG GTG TTC-3' and 5'-ACA CTC GGA CCA CGT CTT TC-3' for the amplification of Oct-4. For this, the embryos were washed with PBS, transferred to 30 µL of cold cell lysis buffer and incubated in a thermal cycler at 75�C for 10 min. The cell lysate was treated with DNase-I at 37�C for 30 min to degrade genomic DNA and then heated at 75�C for 5 min to inactivate the DNase-I. The cell lysate (10 µL) was used for making cDNA using random primer. The PCR cycle included heating to 94�C for 2 min, followed by 33 cycles of 94�C for 30 s, 57�C for 30 s, and 72�C for 45 s. A final extension at 72�C for 10 min was carried out to complete the amplification of the Oct-4 gene. Transcripts of Oct-4 were detected at all of the embryonic stages, from the 2-cell through the hatched blastocyst stage in both IVF and parthenogenetically generated embryos. These results indicate that Oct-4, which is believed to be a reliable marker for pluripotency of ESCs in a number of species, is expressed in early cleavage-stage buffalo embryos and continues to be expressed in preimplantation-stage blastocysts.

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

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.

scholarly journals Ultrastructure of in vitro oocyte maturation in buffalo (Bubalus bubalis)

Zygote ◽  
2010 ◽  
Vol 18 (4) ◽  
pp. 309-314 ◽  
Author(s):  
Rafael Gianella Mondadori ◽  
Tiago Rollemberg Santin ◽  
Andrei Antonioni Guedes Fidelis ◽  
Khesller Patrícia Olázia Name ◽  
Juliana Souza da Silva ◽  
...  
Keyword(s):  
Polar Body ◽  
Bubalus Bubalis ◽  
Ultrastructural Changes ◽  
Oocyte Nucleus ◽  
Cortical Granules ◽  
Immature Oocytes ◽  
Transmission Electron ◽  
High Lipid ◽  
First Polar Body

SummaryThe objective of the present study was to describe ultrastructural changes in the nucleus and cytoplasmic organelles during in vitro maturation (IVM) of buffalo cumulus–oocyte complexes (COCs). The structures were collected by ovum pick-up (OPU). Some COCs, removed from maturation medium at 0, 6, 12, 18 and 24 h, were processed for transmission electron microscopy. The average number of COCs collected by OPU/animal/session was 6.4, and 44% of them were viable. Immature oocytes had a peripherally located nucleus, Golgi complex and mitochondrial clusters, as well as a large number of coalescent lipid vacuoles. After 6 h of IVM, the oocyte nucleus morphology changed from round to a flatter shape, and the granulosa cells (GC) lost most of their contact with zona pellucida (ZP). At 12 h the first polar body was extruded and the aspect of lipid droplet changed to dark, probably denoting lipid oxidation. Cortical granules were clearly visible at 18 h of maturation, always located along the oocyte periphery. At 24 h of IVM the number of cortical granules increased. Ultrastructure studies revealed that: (1) immature oocytes have a high lipid content; (2) the perivitelline space (PS) increases during IVM; (3) Golgi complexes and mitochondrial clusters migrate to oocyte periphery during IVM; (4) 6 h of IVM are enough to lose contact between GC and ZP; (5) the oocyte lipid droplets’ appearance changes between 6 and 12 h of IVM.

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.

scholarly journals The Role of Ca2 + in Maturation and Reprogramming of Bovine Oocytes: A System Study of Low-Calcium Model

2021 ◽  
Vol 9 ◽  
Author(s):  
Lin Meng ◽  
Hongmei Hu ◽  
Zhiqiang Liu ◽  
Luyao Zhang ◽  
Qingrui Zhuan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Polar Body ◽  
Rna Seq ◽  
Low Calcium ◽  
Maturation Medium ◽  
First Polar Body ◽  
Significant Difference ◽  
Bovine Oocytes

[Ca2+]i is essential for mammalian oocyte maturation and early embryonic development, as those processes are Ca2+ dependent. In the present study, we investigated the effect of [Ca2+]i on in vitro maturation and reprogramming of oocytes in a lower calcium model of oocyte at metaphase II (MII) stage, which was established by adding cell-permeant Ca2+ chelator BAPTA-AM to the maturation medium. Results showed that the extrusion of the first polar body (PB1) was delayed, and oocyte cytoplasmic maturation, including mitochondrial and endoplasmic reticulum distribution, was impaired in lower calcium model. The low-calcium-model oocytes presented a poor developmental phenotype of somatic cell nuclear transfer (SCNT) embryos at the beginning of activation of zygotic genome. At the same time, oxidative stress and apoptosis were observed in the low-calcium-model oocytes; subsequently, an RNA-seq analysis of the lower-calcium-model oocytes screened 24 genes responsible for the poor oocyte reprogramming, and six genes (ID1, SOX2, DPPA3, ASF1A, MSL3, and KDM6B) were identified by quantitative PCR. Analyzing the expression of these genes is helpful to elucidate the mechanisms of [Ca2+]i regulating oocyte reprogramming. The most significant difference gene in this enriched item was ID1. Our results showed that the low calcium might give rise to oxidative stress and apoptosis, resulting in impaired maturation of bovine oocytes and possibly affecting subsequent reprogramming ability through the reduction of ID1.

267 TARGETED SUPPRESSION OF THE EXPRESSION OF MATERNAL AND EMBRYONIC GENES DURING IN VITRO DEVELOPMENT OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 241
Author(s):  
D. Tesfaye ◽  
K. Nganvongpanit ◽  
F. Rings ◽  
M. Gilles ◽  
D. Jennen ◽  
...  
Keyword(s):  
Free Water ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
Bovine Oocytes ◽  
E Cadherin

Despite enormous advances in the identification and sequencing of developmentally relevant bovine genes, the function of the majority of these transcripts is not yet known. Here we aimed to apply the RNA interference (RNAi) approach to suppress the expression of the maternal transcript c-mos (AY630920) and embryonic transcripts E-cadherin (AY508164) and Oct-4 (AY490804) during in vitro development of bovine embryos using microinjection of sequence-specific double-stranded RNA (dsRNA). For this 435-, 341- and 341-bp-long dsRNA specific to the coding sequences of c-mos, E-cadherin and Oct-4 transcripts, respectively, were synthesized using Promega RiboMax" T7 system (Promega, Madison, WI, USA), where sense and antisense strands were transcribed from the target DNA template. Slaughterhouse ovaries were used to aspirate bovine oocytes, which were matured in TCM-199 with 12% estrus cow serum (ECS), fertilized in Fert-TALP, and cultured in CR1 medium at 39�C under humidified atmosphere of 5% CO2 in air. In Experiment 1, immature oocytes were categorized into three groups, each containing 50-60 oocytes: those injected with c-mos dsRNA, those injected with RNase-free water, and uninjected controls. In Experiment 2, zygotes were categorized into four groups, each containing 50-60 zygotes: those injected with E-cadherin dsRNA, those injected with Oct-4 dsRNA, those injected with RNase-free water, and uninjected controls. Each experiment was repeated four times. The effect of dsRNA on in vitro development of oocytes or embryos was assessed after microinjection during culture. The level of mRNA and protein expression was investigated using real-time PCR and western blot analysis, respectively. Data were analyzed using SAS, version 8 (SAS Institute Inc., Cary, NC, USA). Microinjection of c-mos dsRNA resulted in a 70% reduction of c-mos transcript abundance after maturation compared to the water-injected and uninjected controls (P < 0.05). Similarly, microinjection of E-cadherin and Oct-4 dsRNA at the zygote stage resulted in 80% and 60% reduction in transcript abundance at the blastocyst stage, respectively, compared to the uninjected controls (P < 0.05). Decreases in the c-mos (39 kDa) and E-cadherin proteins (119 kDa) were observed in the c-mos and E-cadherin dsRNA-injected groups, respectively, compared to the control. A higher proportion of oocytes (75%) showed first polar body extrusion after maturation in c-mos dsRNA-injected groups, compared to 52% in water-injected and 57% in uninjected controls. Only 22% from E-cadherin dsRNA- and 24% from Oct-4 dsRNA-injected zygotes developed to the blastocyst stage compared to 39 and 37% blastocyst rates in water-injected and uninjected control groups, respectively. In conclusion, injection of sequence-specific dsRNA in bovine oocytes and embryos resulted in suppression of mRNA and their protein products, thereby affecting in vitro development of bovine embryos.

355 INFLUENCE OF NITRIC OXIDE AND CYCLIC GMP SIGNALING PATHWAY ON THE IN VITRO MATURATION OF BOVINE OOCYTES: PRELIMINARY RESULTS

2010 ◽  
Vol 22 (1) ◽  
pp. 334
Author(s):  
K. R. L. Schwarz ◽  
T. H. C. de Bem ◽  
P. R. L. Pires ◽  
L. G. Mesquita ◽  
L. Remy ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathway ◽  
Polar Body ◽  
Calf Serum ◽  
Guanosine Monophosphate ◽  
No Donor ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Bovine Oocytes

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthase enzyme (NOS) and has been shown to be involved in oocyte maturation. NO is known to act through the guanylate cyclase (GC) signaling pathway, stimulating the production of cyclic guanosine monophosphate (cGMP), which in turn activates protein kinase G (PKG). The objective of the present study was to investigate the involvement of NO and GC/cGMP/PKG pathway on the IVM of bovine oocytes. Slaughterhouse ovaries were transported to the laboratory and oocytes were aspirated from 2 to 8 mm follicles. Oocytes were submitted to IVM (TCM-199+10% fetal calf serum + hormones) for 24 h (38.5°C and 5% CO2 in air) and were assessed for nuclear maturation by acetic-orcein (1%) staining. Maturation rates were analyzed by ANOVA. Five replicates were performed with 20 oocytes per group per replicate. When the oocytes were matured with the NO donor [(0, 10-9, 10-8 and 10-7M S-nitroso-N-acteyl-D,L- penicillamine (SNAP)] germinal vesicle break down (GVBD) rates after 7 h in IVM were 36, 31, 42, and 24%, respectively (P > 0.05). Maturation rates after 24 h IVM ranged from 80 to 85% (P > 0.05). The inhibition of GC [(0, 0.1, 10, and 100 μM 1, H-[1, 2, 4]oxadiazole[4, 3-a]quinoxalon-1-one (ODQ)] and PKG (0, 1, 10, and 100 μM KT5823) did not affect (P > 0.05) the ability of oocytes to form the first polar body (average of 83 and 88%, respectively). When the cGMP-analogue (0, 1, 2, and 4 mM 8-Bromo-cGMP) and the GC-stimulator (0, 5, 10, and 50 μM Protoporphyrin IX) were used during IVM, maturation rates were over 85% in all groups (P > 0.05). To confirm the lack of effect of the inhibitors, another evaluation with higher concentrations of inhibitors in semi-defined IVM medium (TCM-199 + 0.04% BSA) was carried out. Maturation rates were 70 to 75% (P > 0.05) with ODQ and 57 to 76% (P > 0.05) with KT5823. The evaluation with the GC stimulator and the cGMP analogue in semi-defined medium is currently underway. In conclusion, under the conditions studied, the GC/cGMP/PKG signaling pathway is not involved in the nuclear maturation of bovine oocytes. Supported by FAPESP, Brazil.

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