|
| ||
|
|
||
Bovine Oocytes (bovine + oocyte)
Selected AbstractsEffect of Alpha-Tocopherol and Ascorbic Acid on Bovine Oocyte in Vitro MaturationREPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2005G Dalvit Contents In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus,oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence. [source] Expression Pattern of Apoptotic Genes in Vitrified-Thawed Bovine OocytesREPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2010VM Anchamparuthy Contents This study describes a method for quantification of transcripts from low numbers of bovine oocytes using real time RT-PCR. The objective was to evaluate the expression pattern of apoptotic genes (Fas, FasL, Bax and Bcl-2) in vitrified-thawed oocytes. Oocytes were evaluated at germinal vesicle stage; at 15 h of maturation; after vitrification and warming at 15 h of maturation and at 9 h of additional maturation. All transcripts showed an increase in at least 1.2-fold change post-vitrification warming, but the levels tended to decrease at 9 h of maturation post-vitrification warming. Transcript abundance for Fas mRNA was 1.4-fold for oocytes after vitrification and warming. The level of Fas mRNA upon maturation was 0.8-fold. The increase in the abundance of FasL mRNA was 2.1, while it was 0.5-fold relative to control. Vitrification resulted in 1.5-fold change in Bax mRNA expression in oocytes. After 9 h of maturation post-vitrification warming, the level for Bax mRNA was 0.6-fold. The mRNA for Bcl-2 was nearly the same after vitrification and warming. The abundance of mRNA for Bcl-2 was 1.2-fold in vitrified oocytes and fell (p = 0.05) to 0.5 at 9 h of maturation post-vitrification and warming. The up-regulation of apoptotic genes in vitrified oocytes may be an early indicator of reduced developmental competence following vitrification. Yet, results from terminal deoxynucleotidyl transferase dUTP nick end labelling and caspase assays did not support the evidence of apoptosis in embryos derived from large numbers of vitrified oocytes. [source] Use of Fetuin Before and During Vitrification of Bovine OocytesREPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2008G Horvath Contents After vitrification of oocytes, fertilization rates and subsequent development are unsatisfactory, possibly due in part to zona hardening. Foetal calf serum (FCS) can prevent zona hardening because of its fetuin content, but FCS composition varies among batches, and may contain viruses. In this study, we therefore compared media supplemented with different sources of macromolecules, 2% bovine serum albumin (BSA), 2% BSA + 1 mg/ml fetuin and 20% FCS, for handling oocytes for 10,30 min prior to vitrification. None of the treatments resulted in developmental rates comparable with the non-vitrified controls, but FCS inclusion in pre-vitrification handling medium resulted in higher blastocyst production per oocyte (p < 0.05) (10.8%) on day 9 of culture than BSA (5.3%) or BSA + fetuin (6.4%). Blastocysts developing from oocytes from all vitrification treatments were somewhat retarded relative to those developed from non-vitrified oocytes. We also tested the use of fetuin during vitrification as well as two different exposure times with cryoprotectants, 180 and 30 s. There was no significant effect of fetuin or exposure time on rates of subsequent blastocyst production. [source] Mitochondrial Activity, Distribution and Segregation in Bovine Oocytes and in Embryos Produced in VitroREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2006AM Tarazona Contents Bovine oocytes and embryos produced in vitro were studied to determine the mitochondrial pattern of distribution, segregation and activity using DIOC 6 and Jc-1 fluorescence. The highest fluorescence level observed in mature oocytes was taken as 100% activity and six activity levels were estimated as follows: (1) 0%, (2) 1,15%, (3) 16,30%, (4) 31,50%, (5) 51,75% and (6) 76,100%. Three patterns of mitochondrial distribution were found: (1) diffused throughout the cytoplasm in oocytes and embryos, (2) pericytoplasmic in oocytes and embryos, and (3) perinuclear only in embryos. The segregation of mitochondria in blastomeres showed two distinct patterns: (1) symmetrical with an even mitochondrial population, and (2) asymmetrical with different numbers of mitochondria in each blastomere. In immature oocytes, mitochondrial activity was very low and the distribution was diffuse or negligible, while in mature oocytes the activity was high and the distribution was diffuse or pericytoplasmic. Competent embryos up to the 16-cell stage showed intermediate levels of activity (16,50%) but activity decreased thereafter up to the blastocyst stage. Non-competent embryos showed low levels of activity (1,15%) at all stages. These results suggest that mitochondria might play an important role during early development and that a minimum threshold of activity regulates the potential competence for reaching the blastocyst stage. [source] Histone H1 and MAP Kinase Activities in Bovine Oocytes following Protein Synthesis InhibitionREPRODUCTION IN DOMESTIC ANIMALS, Issue 3-4 2001B Meinecke In vitro nuclear maturation is associated with known activity profiles of the M-phase promoting factor (MPF) and the mitogen-activated protein (MAP) kinases, which are two key regulators of mitotic and meiotic cell cycles. Initiation of meiotic resumption in vitro can be prevented by cycloheximide treatment and after removal of the inhibitor germinal vesicle breakdown takes place nearly twice as fast as in untreated controls. In this study experiments were conducted in order to examine the chromosome condensation status and the dynamics of MPF and MAP kinase activities after cycloheximide treatment (10 ,g/ml) of cumulus-enclosed oocytes for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium for various times. Bovine oocytes displayed variations in the degree of chromosome condensation at the end of the inhibitor treatment phase. Following removal of the inhibitor germinal vesicle breakdown occurred after 4,5 h of subsequent culture in inhibitor-free medium. MPF and MAP kinase exhibited low activities during the first 1,3 h following cycloheximide treatment. Increasing levels of enzyme activities were detected 4,7 h following cycloheximide treatment for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium. The patterns of enzyme activities corresponded with the accelerated nuclear maturation process. It can be concluded that cycloheximide treatment does not lead to a more synchronous course of nuclear maturation and that the activities of both, MPF and MAP kinase are initiated at least 2,5 h earlier in comparison with untreated oocytes. [source] Mitochondrial Activity, Distribution and Segregation in Bovine Oocytes and in Embryos Produced in VitroREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2006AM Tarazona Contents Bovine oocytes and embryos produced in vitro were studied to determine the mitochondrial pattern of distribution, segregation and activity using DIOC 6 and Jc-1 fluorescence. The highest fluorescence level observed in mature oocytes was taken as 100% activity and six activity levels were estimated as follows: (1) 0%, (2) 1,15%, (3) 16,30%, (4) 31,50%, (5) 51,75% and (6) 76,100%. Three patterns of mitochondrial distribution were found: (1) diffused throughout the cytoplasm in oocytes and embryos, (2) pericytoplasmic in oocytes and embryos, and (3) perinuclear only in embryos. The segregation of mitochondria in blastomeres showed two distinct patterns: (1) symmetrical with an even mitochondrial population, and (2) asymmetrical with different numbers of mitochondria in each blastomere. In immature oocytes, mitochondrial activity was very low and the distribution was diffuse or negligible, while in mature oocytes the activity was high and the distribution was diffuse or pericytoplasmic. Competent embryos up to the 16-cell stage showed intermediate levels of activity (16,50%) but activity decreased thereafter up to the blastocyst stage. Non-competent embryos showed low levels of activity (1,15%) at all stages. These results suggest that mitochondria might play an important role during early development and that a minimum threshold of activity regulates the potential competence for reaching the blastocyst stage. [source] Histone H1 and MAP Kinase Activities in Bovine Oocytes following Protein Synthesis InhibitionREPRODUCTION IN DOMESTIC ANIMALS, Issue 3-4 2001B Meinecke In vitro nuclear maturation is associated with known activity profiles of the M-phase promoting factor (MPF) and the mitogen-activated protein (MAP) kinases, which are two key regulators of mitotic and meiotic cell cycles. Initiation of meiotic resumption in vitro can be prevented by cycloheximide treatment and after removal of the inhibitor germinal vesicle breakdown takes place nearly twice as fast as in untreated controls. In this study experiments were conducted in order to examine the chromosome condensation status and the dynamics of MPF and MAP kinase activities after cycloheximide treatment (10 ,g/ml) of cumulus-enclosed oocytes for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium for various times. Bovine oocytes displayed variations in the degree of chromosome condensation at the end of the inhibitor treatment phase. Following removal of the inhibitor germinal vesicle breakdown occurred after 4,5 h of subsequent culture in inhibitor-free medium. MPF and MAP kinase exhibited low activities during the first 1,3 h following cycloheximide treatment. Increasing levels of enzyme activities were detected 4,7 h following cycloheximide treatment for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium. The patterns of enzyme activities corresponded with the accelerated nuclear maturation process. It can be concluded that cycloheximide treatment does not lead to a more synchronous course of nuclear maturation and that the activities of both, MPF and MAP kinase are initiated at least 2,5 h earlier in comparison with untreated oocytes. [source] Male and Female Effects on the In Vitro Production of Bovine EmbryosANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 5 2004G. A. Palma Summary A 3-year study was carried out to evaluate male and female effects on the efficiency of an in vitro fertilization (IVF) programme. The semen of different bulls used for artificial insemination was tested for the in vitro production of transferable blastocysts. The fertilization capacity was recorded for each bull. Bovine oocytes were matured in vitro, fertilized with frozen/thawed semen of 63 individual bulls and cultured during 8 days. The semen of one bull was used as control. The percentage of cleavage (36.3,93.4%) and blastocysts on day 7 (6.9,51.2%) varied from bull to bull. Despite high variability, blastocysts were produced with the semen of all bulls in the first trial. Moreover, oocytes fertilized with 85% of tested bulls reached a blastocyst rate not different to the control bull. The correlation coefficients of six bulls showed no significant male effect but an influence of oocytes on the cleavage rate (F -value 0.38, P > 0.05, and 12.4, P < 0.001, respectively). The development to blastocysts on day 7 was significantly influenced by sperms and also oocytes and session (P < 0.01), but no combined interaction was observed between female and male. It is concluded that transferable embryos can be produced in vitro in the first trial with frozen/thawed semen of 63 tested bulls. The results show different capacities of bulls to produce embryos and high male and female effects on the efficiency of an IVF programme. [source] Effect of Alpha-Tocopherol and Ascorbic Acid on Bovine Oocyte in Vitro MaturationREPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2005G Dalvit Contents In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus,oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence. [source] Behaviors of ATP-dependent chromatin remodeling factors during maturation of bovine oocytes in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2010Gabbine Wee The mammalian oocyte undergoes dynamic changes in chromatin structure to reach complete maturation. However, little known is about behaviors of ATP-dependent chromatin remodeling factors (ACRFs) during meiosis. Here, we found that respective ACRFs may differently behave in the process of oocyte maturation in the bovine. All ACRFs interacted with oocytic chromatin at the germinal vesicle (GV) stage. Mi-2 and hSNF2H disappeared from GV-chromatin within 1,hr of in vitro culture whereas Brg-1 and BAF-170 were retained throughout germinal vesicle break down (GVBD). Brg-1 was localized on the condensed chromatin outside, whereas BAF-170 was entirely excluded from condensed chromatin. Thereafter, Brg-1 and BAF-170 interacted with metaphase I and metaphase II chromosomes. These results imply that Mi-2 and hSNF2H may initiate the meiotic resumption, and Brg-1 and BAF-170 may support chromatin condensation during meiosis. In addition, DNA methylation and methylation of histone H3 at lysine 9 (H3K9) seem to be constantly retained in the oocyte chromatin throughout in vitro maturation. Inhibition of ACRF activity by treatment with the inhibitor apyrase led to retarded chromatin remodeling in bovine oocytes, thereby resulting in poor development of fertilized embryos. Therefore, these results indicate that precise behaviors of ACRFs during meiosis are critical for nuclear maturation and subsequent embryonic development in the bovine. Mol. Reprod. Dev. 77: 126,135, 2010. © 2009 Wiley-Liss, Inc. [source] Na+/K+ATPase regulates sperm capacitation through a mechanism involving kinases and redistribution of its testis-specific isoformMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2010Larissa D. Newton Incubation of bovine sperm with ouabain, an endogenous cardiac glycoside that inhibits both the ubiquitous (ATP1A1) and testis-specific ,4 (ATP1A4) isoforms of Na+/K+ATPase, induces tyrosine phosphorylation and capacitation. The objectives of this study were to investigate: (1) fertilizing ability of bovine sperm capacitated by incubating with ouabain; (2) involvement of ATP1A4 in this process; and (3) signaling mechanisms involved in the regulation of sperm capacitation induced by inhibition of Na+/K+ATPase activity. Fresh sperm capacitated by incubating with ouabain (inhibits both ATP1A1 and ATP1A4) or with anti-ATP1A4 immunoserum fertilized bovine oocytes in vitro. Capacitation was associated with relocalization of ATP1A4 from the entire sperm head to the post-acrosomal region. To investigate signaling mechanisms involved in oubain-induced regulation of sperm capacitation, sperm preparations were pre-incubated with inhibitors of specific signaling molecules, followed by incubation with ouabain. The phosphotyrosine content of sperm preparations was determined by immunoblotting, and capacitation status of these sperm preparations were evaluated through an acrosome reaction assay. We inferred that Na+/K+ATPase was involved in the regulation of tyrosine phosphorylation in sperm proteins through receptor tyrosine kinase, nonreceptor type protein kinase, and protein kinases A and C. In conclusion, inhibition of Na+/K+ATPase induced tyrosine phosphorylation and capacitation through multiple signal transduction pathways, imparting fertilizing ability in bovine sperm. To our knowledge, this is the first report documenting both the involvement of ATP1A4 in the regulation of bovine sperm capacitation and that fresh bovine sperm capacitated by the inhibition of Na+/K+ATPase can fertilize oocytes in vitro. Mol. Reprod. Dev. 77: 136,148, 2010. © 2009 Wiley-Liss, Inc. [source] BH4 peptide derived from Bcl-xL and Bax-inhibitor peptide suppresses apoptotic mitochondrial changes in heat stressed bovine oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2009Paolete Soto Mitochondria play an important role in the integration and transmission of cell death signals mediated by the Bcl-2 family proteins. Experiments were conducted to determine whether the anti-apoptotic peptides BH4 domain of Bcl-xL (TAT-BH4) and Bax inhibitor peptide (BIP) suppresses heat stress (HS) injury in oocytes by reduction of apoptotic-like events. Cumulus,oocyte complexes (COCs) were matured at 39°C (control) or 41°C (HS) for 21 hr then placed in maturation medium containing 0 or 100 µM BIP in water and 0 or 1 µM TAT-BH4 in dimethyl sulfoxide (DMSO), or a combination of both peptides (BIP,+,BH4). Peptide effects on embryo development, DNA fragmentation, mitochondrial membrane potential (,,m), and mitochondrial DNA (mtDNA) copy number were measured. All groups were fertilized and cultured in vitro at 39°C for 8 days. Compared to control, HS-treated oocytes induced a decrease in embryo development (P,<,0.05), increase in proportion of TUNEL-positive chromatin in oocytes and blastocysts (P,<,0.05), and loss of oocyte ,,m (P,<,0.001). In the presence of BIP or BIP,+,BH4, development of HS-treated oocytes into blastocysts was increased (P,<,0.05). Conversely, COCs matured with TAT-BH4 at 41°C showed reduced embryonic development (P,<,0.05). Exposure of HS-treated to each or both peptides resulted in a reduction of TUNEL frequency in oocytes and blastocysts cells derived from these oocytes (P,<,0.05). The loss of ,,m in HS-treated oocytes was not restored by exposure to BIP,+,BH4 and there was no effect in mtDNA copy number. In conclusion, the present results show that HS-induced apoptosis in bovine oocytes involves Bax and BH4 domain-dependent pathways. Mol. Reprod. Dev. 76: 637,646, 2009. © 2008 Wiley-Liss, Inc. [source] Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrificationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2008Roser Morató Abstract Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 ,M Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 ,M Taxol to the vitrification solution. All oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well as both cow and calf Taxol/CPAs oocytes showed well-organized metaphase plates and normal spindle morphology. All calf OPS and calf Taxol/OPS oocytes displayed a single block of chromatin and no microtubules could be observed around the chromosomes. In conclusion, treatment with 1 µM Taxol before and during vitrification did not induce adverse changes in the oocyte cytoplasm or metaphase spindles in adult bovine oocytes, but stabilized the metaphase and spindle morphology. Mol. Reprod. Dev. 75: 1318,1326, 2008. © 2008 Wiley-Liss, Inc. [source] Large-scale chromatin remodeling in germinal vesicle bovine oocytes: Interplay with gap junction functionality and developmental competenceMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2007Valentina Lodde Abstract In mammals, oocyte acquires a series of competencies sequentially during folliculogenesis that play critical roles at fertilization and early stages of embryonic development. In mouse, chromatin in germinal vesicle (GV) undergoes dynamic changes during oocyte growth and its progressive condensation has been related to the achievement of developmental potential. Cumulus cells are essential for the acquisition of meiotic competence and play a role in chromatin remodeling during oocyte growth. This study is aimed to characterize the chromatin configuration of growing and fully grown bovine oocytes, the status of communications between oocyte and cumulus cells and oocyte developmental potential. Following nuclear staining, we identified four discrete stages of GV, characterized by an increase of chromatin condensation. GV0 stage represented 82% of growing oocytes and it was absent in fully grown oocytes. GV1, GV2, and GV3 represented, respectively, 24, 31, and 45% of fully grown oocytes. Our data indicated a moderate but significant increase in oocyte diameter between GV0 and GV3 stage. By dye coupling assay the 98% of GV0 oocytes showed fully open communications while the number of oocytes with functionally closed communications with cumulus cells was significantly higher in GV3 group than GV1 and GV2. However, GV0 oocytes were unable to progress through metaphase II while GV2 and GV3 showed the highest developmental capability. We conclude that in bovine, the progressive chromatin condensation is related to the sequential achievement of meiotic and embryonic developmental competencies during oocyte growth and differentiation. Moreover, gap-junction-mediated communications between oocyte and cumulus cells could be implicated in modulating the chromatin remodeling process. Mol. Reprod. Dev. 74: 740,749, 2007. © 2006 Wiley-Liss, Inc. [source] Impact of in vitro production techniques on the expression of X-linked genes in bovine (bos taurus) oocytes and pre-attachment embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2007Maria I. Nino-Soto Abstract Our previous studies showed that expression patterns of X-linked genes in cultured cells are different from those of their tissues of origin. This investigation analyses the transcription pattern of the X-linked genes BIRC4, GAB3, MECP2, RPS4X, SLC25A6, and XIST in bovine in vitro matured oocytes and in vitro fertilized embryos, and their in vivo counterparts. In vitro-derived pools of mature oocytes and pre-attachment embryos were obtained by: (a) TCM-199/serum with bovine oviductal epithelial cells as co-culture, and (b) synthetic oviductal fluid/BSA. Pools of in vivo-derived morulae and blastocysts were provided by a commercial embryo transfer operation. Total RNA was extracted for quantification of gene-specific transcript levels using real-time quantitative PCR. Statistical analysis was performed using a mixed model factorial ANOVA with ,,=,0.05. The effect of the in vitro environmental conditions on X-linked gene transcription was most evident during the fourth cell cycle, at the period of activation of the embryonic genome, and seemed to be less pronounced at later developmental stages, with the exception of BIRC4. The levels of X-linked genes transcripts in in vivo-derived embryos were lower relative to their in vitro counterparts for all genes tested. Finally, the pattern of expression of XIST in bovine oocytes and embryos was similar to that reported in humans. These results highlight the possibility that X-linked gene expression analysis is a useful tool to monitor the impact of reproductive biotechnologies on the developmental potential of embryos and aid in their improvement. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, ,-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, ,-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2006AnilKumar Bettegowda Abstract Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, ,-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, ,-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Specific maternal transcripts in bovine oocytes and cleavaged embryos: Identification with novel DDRT-PCR methodsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2005Kyu-Chan Hwang Abstract We used annealing control primer (ACP)-based differential display reverse transcription polymerase chain reaction (DDRT-PCR) to isolate differentially expressed amplicons in bovine germinal vesicle (GV) stage oocytes, 8-cell stage embryos produced in vitro, and blastocyst stage embryos produced in vitro. Four expressed sequence tags (ESTs) of genes that were specifically and predominantly expressed in GV oocytes were cloned and sequenced. We have used a fluorescence monitored real-time quantitative PCR (qPCR) to quantify and analyzed the temporal expression of the target differentially expressed transcripts throughout the preimplantation stages from oocytes to blastocysts. The cloned genes or ESTs all exhibited significant sequence similarity with known bovine genes (98%,100%; DNCL1 and ZP2) or ESTs (81%,97%; FANK1 and GTL3) of other species. As revealed by real-time qRT-PCR, DNCL1, FANK1, GTL3, and ZP2 transcripts were observed in the GV stage oocytes and expression gradually decreased up to the 8-cell stage embryo and the transcripts were not detected in later stages. Similarly, upregulation was observed in GV stage mouse oocytes and metaphase II, suggesting that these four differentially expressed orthologous genes play important roles in early preimplantation, as maternally-derived transcripts. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Protein synthesis and mRNA storage in cattle oocytes maintained under meiotic block by roscovitine inhibition of MPF activityMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Céline Vigneron Abstract Roscovitine, a specific inhibitor of MPF kinase activity, has been shown to block efficiently and reversibly the meiotic resumption of oocytes from different species, including cattle. In view to verify that oocytes maintain germinal vesicle like molecular activities under roscovitine treatment, we compared in the present study the M-phase Promoting Factor (MPF) and Mitogen Activated Protein (MAP) kinase activities; protein synthesis and phosphorylation patterns in oocytes and cumulus cells; and CDK1 and Cyclin B messengers storage under control culture and under roscovitine inhibition. We observed that roscovitine induced a full and reversible inhibition of MPF kinase activity and of the activating phosphorylation of both ERK1/2 MAPK. During in vivo maturation, there was a highly significant increase in the relative mRNA level of both cyclin B1 and CDK1 whereas during in vitro culture, the relative amount of CDK1 messenger was reduced. These messengers may be used as markers for the optimization of in vitro maturation treatment. Roscovitine reversibly prevented this drop in relative quantities of CDK1 messenger. Oocytes cultured in the presence of roscovitine maintained a GV like profile of protein synthesis except that two proteins of 48 and 64 kDa specific of matured oocytes also appeared under roscovitine treatment. However, roscovitine did not prevent most of the modifications of protein phosphorylation pattern observed during maturation. In conclusion, results of this study revealed that the use of roscovitine did not prevent all the events related to maturation of bovine oocytes. Mol. Reprod. Dev. 69: 457,465, 2004. © 2004 Wiley-Liss, Inc. [source] Expression of peroxiredoxins in bovine oocytes and embryos produced in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2004Gregory Leyens Abstract Peroxiredoxins (PRDXs) form a family of peroxidases involved in antioxidant protection and cell signaling. Due to their peroxide reductase activity, these enzymes might be involved in fine-tuning peroxide levels in embryos during in vitro production. In this study, RT-PCR was used to examine the expression of the six PRDX isoforms (PRDX1 to PRDX6) in bovine oocytes and embryos. PRDXs were detected in oocytes both before and after in vitro maturation. Besides, PRDX6 was up-regulated after maturation. Single embryos were analyzed from the two-cell to the blastocyst stages. PRDX1 and PRDX5 transcripts were detected throughout development. PRDX2, PRDX3, and PRDX6 were not expressed around the 9- to 16-cell stage. PRDX4 transcripts were weakly detected in pools of embryos from the 9- to 16-cell stage onwards. In situ immunodetection of PRDX5, which was previously reported to exhibit the widest subcellular distribution among PRDXs in adult mammalian cells, showed a mitochondrial distribution pattern in the bovine embryo. Finally, the potential modulation by oxidative stress of PRDX expression around the major embryonic genome activation was evaluated by culturing embryos under 20% O2 instead of 5%. No significant difference in the pattern of PRDX expression was observed under 20% O2. In conclusion, our data show for the first time that PRDXs are expressed in mammalian oocytes and early embryos. Moreover, the bovine transcripts exhibit various patterns of expression that might be related to the potential role of PRDXs in oocyte maturation and embryo development. Mol. Reprod. Dev. 69: 243,251, 2004. © 2004 Wiley-Liss, Inc. [source] Comparison by restriction fragment differential display RT‐PCR of gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serumMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001S. Jacek Rzucidlo Abstract A novel restriction fragment differential display (RFDD) RT‐PCR has been used to compare patterns of mRNA expression in bovine oocytes matured in vitro in the presence (10%) or absence of fetal calf serum (FCS). Total RNA extracted from matured and denuded oocytes was processed using display Profile kit (Display System Biotech). RFDD RT‐PCR products were separated on 6% polyacrylamide gel and analyzed using a Storm 860 scanner. Selected bands representing potentially differentially expressed fragments were excised from the gel and re‐amplified. Re‐amplified fragments with size matched to the original fragment were cloned into the TA vector and sequenced. Initially, 10 and 15 differentially expressed fragments were isolated from oocytes matured in the presence and absence of FCS, respectively. Eight out of 10 and 10 out of 15 fragments were re‐amplified successfully as evidenced bysize similarity to the original fragments. Finally, the size of six inserts sequenced from each group matched the size of corresponding original as well as re‐amplified fragments. Sequence comparison search revealed similarity of some isolated fragments to 18s ribosomal RNA, bovine apolipoprotein A‐I, bovine mitochondrion DNA, human CGI‐79 mRNA, human Ab1‐interactor protein, and bovine satellite DNA. The other sequenced fragments may represent novel genes. We showed that RFDD RT‐PCR can be effectively applied to contrast gene expression pattern in bovine oocytes and that presence or absence of FCS during maturation interval affects gene expression pattern in matured bovine oocytes. Mol. Reprod. Dev. 59:90–96, 2001. © 2001 Wiley‐Liss, Inc. [source] Expression Pattern of Apoptotic Genes in Vitrified-Thawed Bovine OocytesREPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2010VM Anchamparuthy Contents This study describes a method for quantification of transcripts from low numbers of bovine oocytes using real time RT-PCR. The objective was to evaluate the expression pattern of apoptotic genes (Fas, FasL, Bax and Bcl-2) in vitrified-thawed oocytes. Oocytes were evaluated at germinal vesicle stage; at 15 h of maturation; after vitrification and warming at 15 h of maturation and at 9 h of additional maturation. All transcripts showed an increase in at least 1.2-fold change post-vitrification warming, but the levels tended to decrease at 9 h of maturation post-vitrification warming. Transcript abundance for Fas mRNA was 1.4-fold for oocytes after vitrification and warming. The level of Fas mRNA upon maturation was 0.8-fold. The increase in the abundance of FasL mRNA was 2.1, while it was 0.5-fold relative to control. Vitrification resulted in 1.5-fold change in Bax mRNA expression in oocytes. After 9 h of maturation post-vitrification warming, the level for Bax mRNA was 0.6-fold. The mRNA for Bcl-2 was nearly the same after vitrification and warming. The abundance of mRNA for Bcl-2 was 1.2-fold in vitrified oocytes and fell (p = 0.05) to 0.5 at 9 h of maturation post-vitrification and warming. The up-regulation of apoptotic genes in vitrified oocytes may be an early indicator of reduced developmental competence following vitrification. Yet, results from terminal deoxynucleotidyl transferase dUTP nick end labelling and caspase assays did not support the evidence of apoptosis in embryos derived from large numbers of vitrified oocytes. [source] Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo DevelopmentREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010FS Gonçalves Contents Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 ,m,-mercaptoethanol (,-ME) and 50 ,m cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for ,-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage. [source] Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm,Egg Binding, Fertilization and Embryo DevelopmentREPRODUCTION IN DOMESTIC ANIMALS, Issue 6 2008RF Gonçalves Contents Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 × 105 frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events. [source] Mapping and transcription profiling of CASP1, 3, 6, 7 and 8 in relation to caspase activity in the bovine cumulus,oocyte complexANIMAL GENETICS, Issue 3 2004Y. Q. Yuan Summary So far 12 caspases have been described in mouse and human while only one (CASP13) is known in cattle. The aim of this study was to (1) search for other bovine caspases by reverse transcription and polymerase chain reaction (RT,PCR) and (2) examine the presence of bovine caspase mRNA and active protein in the cumulus,oocyte complex. Five caspases (1, 3, 6, 7 and 8) were identified, partially cloned and sequenced. Four of them were mapped. Differential transcription of the caspase genes was detected, but no active caspase protein was found in diverse bovine oocytes. Cumulus granulosa cells (CGC) contain CASP1, 6, 7 and 8 mRNA and active caspase protein. The presence of caspase mRNA and active caspase proteins in CGCs suggests the occurrence of apoptosis in cumulus,oocyte complex, while caspase activation is blocked in fresh oocytes and therefore caspase transcription cannot be used to predict the oocyte developmental capacity. [source] | ||||||||||