Pools Of Embryos Research Articles

Cloning and characterization of microRNAs from rainbow trout (Oncorhynchus mykiss): Their expression during early embryonic development

BackgroundCurrent literature and our previous results on expression patterns of oocyte-specific genes and transcription factors suggest a global but highly regulated maternal mRNA degradation at the time of embryonic genome activation (EGA). MicroRNAs (miRNAs) are small, non-coding regulatory RNAs (19–23 nucleotides) that regulate gene expression by guiding target mRNA cleavage or translational inhibition. These regulatory RNAs are potentially involved in the degradation of maternally inherited mRNAs during early embryogenesis.ResultsTo identify miRNAs that might be important for early embryogenesis in rainbow trout, we constructed a miRNA library from a pool of unfertilized eggs and early stage embryos. Sequence analysis of random clones from the library identified 14 miRNAs, 4 of which are novel to rainbow trout. Real-time PCR was used to measure the expression of all cloned miRNAs during embryonic development. Four distinct expression patterns were observed and some miRNAs showed up-regulated expression during EGA. Analysis of tissue distribution of these miRNAs showed that some are present ubiquitously, while others are differentially expressed among different tissues. We also analyzed the expression patterns of Dicer, the enzyme required for the processing of miRNAs and Stat3, a transcription factor involved in activating the transcription of miR-21. Dicer is abundantly expressed during EGA and Stat3 is up-regulated before the onset of EGA.ConclusionThis study led to the discovery of 14 rainbow trout miRNAs. Our data support the notion that Dicer processes miRNAs and Stat3 induces expression of miR-21 and possibly other miRNAs during EGA. These miRNAs in turn guide maternal mRNAs for degradation, which is required for normal embryonic development.

Preimplantation genetic screening (PGS) for aneuploidy in couples undergoing donor egg in vitro fertilization cycles

Preimplantation genetic screening (PGS) is used in an effort to decrease spontaneous miscarriage, prevent the birth of aneuploid offspring and to increase the delivery rate by removing genetically abnormal embryos from the pool of embryos to be transferred. The most currently available information regarding aneuploidy rates among human preimplantation embryos comes from PGS of embryos from patients with impaired fertility. PGS for aneuploidy is not a common recommendation for the donor egg patient group.

Histoblood Groups Other Than HLA in Organ Transplantation

Immunological matching of a living related donor and recipient of an allograft is precise, but for cadaver organs matching is controversial, including at least detection of specific sensitization in the recipient against the donor, especially for HLA-DR. With the publication of some cases of ABO histoblood group incompatible transplantations with favorable outcomes, transplantation immunologists now focus on many of the 29 International Society of Blood Transfusion-approved histoblood group systems. So far, research lags behind knowledge about which system occurs in which organ, but modern molecular biology tests, like basic local alignment search tools (BLAST) and the recent inclusion of some systems into the CD classification, make possible the tracking of some histoblood group epitopes to specific tissue components. We have conducted such a search. With respect to tissue distribution, mRNA transcripts, and expressed sequence tags (EST), we observed a huge variety of distribution patterns. The total number of EST in the embryo pool was 752,991 and in the adult pool 1,227,835. Representative results were described for umbilical cord, bone marrow, peripheral stem cells, the nervous system, and the embryo. The ABO histoblood group systems maintain high priority for matching, because of the occurrence of naturally occurring anti-A/B antibodies. Substantial progress has been made in monitoring their levels and immunoglobulin isotypes in recipients, which, beyond hemagglutination, can now be quantitated using ELISA or cytofluorometry. A picture of ever-improving compatibility matching in solid organ and stem cell transplantation beyond mere HLA typing is the consequence.

178 RELATIVE ABUNDANCE OF Oct-4, Nanog, AND Sox-2 TRANSCRIPTS IN PORCINE OOCYTES AND CLEAVAGE-STAGE EMBRYOS PRODUCED VIA FERTILIZATION IN VITRO OR PARTHENOGENESIS

Parthenogenetic embryos obtained by electroactivation of mature oocytes have been used as models in developmental studies. The correct gene expression in early cleavage embryos is essential to sustain embryo development. The precise regulation of genes involved in pluripotency (Oct-4, Sox-2, and Nanog) is crucial to the formation of inner cell mass and trophoblast cells. Failure to do so can contribute to impaired development. We hypothesized that porcine embryos produced by fertilization in vitro and parthenogensis would possess a similar pattern of expression of Oct-4, Nanog, and Sox-2 during cleavage development. The objective of this study was to determine the developmental expression pattern of these three transcription factors in porcine oocytes and cleavage-stage embryos produced by either fertilization or parthenogenesis. Messenger RNAwas isolated from pools of 40-150 germinal vesicle (GV)- and MII-arrested oocytes and pools of 2-cell (2c), 4-cell (4c), 8-cell (8c), and blastocyst-stage embryos produced by in vitro fertilization (IVF) or electroactivation. Quantitative real-time PCR was performed following cDNA synthesis. Transcripts for Oct-4, Nanog, Sox-2, andYWHAG (housekeeping gene control) were amplified in duplicate across three to five experimental replicates. Transcripts were quantified using the comparative CT method using YWHAG as internal control and GV stage as normalizing stage. Fold activation and repression were analyzed with ANOVA and Tukey's post-hoc test. Our results show that porcine embryos produced by either IVF or electroactivation possess a similar pattern of pluripotent gene expression during cleavage-stage development. Oct-4 was found to be present in high abundance in the 2-cell parthenogenetic embryos and then repressed at the 8-cell stage (10-fold; P < 0.05, 2c v. 8c). In IVF embryos, Oct-4 was found in significantly higher amount at the 2-cell stage (35-fold; P < 0.05, 2c v. GV). Nanog transcripts were present at low levels from the GV oocyte until the 4-cell stage in both IVF and parthenogenetic embryos and then upregulated 10 000-fold at the 4-cell stage (P < 0.0001, GV v. 4c); at the blastocyst stage, Nanog transcript levels were similar to the levels found in the GV stage oocytes. Sox-2 transcripts were lower in MII oocytes and were significantly upregulated in 8-cell-stage embryos produced by either IVF or electroactivation (9- and 20-fold; P < 0.01, P < 0.0001, MII v. 8c, respectively). In addition, Sox-2 transcripts were significantly higher in parthenogenetic blastocysts compared to IVF-derived blastocysts (P < 0.05). This work demonstrates that cleavage-stage porcine embryos, produced by either electroactivation or IVF, undergo a similar pattern of activation of key regulatory genes; however, the activation method can have an influence on the transcript abundance of specific genes at defined stages.

152 TRANSMISSION OF BOVINE VIRAL DIARRHEA VIRUS BY IN VITRO-FERTILIZED EMBRYOS UNDER EXPERIMENTAL CONDITIONS

Bovine viral diarrhea virus (BVDV) infection affects cattle throughout the world. It causes significant economic losses in the cattle industry. The potential for transmission of a cytopathic biotype of BVDV by in vivo-derived embryos has been thought to be negligible. However, there is no study to prove non-transmission of the most common field isolate of noncytopathic biotype (NCPB) of BVDV by IVF embryos. Here we report on the preliminary outcome of embryo transfer (ET) of IVF embryos exposed in vitro to type-1 (NY-1) and type-2 (P-131) genotypes of NCPB of BVDV. For this experiment, IVF embryos were generated using standard methods which briefly involve: maturation of cumulus–oocyte complexes in TCM medium, fertilization of oocytes with BVDV-free semen, and culture of zygotes to the blastocyst stage in SOF medium without somatic cells. Day 7 blastocysts were exposed for 1 h to NY-1 or P-131 (103–107 TCID50 mL–1) BVDV strains before being washed (without trypsin) as recommended by IETS. Two embryos were transferred on each occasion. Embryo recipients were virus-free and anti-BVDV antibody-free prior to ET. The recipients remained individually in isolation premises after ET. In total, 126 ET procedures were performed resulting in 57 pregnancies and 34 calves born free of the infectious virus and BVDV antibodies (5 pregnancies are still pending). In total, 23 pregnancies were lost after 30 days. Exposure of embryos to type-2 BVDV resulted in a loss of 46% (17/37) of pregnancies after 30 days post-ET and 20 recipients seroconverted to BVDV. Within seroconverted and pregnant animals (n = 14), only 2 recipients maintained pregnancy and delivered uninfected calves at term. In contrast, exposure of embryos to type-1 caused 30% (6/20) of the pregnancy losses after 30 days and did not cause any seroconversion in ET recipients. After washing, 33% (3/9) and 38% (17/44) single embryos from the infected pool of IVF embryos tested positive for the BVDV. In conclusion, under these experimental conditions, a proportion of recipients was apparently infected after receipt of BVDV-exposed embryos. However, all of the calves that survived to term were BVDV-free and anti-BVDV antibody free.

Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19gene expression in individual mouse embryos

BackgroundIn the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos.In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 promoter was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data.ResultsWe show that:1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium.2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium.3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16.ConclusionCompared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the H19 ICR and H19 promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the epigenetic disturbance of the utilized techniques.

The Effect of Nitric Oxide Inhibition and Temporal Expression Patterns of the mRNA and Protein Products of Nitric Oxide Synthase Genes During In Vitro Development of Bovine Pre‐implantation Embryos

This study was conducted to determine the effect of Nitric oxide (NO) inhibition in bovine in vitro development and expression analysis of the three Nitric oxide synthase (NOS) isoforms: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS), mRNA and protein in bovine oocytes and embryos. Selective inhibitor of NOS, N-omega-nitro-l-arginine methyl ester (l-NAME) was applied at different doses (0, 0.1, 1 and 10 mm) in maturation (experiment 1A), culture medium (experiment 1B) and in both maturation and culture media (experiment 1C). No significant differences were observed in cleavage and blastocyst rates when oocytes were matured in the presence of l-NAME as long as the inhibitor was omitted during fertilization and culture. However, significantly lower blastocyst rates were observed when l-NAME was present at higher level (10 mm) in culture medium alone and in both maturation and culture media. In experiment 2, mRNA isolated from triplicate pools of oocytes and embryos (n = 15-20) was subjected to quantitative real time reverse transcription polymerase chain reaction to investigate the expression of eNOS, iNOS and nNOS mRNA in normal IVP bovine oocytes and embryos. While eNOS and iNOS transcripts were detected at higher level in oocytes (immature and mature), two-cell and four-cell stage embryos, the nNOS was detected only in immature oocyte, two-cell and morula stages. In experiment 3, eNOS and iNOS protein expression analysis was performed in IVP oocytes and embryos and both proteins were detected in the cytoplasm and the nuclei (weak) of oocytes and embryos. These data provide the first evidence for the role of NO production and the presence of mRNA and protein products of NOS isoforms during bovine embryogenesis.

Impact of in vitro production techniques on the expression of X‐linked genes in bovine (bos taurus) oocytes and pre‐attachment embryos

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 alpha = 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.

Maturation medium supplements affect transcript level of apoptosis and cell survival related genes in bovine blastocysts produced in vitro

The aim of the present study was to investigate whether protein or macromolecule supplements to in vitro maturation media affect transcript abundance of seven genes (Bax, Bcl2, Hsp70, IGF1, IGF1R, IGF2, and IGF2R) in oocytes and blastocysts. Cumulus-oocyte complexes aspirated from slaughterhouse ovaries were matured in TCM199 medium supplemented either with 10% FBS, 6% fatty acid free BSA (fafBSA) or 4% PVP40, then inseminated and cultured in vitro for 9 days. Transcript abundance analysis was carried out on immature and in vitro matured oocytes, as well as on blastocysts. Total RNA was isolated from pools of oocytes and embryos, reverse transcribed into cDNA and subjected to transcript analysis by real-time PCR. No transcript of IGF1 gene was detected either in oocytes or in blastocysts. Maturation conditions significantly affected transcript levels of investigated loci in blastocysts but not in matured oocytes, with one exception. Only relative abundance (RA) of IGF2 gene was higher in oocytes matured with fafBSA. Moreover, oocyte maturation with fafBSA elevated transcript abundance of IGF1R, IGF2, and IGF2R genes in resulting blastocysts, whereas Hsp70 transcription was stimulated by FBS supplementation. Thus, under described conditions, fafBSA may be the optimal supplement to IVM medium due to higher transcript level of growth factor coding genes accompanied by a lower transcript level of Hsp70.

273 EXPRESSION PATTERN OF CONNEXIN 43 (Cx43) AND POLY-(A) POLYMERASE (PAP) GENES IN BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED IN VITRO

Polyadenylation of pre-mRNA is carried out by poly-(A) polymerase (PAP), and study of the transcription pattern of this gene is said to indicate the developmental competence of the embryos. Connexin 43 (Cx43) is one of the important gap junction proteins that controls growth, cellular differentiation, and embryonic development. The objective of the present investigation was to study the expression pattern of PAP and Cx43 genes in buffalo (Bubalus bubalis) embryos produced in vitro. Embryos were produced from the slaughterhouse ovaries using standard IVMFC protocol (Rajhans et al. 2006 J. Reprod. Fertil. Dev. 18, 253–254). Briefly, oocytes were aspirated from follicles (2–8 mm in diameter) and matured in vitro in TCM-199 supplemented with 10% FCS and epidermal growth factor (20 ng mL-1) for 24 h. Presumptive zygotes after 18 h of fertilization were cultured in mSOF containing insulin-like growth factor-1 (100 ng mL-1) and β-mercaptoethanol (100 �M) for 9 days or until blastocyst formation, whichever was earlier. Pools of immature (n = 200), in vitro-matured (n = 200), oocytes and embryos (2–4 cell, n = 83; 8–16 cell, n = 80; morula, n = 77), and blastocysts (n = 40) were collected for mRNA isolation. Immature and in vitro-matured oocytes were treated with 1X trypsin-EDTA solution to remove the attached cumulus and then washed with TCM-199 before mRNA isolation to avoid any contamination of these cells during RNA isolation. mRNA from each pool was isolated using a commercially available direct mRNA isolation kit (Oligotex Direct mRNA kit; Qiagen, Valencia, CA, USA). cDNA was prepared using specific reverse primer and M-MLV RT in 20 �L reaction volume following manufacturer's instructions. Polymerase chain reaction was done for 35 cycles with annealing temperatures of 60�C and 58�C for 252 bp of PAP and 425 bp of Cx43, respectively. Amplicons were subjected to restriction endonuclease digestion for further confirmation of expressed genes. RT-PCR amplicon of PAP was digested with HaeIII to obtain characteristic band patterns at 119 bp and 133 bp, and Cx43 RT-PCR amplicon was digested with EcoR1 to obtain characteristic band patterns at 137 bp and 288 bp. While PAP expression could be detected in all stages of developing embryos starting from immature oocytes to blastocyst stage, Cx43 mRNA was detected in immature oocytes to morula stage but not in blastocyst-stage embryos. It could be concluded that the expression patterns of PAP and Cx43 genes in buffalo embryos produced in vitro are similar to those of cattle embryos.

Relationship between pre-embryo pronuclear morphology (zygote score) and standard day 2 or 3 embryo morphology with regard to assisted reproductive technique outcomes

To test the hypothesis that pregnancy rates are low if grade Z1 pre-embryos are not available for transfer and to determine if pronuclear morphology is a better predictor of pregnancy than traditional embryo morphology. Prospective clinical study. Academic human reproduction laboratory. One hundred couples undergoing IVF with conventional insemination or ICSI. Embryo quality was assessed using both pre-embryo pronuclear morphology (zygote scoring or Z-scoring) at the time of fertilization evaluation and standard day 2 and day 3 embryo morphology (number of blastomeres and grading based on degree of fragmentation and blastomere size). We tested two decision models, one based on Z-scores and another on morphology, to determine which grading system better predicted pregnancy outcomes in assisted reproductive technique. Zygote score and embryo morphology were measured for all embryos and the transferred embryo pool. Implantation and pregnancy rates resulting from the embryo transfers of all cycles were calculated. The Z-score distribution of 552 embryos was 27% Z1, 8% Z2, 50% Z3, and 15% Z4. Z1 and Z3 embryos had significantly (P approximately .03) higher quality over Z2 and Z4 embryos. Using the Z-score decision model with Z1 embryos having highest priority for transfer, pregnancy rates were similar between Z1 and Z3 embryos. Using embryo morphology as a decision model, pregnancy rates were highest in transfers containing one or two "best"-quality embryos. Z1 and Z3 embryos had similar morphology and pregnancy rates. The decision model based on the Z-score model was not better than standard embryo morphology in predicting pregnancy outcome.

Observations on Day 3 Embryo Morphology and Chromosomal Status in the Older Patient

Preimplantation genetic diagnosis (PGD) has been an invaluable tool in the study of increased aneuploidy rates with oocytes from older patients. The technique is however labor intensive , requires expertise and necessitates growth of the embryo in vitro to the blastocyst stage. Moreover, embryonic mosaicism may result in misdiagnosis and discarding of normal embryos. Thus, the use of preimplantation genetic diagnosis for aneuploidy has been recommended primarily for patients with a larger pool of embryos.

Preimplantation Embryo Aneuploidy Rates in Couples Undergoing Donor Egg In Vitro Fertilization Cycles

Preimplantation genetic diagnosis (PGD) is used in an effort to decrease spontaneous abortion, prevent the birth of aneuploid offspring and to potentially increase implantation by removing genetically abnormal embryos from the pool of embryos to be transferred. All currently available information regarding rates of aneuploidy among human preimplantation embryos comes from PGD of embryos of people with impaired fertility. In this population, aneuploidy screening is suggested for women with recurrent pregnancy loss, the transfer of good quality embryos without achieving a viable pregnancy or women of advanced maternal age.

Patients' attitudes towards donation of surplus cryopreserved embryos for treatment or research.

The aim of the study was 2-fold: first, to investigate couples' reasons for not using cryopreserved embryos within the maximum storage period; second, to study their attitudes towards potential embryo donation for specific purposes. A questionnaire was sent to 284 IVF/ICSI couples who experienced destruction of their cryopreserved embryos (n=1180) because the cryopreservation period exceeded the Danish legislative limit of 24 months. Seventy-four per cent of the couples responded. The main reasons for not utilizing surplus embryos was 'successful delivery' (85%), 'consider family completed' (61%) and 'too short legislative limit for cryopreservation' (59%). Sixty per cent of the couples agreed to the concept of donation of cryopreserved embryos for infertility research, 57% responded affirmatively to donation for stem cell research and 49% for stem cell treatment, but only 29% agreed to the concept of donation to infertile couples. Multiple logistic regression analysis showed that delivery of a child after IVF treatment (OR 3.8, 95% CI 1.4-10.2) and female age <35 years (OR 2.2, 95% CI 1.3-6.0) were predictive of agreement to the idea of donation for stem cell research and stem cell treatment respectively; however, male age, duration of infertility, mode of conception (IVF or ICSI) and having IVF children were not significant predictors. The following predictive variables were entered into the analysis: female and male age, duration of infertility, IVF versus ICSI, donor semen and +/- IVF children. This study shows that 23% of all couples having cryopreserved embryos do not utilize them for further treatment within the legislative storage period of 2 years. A major reason is successful delivery. More than half of these patients agreed to the concept of donation of surplus outdated embryos for research, whereas less than one-third agreed to donation to other infertile couples. Based on these figures, an alternative utilization of surplus embryos for stem cell research would require a 100-fold larger pool of available embryos to provide a realistic basis for this purpose.

Expression of peroxiredoxins in bovine oocytes and embryos produced in vitro.

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.

238ESTROGEN RECEPTOR ALPHA AND PROGESTERONE RECEPTOR EXPRESSION FROM REPRODUCTIVE TISSUE AND IN VITRO PRODUCED EMBRYOS OF THE DOMESTIC CAT

The in vitro production of cat embryos has been reported by several laboratories, and kittens have been born after transfer of embryos derived by IVF, ICSI, and NT. However, evidence accumulating in other species indicates that in vitro-derived embryos exhibit altered gene expression of developmentally important genes. Because the domestic cat genome is not well defined, the lack of primer sequence information poses a challenge for gene expression profiling. Estrogen, in addition to its essential role in the development and function of the female reproductive tract, is important for maturation of the oocyte. The aim of this preliminary study was to evaluate the expression profile of estrogen receptor alpha (ERα) and progesterone receptor (PR) in domestic cat female reproductive tissue and in vitro-produced (IVP) embryos. Embryos were produced in vitro as described by Gomez et al. (2003 Theriogenology 60, 239–251). mRNA was isolated from pools (n=35–50) of IVP domestic cat embryos at the morula and blastocyst stages (Days 7 and 8) using a modified protocol of the PolyAT Tract System (Promega, Madison, WI, USA). Uterus and ovaries were collected from hystorectomized cats and total RNA was isolated from these estrogen-targeted organs (RNeasy standard protocol, QIAGEN, Valencia, CA, USA). All RNA was reverse-transcribed and subjected to real-time PCR to evaluate expression of ERα and PR. The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as a control for expression in all tissues. Using primers designed to amplify human sequences or multispecies primers, we successfully amplified all three genes from as little as ∼100ng of ovarian or uterine mRNA. We were also able to detect ERα and GAPDH from pools of IVP embryos. Transcripts were cloned and confirmed by sequencing to be homologous to known sequences of the respective genes in a variety of species, including human, mouse, and pig. A 234-bp transcript of ERα (accession #AY349164;; GenBank) corresponding to exons 5 through 7 of the human ERα gene (hormone-binding domain) was identified along with a 110-bp sequence of PR and 98-bp sequence of GAPDH. To our knowledge, this is the first description of ERα or PR cloning in the domestic cat. In summary, we have demonstrated that highly sensitive real-time PCR is effective for the assessment of gene expression in cat ovarian and uterine tissue, as well as in embryos, the latter, of which, are known to have a low transcript copy number. Furthermore, these experiments provide the basis for future studies on the effect of exogenous estrogen on gene expression in cat IVM oocytes and IVP embryos.

244REAL TIME PCR EVALUATION OF EXPRESSION PROFILES OF SOME MATERNAL-SOURCED GENE TRANSCRIPTS IN IN VITRO PRODUCED PRE IMPLANTATION STAGE BOVINE EMBRYOS

Gene expression profiling data collected in a time series and quality related parameters are important for understanding the developmental mechanisms carried out in a developing embryo, and are also a source to enrich the knowledge base of embryo development. However, such data are frequently constrained by limitation and handling of the sample as well as cost associated with generating such data. Cumulatively, these factors have contributed to the existing insufficient data compared to the large need stemming from a drive to control and guide optimum embryo development. In this ongoing study, with objectives to quantify and evaluate gene transcripts identified from certain developmental stages, expression profiles of two ESTs (C256 and C112), derived from an oocyte cDNA library, were analyzed from the above perspectives to understand the change in the level of these gene transcripts throughout the pre-implantation stage of embryo development. For this analysis, pools of oocytes and embryos were prepared by balancing the amount proportional to the number of cells present. mRNA was isolated separately from each pool of matured oocytes, 2-cell, 4-cell, 8-cell, and 16-cell stages, as well as morula and blastocyst stages by using Dynal beads Oligo (dt)25 (Dynabeads, Dynal Biotech, Oslo, Norway) following the manufacturer’s recommendations. These mRNAs were checked for DNA contamination and, when proved free, first-strand cDNA was synthesised by reverse transcribtion at 42°C for 2h following standard laboratory procedures. Transcript quantification was performed by real-time PCR using gene-specific primers, equal amounts of cDNA from each sample and SYBR Green universal master mix. Following this analysis, both transcripts were found to be expressed in a wave-like manner being highly expressed in mature oocytes, declining gradually as the development stage advanced, with the lowest level at the 16-cell stage, and then reviving in level thereafter until it reached blastocyst stage. Taking the 16-cell stage as calibrator for both, C256 was 26.4, 23.2, 8.5, 1.7. 2.4 and 2.7 times more expressed in oocyte, 2-cell, 4-cell, 8-cell, morula and blastocyst stages, respectively. Similarly, C112 was 110.7, 169.2, 9.8, 2.5, 4.1 and 7.3 times more expressed in oocyte, 2-cell, 8-cell, morula and blastocyst stages, respectively. These expression patterns suggest the probable origin of these transcripts initially to be maternal. C256 is strongly similar to human retinoid X receptor beta (RXRb) gene (NM_021976.3), which is involved in transcriptional functions and in increasing DNA binding, whereas C112 is strongly similar to TATA box-binding protein-associated factor gene (AY189986.1), which is also involved in transcriptional functions. As seen from their functions, these transcripts can be vital for developing the embryo and the variations at different developmental stages shows their most probable role as part of genes contributing to developmental competence in pre-implantation development stages.

Expression of growth hormone and its transcription factor, Pit-1, in early bovine development.

During bovine embryogenesis, bovine growth hormone (bGH) contributes to proliferation, differentiation, and modulation of embryo metabolism. Pituitary-specific transcription factor-1 (Pit-1) is a transcription factor that binds to promoters of GH, prolactin (PRL), and thyroid-stimulating hormone-beta (TSHbeta) encoding genes. A polymorphism in the fifth exon of the bGH gene resulting in a leucine (Leu) to valine (Val) substitution provides an Alu I restriction site when the Leu allele is present. To determine the onset of embryonic expression of the bGH gene, oocytes derived from ovaries homozygous for Leu alleles were fertilized in vitro with spermatozoa obtained from a Val homozygote. For each developmental stage examined, three separate pools of embryos composed of approximately 100 cell samples underwent RNA isolation, reverse transcription to cDNA, and amplification by nested PCR (nPCR). Bovine GH gene transcripts were identified at 2- to 4-cell (n = 162), 8- to 16-cell (n = 73), morulae (n = 51), and blastocyst (n = 15) stages. Likewise, transcripts for Pit-1 were detected at 2-cell (n = 125), 4-cell (n = 114), 8-cell (n = 56), 12-to-32-cell (n = 32), morulae (n = 68), and blastocyst (n = 14) stages. After digestion with Alu1, bGH cDNA was genotyped by restriction fragment length polymorphism (RFLP) analysis. Bovine GH mRNA was present in all pools of stages examined. Both Leu and Val alleles (maternal and paternal) were only detected in pools of embryos that had reached 8- to 16-cell stage. Results suggest that transcription of the bGH gene begins at the 8- to 16-cell stage in bovine embryos, possibly under control of the transcription factor, Pit-1, and that RFLP analysis of the bGH gene can be used to determine parental origin of transcripts in early embryonic development.

The potential association between blastocyst transfer and monozygotic twinning

The potential association between blastocyst transfer and monozygotic twinning

Expression of Cu/Zn and Mn superoxide dismutases during bovine embryo development: influence of in vitro culture.

Temporal pattern of expression of Cu/Zn and Mn superoxide dismutases (SODs) was investigated in bovine oocytes and embryos produced in vitro in two different culture conditions and in vivo after superovulation. SODs were examined at a transcriptional level in single oocytes and embryos by reverse transcriptase-polymerase chain reaction (RT-PCR) and, at a protein level, by Western blotting on pools of embryos. mRNA encoding Cu/Zn SOD were detected in in vitro bovine embryos throughout preattachment development as well as in in vivo derived morulae and blastocysts. Transcripts for Mn SOD gene were detected in most immature and in vitro matured oocytes as well as in some zygotes and 5- to 8-cell embryos while no transcript was found at the 9-to 16-cell stage in both culture conditions. In vitro embryonic expression of Mn SOD was detected earlier in the presence of serum. Half of the morulae showed the transcript if cultured with 5% serum while none without serum. At the blastocyst stage Mn SOD could be detected independently of culture conditions. For in vivo-derived embryos Mn SOD transcripts were detected both in morulae and blastocysts. Immunoblotting analyses revealed that Cu/Zn SOD and Mn SOD were also present at a protein level in in vitro-derived zygotes and blastocysts. Together these data demonstrate, for the first time, that Mn SOD is transcribed and that Cu/Zn and Mn SOD proteins are expressed in preimplantation bovine embryos. Finally, they suggest that Mn SOD transcription is altered by in vitro culture conditions.