Individual bull variations on sperm acrosome reaction, sperm-zona binding, in-vitro embryo production, and preimplantation embryo apoptosis and gene expression

The overall objective of this study was to develop in-vitro tests to predict fertility of bulls in the field. The specific objectives were to investigate 1) the bull effect on sperm acrosome reaction, sperm-zona binding and in vitro embryo production (experiment 1), 2) the effect of sperm pre-incubation time and sperm concentration of bulls on in-vitro fertilization (experiment 2), 3) the bull effect on apoptosis and expression of Bcl-2, Bax, p53, interferon tau and heat shock protein-70 genes in bovine preimplantation embryos produced in-vitro (experiment 3), and 4) the correlation between the above in-vitro tests and the field fertility data. In the first experiment, prefreeze motility, acrosome reaction at 0 h, increase in acrosome reaction at 4 h and sperm-zona binding were different (p<0.05) among bulls. Significant correlations were observed between individual sperm parameters. None of the in-vitro tests was correlated with non-return rates (field fertility data). In the second experiment, significant bull effects were observed on fertilization, when using short and long sperm pre-incubation time with normal and high sperm:oocyte ratio. When using normal sperm:oocyte ratio (25,000:1), the percent difference in normally fertilized zygotes between short and long sperm pre-incubation times showed high degree of correlation with non-return rates (r = 0.90; p<0.05) of the experimental bulls. In the third experiment, significant bull effects (P<0.01) were observed on cleavage and morula to blastocyst development rates; percentage of apoptotic, live and dead cells; and expression levels of heat shock protein 70 and interferon tau genes in morula to blastocyst stage embryos. The expression levels of Bax, Bcl-2 and p53 genes in morula to blastocyst stage embryos were not different among bulls. The field fertility measured by 60-90 day non-return rate was highly correlated with relative abundance of Bcl-2 mRNA transcripts (r = -0.93) and the ratio of Bax to Bcl-2 gene expression (r = 0.84). The findings of this study conclude that variations exist among individual bulls in sperm acrosome reaction, sperm-zona binding and in-vitro embryo production, apoptosis, and interferon tau and heat shock protein 70 gene expression. Combinations of some of these sperm parameters may be potentially useful for the accurate prediction of bull fertility in the field.Land and Food Systems, Faculty ofGraduat

Bovine in vitro embryo production and oocyte preservation

Several experiments were done with the overall objective to improve the production of in vitro embryos. In the first experiment, the optimum period for ovary removal and the effect of FSH priming at standing estrus were investigated to maximize viable oocyte yield from culled cows. Animals were ovariectomized either 2 d after induced estrus, 2 d after a single dose of FSH on the day of standing estrus or randomly irrespective of the stage of the estrous cycle. Oocyte recovery and, cleavage and blastocyst formation rates were recorded for each treatment. Highest oocyte recovery was obtained from FSH-primed cows. Cleavage and blastocyst formation rates were not significantly different among treatments. These results indicated that FSH treatment increases oocyte yield in culled cows. The second experiment examined methods for the short-term preservation of bovine oocytes. Oocytes were matured in either i) straws containing maturation media (MM) at 38.5 °C without 5% C02, ii) straws containing MM at room temperature without 5% C02 or iii) culture plates containing MM at 38.5 °C and 5% C02. After 24 h, oocytes were fertilized and cultured in vitro. Oocytes, which were matured at room temperature without 5% C02, showed significantly lower maturation, fertilization, cleavage, and blastocyst formation rates than those matured at 38.5 °C. Oocytes matured at 38.5 °C with or without 5% C02 showed similar maturation, fertilization and cleavage rates, however, those with 5% C02 showed a significantly higher blastocyst formation rate. Therefore, 38.5 °C and a 5% C02 environment are optimal for oocyte maturation and embryo development. The third experiment was done to find a suitable cryopreservation method for the long-term preservation of bovine oocytes. Developmental competency of immature and mature oocytes undergoing slow freezing or vitrification was measured by cleavage and blastocyst formation rates. None of the slow frozen mature oocytes or vitrified immature and mature oocytes cleaved 72 h after insemination or developed to the blastocyst stage. Although some slow frozen immature oocytes eventually cleaved, the cleavage rate was significantly lower than that of control oocytes. This indicated that both cryoprotectant and cooling procedure affect the developmental competency of bovine oocytes. [Scientific formulae used in this abstract could not be reproduced.]Land and Food Systems, Faculty ofGraduat

Human embryonic stem cells derived from embryos at different stages of development share similar transcription profiles

We have derived hESC from biopsied blastomeres of cleavage stage embryos under virtually the same conditions we used for the derivation of hESC lines from inner cell mass of blastocyst stage embryos. Blastomere-derived hESC lines exhibited all the standard characteristics of hESC including undifferentiated proliferation, genomic stability, expression of pluripotency markers and the ability to differentiate into the cells of all three germ layers both in vitro and in vivo. To examine whether hESC lines derived from two developmental stages of the embryo differ in gene expression, we have subjected three blastomere-derived hESC lines and two ICM-derived hESC lines grown under identical culture conditions to transcriptome analysis using gene expression arrays. Unlike previously reported comparisons of hESC lines which demonstrated, apart from core hESC-associated pluripotency signature, significant variations in gene expression profiles of different lines, our data show that hESC lines derived and grown under well-controlled defined culture conditions adopt nearly identical gene expression profiles. Moreover, blastomere-derived and ICM-derived hESC exhibited very similar transcriptional profiles independent of the developmental stage of the embryo from which they originated. Furthermore, this profile was evident in very early passages of the cells and did not appear to be affected by extensive passaging. These results suggest that during derivation process cells which give rise to hESC acquire virtually identical stable phenotype and are not affected by the developmental stage of the starting cell population

Behavior and Brain Gene Expression Changes in Mice Exposed to Preimplantation and Prenatal Stress

Preimplantation culture of mouse embryos has been suggested to result in reduced anxiety-like behavior in adulthood. Here, we investigated the effects of in vitro fertilization (IVF), embryo culture, and different diets on anxiety-like behavior using the elevated plus maze (EPM). We hypothesized that exposure to suboptimal conditions during the preimplantation stage would interact with the suboptimal diet to alter behavior. The expression of genes related to anxiety was then assessed by quantitative real-time polymerase chain reaction in various brain regions. When fed a normal diet during gestation and a moderately high-fat Western diet (WD) postnatally, naturally conceived (NC) and IVF mice showed similar anxiety-like behavior on the EPM. However, when fed a low-protein diet prenatally and a high-fat diet postnatally (LP/HF), NC mice showed a modest increase in anxiety-like behavior, whereas IVF mice showed the opposite: a strongly reduced anxiety-like behavior on the EPM. The robust reduction in anxiety-like behavior in IVF males fed the LP/HF diets was, intriguingly, associated with reduced expression of MAO-A, CRFR2, and GABA markers in the hypothalamus and cortex. These findings are discussed in relation to the developmental origin of health and disease hypothesis and the 2-hit model, which suggests that 2 events, occurring at different times in development, can act synergistically with long-term consequences observed during adulthood

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification.

Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines