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The overall aim was to evaluate a non-invasive technique for the assessment of oocytes and embryos using polarized light microscopy (PolScope-LC) with the goal of improving success rates in IVF. A literature review revealed little validation of the PolScope techniques in published work. A reproducible and accurate method for measuring the zona pellucida (ZP) thickness and density involving the PolScope computer software was validated by achieving low coefficient of variance and small inter/intra observer errors. Utilizing this method, 1477 oocytes from 211 stimulated cycles were analysed in this thesis. Results showed that increasing age has an adverse effect on the ZP thickness and density. Study of extended culturing of embryos showed that the ZP starts thinning as early as day 3 and embryos tend to have denser zonas over time. Standardisation of timing of PolScope observations in relation to the meiotic spindle was studied. Metaphase II oocytes were examined sequentially in culture from aspiration until microinjection using the PolScope The spindle is a highly dynamic structure that can appear and disappear over time in culture. A visible spindle was detected in 58% of the oocytes immediately after aspiration. This percentage increased until it stabilised at 39-40hrs post hCG and then declined significantly. Average spindle signal intensity increased over time reaching its peak at 39-40hrs post hCG, then declined significantly by 40.5hrs post hCG. The importance of spindle presence and morphology was investigated by following up embryos created after sperm injection at 39-40hrs post hCG. There was a significant relationship between normal meiotic spindle shape and density and embryo quality. A higher percentage of ???usable??? embryos, and all of pregnancies, arose from oocytes with a normal barrel shaped spindle. Finally, the impact of two issues related to spindle formation - the type of hCG used to trigger oocyte maturation and the site of microinjection during ICSI were assessed using the PolScope. The results showed a biological difference in spindle formation and embryo quality between rhCG and uhCG. In a separate randomised trial embryo quality was better when injecting the sperm in the vegetal pole away from the spindle during ICSI. The results from this thesis suggest that PolScope, if appropriately applied, may assist in improving IVF outcome.

The overall aim was to evaluate a non-invasive technique for the assessment of oocytes and embryos using polarized light microscopy (PolScope-LC) with the goal of improving success rates in IVF. A literature review revealed little validation of the PolScope techniques in published work. A reproducible and accurate method for measuring the zona pellucida (ZP) thickness and density involving the PolScope computer software was validated by achieving low coefficient of variance and small inter/intra observer errors. Utilizing this method, 1477 oocytes from 211 stimulated cycles were analysed in this thesis. Results showed that increasing age has an adverse effect on the ZP thickness and density. Study of extended culturing of embryos showed that the ZP starts thinning as early as day 3 and embryos tend to have denser zonas over time. Standardisation of timing of PolScope observations in relation to the meiotic spindle was studied. Metaphase II oocytes were examined sequentially in culture from aspiration until microinjection using the PolScope The spindle is a highly dynamic structure that can appear and disappear over time in culture. A visible spindle was detected in 58% of the oocytes immediately after aspiration. This percentage increased until it stabilised at 39-40hrs post hCG and then declined significantly. Average spindle signal intensity increased over time reaching its peak at 39-40hrs post hCG, then declined significantly by 40.5hrs post hCG. The importance of spindle presence and morphology was investigated by following up embryos created after sperm injection at 39-40hrs post hCG. There was a significant relationship between normal meiotic spindle shape and density and embryo quality. A higher percentage of ???usable??? embryos, and all of pregnancies, arose from oocytes with a normal barrel shaped spindle. Finally, the impact of two issues related to spindle formation - the type of hCG used to trigger oocyte maturation and the site of microinjection during ICSI were assessed using the PolScope. The results showed a biological difference in spindle formation and embryo quality between rhCG and uhCG. In a separate randomised trial embryo quality was better when injecting the sperm in the vegetal pole away from the spindle during ICSI. The results from this thesis suggest that PolScope, if appropriately applied, may assist in improving IVF outcome.

The major source of calcium for the developing chick embryo is the eggshell. However, the actual mechanism of calcium solubilization from the shell is unknown. The temporal correlation of carbonic anhydrase activity to calcium movement implies that an acidic environment is essentail. To clarify the role of carbonic anhydrase in calcium solubilization and uptake by the chorioallantoic membrane of the chick embryo treatment effects on embryo development, carbonic anhydrase activity and calcium and pH levels were investigated. The treatments consisted of solutions of acid, base, calcium and strontium chlorides, and the enzyme inhibitor-acetazolamide.Experimental sampling was conducted from eleven to sixteen days of incubation. The treatments were administered daily by dipping the eggs into a treatment solution with subsguent sampling on the following day. The acid treatment solution produced a significant (P≤0.05) increase in calcium solubilization and a decrease in carbonic anhydrase activity from the control levels. The base treatment solution produced a significant decrease in calcium solubilization and an increase in carbonic anhydrase activity from the control levels. The calcium chloride treatment solution (providing a partial source of a calcium non-carbonate compound) and the strontium chloride treatment solution (providing a partial source of a non-calcium non-carbonate compound) showed no overall effect on embryonic calcium concentrations. However, there was a decrease in carbonic anhydrase activity from that of the control. This decrease did not appear to be due to a calcium mediated process but was more sensitive to changes in bicarbonate or carbonate levels. Treatments with acetazolamide demonstrated that there was a decrease in carbonic anhydrase activity and also a decrease in calcium transported. However, when an acid treatment was combined with the acetazolamide treatment, calcium was transported even though carbonic anhydrase activity was greatly suppressed. These results confirm that carbonic anhydrase activity appears to be fimctioriing in calcium solubilization and maintenance of the embryonic acid-base balance. It does not appear that the enzyme functions primarily in the transport of calcium across the chorioallantoic membrane. / Land and Food Systems, Faculty of / Graduate

Maternally-derived transcripts and proteins support early bovine and ovine embryo development until the 8- to 16-cell stage, at which time embryonic transcripts become essential for continued development. One purported mechanism for the switch from maternal to zygotic control of development (maternal to zygotic genome activation; MZGA) is the appearance of transcription factors that activate specific genes in the embryonic genome. Members of the E26 transformation specific (Ets) family are unique transcription factors involved in development, differentiation, and protease regulation. This study was undertaken to evaluate expression and function of the Ets transcription factors, Ets-1, Ets-2, and Elf-1, in early bovine and ovine embryos from the one-cell stage to Day 15 of pregnancy (Day 0 onset of estrus). In the first experiment, bovine embryos from the one- to 16-cell stages were derived by in vitro maturation, fertilization, and culture. Days 6, 8, 10, 12, and 14 embryos were collected nonsurgically from estrous synchronized and superovulated cows. RNA was extracted at the appropriate time interval and reverse transcribed. The resultant cDNA was amplified by PCR using primers designed for Ets-1, Elf-1, and Ets-2. Ets-1 transcripts were present in both primary and matured oocytes, cleavage stage embryos, and Days 10, 12, and 14 embryos, as well as in the positive control, bovine ovary. Elf-1 transcripts were detected in the matured oocyte, cleavage stage embryos, and Days 6, 10, and 14 embryos. Ets-2 transcripts were not observed in the embryonic stages investigated or the bovine ovary. Ovine embryos were surgically collected from synchronized and superovulated ewes and similarly analyzed for Ets-1 and Elf-1 expression using the same RNA extraction and RT-PCR technique. Embryos expressed both transcripts at Days 13 and 15, but did not show expression at any of the earlier stages evaluated. The second experiment was designed to determine if inhibition of ETS-1 translation would interfere with development and plasminogen activator (PA) production in bovine embryos. Plasminogen activator production was evaluated in Days 5 and 6 embryos nonsurgically collected from superovulated cows and cultured in 1, 2.5, 5, or 10 ��M concentrations of sense or antisense Ets-1 oligonucleotides. In preliminary experiments, 1 ��M antisense was ineffective in suppressing PA production, and 10 ��M oligonucleotides were detrimental to development. Day 5 embryos treated with 2.5 ��M oligonucleotides inhibited developmental effect and total PA production was (P<0.05) lower in antisense treatments when compared to either control or sense treatments. No difference (P>0.10) in PA production was observed between Day 6 embryos treated with 2.5 or 5 ��M sense and antisense oligonucleotides. A significant time effect on PA production was observed in both Day 5 and Day 6 embryos cultured in either 2.5 or 5 ��M concentrations of oligonucleotides. Based on these results, it is unlikely that Elf-1 and Ets-2 are involved in MZGA because the former is constituitively expressed throughout development, and the latter was not observed. There is some uncertainty regarding the expression of Ets-1 during MZGA. This factor may be expressed after MZGA for controlling PA production and other proteases involved in extracellular matrix turnover and early germ layer formation. / Graduation date: 2003

Graduation date: 1997

No description available.

No description available.

No description available.

In the first experiment, estradiol production was measured in cultured embryos collected on Days 7, 9, 11, 13 and 15 post-mating. Estradiol was detected in Day 7 embryos and production increased approximately 50-fold (P<0.05) in medium recovered from Day 13 compared to Day 11 embryos and was greatest (P<0.05) from Day 15 embryos. The second experiment evaluated the luteotrophic effect of estradiol in the llama. Daily injections of vehicle only, 5 or 10 mg estradiol benzoate (EB) in isopropylmyristate were administered on Days 7-15 (Day 0=hCG ovulation induction). Mean progesterone levels were greater (P<0.05) on Days 14, 15, 16, and 17 in llamas injected with 10 mg EB compared to llamas injected with vehicle or 5 mg EB. The final experiment analyzed differences in estrogen receptor expression in the corpus luteum (CL), ovary, and uterus using Reverse Transcription-Polymerase Chain Reaction. A significant linear regression (P<0.05) was detected for both ER�� (increased from Days 7-11), and ER�� (decreased from Days 7-11) in llama CL. ER�� expression in ovary was higher (P<0.05) compared to Days 7 and 9 CL. ER�� expression in CL was lower on Day 9 (P<0.05) than on Days 7 and 11 in pregnant animals. ER�� expression decreased (P<0.01) from Days 7-11 in uterus and expression was lower in pregnant versus non-pregnant uterus (P<0.10). ER�� expression was higher in pregnant versus non-pregnant uterus (P<0.10). No differences (P>0.10) in ER�� or ER�� expression were detected in endometrium due to reproductive status or days post-mating. A decrease (P<0.01) in ER�� expression was detected in pregnant uterus from Days 7-13. ER�� expression was lower (P=0.12) in juvenile uterus versus pubertal females. ER�� expression in non-pregnant uterus was different (P<0.05) by days post-mating and reproductive status. These data suggest that estradiol produced by the embryo may be involved in embryo migration and maternal recognition of pregnancy in the llama, and these events may be mediated by differential expression of ER subtypes / Graduation date: 2000