Thesis (M.S.)--University of Missouri-Columbia, 2008. / "May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

MicroRNAs are small regulatory RNAs that bind to the seeding regions within the 3’-untranslated region (3’-UTR) of their target transcripts to modulate transcript stability and/or inhibit protein translation. MicroRNA Let-7a belonging to the Lethal-7 (Let-7) family is down-regulated at the blastocyst stage, suggesting its suppression is crucial for embryo implantation. Yet, the underlying mechanism on how Let-7a modulates blastocyst implantation remains largely unknown. In silico analysis identified attachment related integrin beta-3, outgrowth related vav3, and the microRNA processing dicer, as Let-7a targets. Therefore, it is hypothesized that down-regulation of Let-7a promotes embryo implantation by stimulating these target proteins. Let-7a is down-regulated during blastulation and at 3-hour post-estradiol activation of the dormant blastocysts. Force-expression of Let-7a in mouse blastocysts suppressed blastocyst attachment, outgrowth on fibronectin-coated plates and compromised pregnancy in vivo. Dual luciferase assay using the 3’-UTR reporter constructs of the integrin beta-3, vav3, and dicer confirmed the interaction between Let-7a and the three targets. Force-expressing or inhibiting Let-7a expression in mouse blastocysts by electroporating the Let-7a precursor or inhibitor respectively illustrated post-transcriptional regulation of Let-7a on integrin beta-3 and vav3, and transcriptional regulation on dicer. Dormant blastocysts retrieved from the delayed implanting mice expressed high Let-7a levels, which was suppressed in the first 12-hours of estradiol activation. Concomitantly, dormant blastocysts expressed low levels of integrin beta-3, vav3, and dicer, yet, their protein expression was up-regulated from 3 hours-post estradiol activation. Furthermore, addition of integrin beta-3 antibody suppressed attachment of trophoblast spheroids (blastocyst surrogate) onto endometrial epithelial cells in a co-culture model and the outgrowth of the spheroids on fibronectin-coated plates. Knockdown of Vav3 with siRNA decreased blastulation, hatching, and outgrowth rates of the embryos in vitro. Although the loss of vav3 activities did not affect embryo implantation, it suppressed trophoblast migration on fibronectin-coated plates and invasion into collagen matrix. In contrast, force-expression of vav3 enhanced blastocyst outgrowth, and promoted cell proliferation. Blocking integrin beta-3 activities in the vav3 knock-down embryos further suppressed blastocyst outgrowth, suggesting the intertwining effect of the integrins and vav3. Dicer knock-down in mouse blastocysts decreased mature Let-7a expression and suppressed blastulation and hatching in vitro and implantation in vivo. Dicer knock-down in estradiol activated mouse blastocysts decreased the epidermal growth factor receptor expression and lowered the affinity of the embryos to EGF, and suppressed the implantation potential to a level similar to that of dormant blastocysts. Taken together, the suppression of Let-7a by estradiol up-regulates integrin beta-3, vav3, and dicer. The increased Itgb3 expression promotes blastocyst attachment and intertwined with the up-regulated vav3 expression to enhance blastocyst outgrowth. The increased vav3 expression further stimulates cell proliferation and confers blastocyst invasion into the collagen matrix. Dicer, by altering microRNA processing, facilitates blastulation and thereby embryo implantation. Thus, the loss of Let-7a biological activities during blastulation is crucial to enhance blastulation and stimulate trophoblast activities for successful embryo implantation. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Traditional Chinese medicine (TCM) plays an important role in the Chinese healthcare system for over five thousand years. It includes the use of herbal medicine, acupuncture, Tui Na (推拿), and diet therapy. TCM helps to maintain a balance of Yin-Yang (阴阳), Five Phases (五行), Meridians (经络) and Qi (气) inside the body. In practise, pregnant women take tocolytic drugs to tonify the blood and qi to provide a continuous supply of nutrients for baby. Traditional Chinese herbal medicines usually prescribed as a complex formula to produce synergistic or agonistic effect to maintain a well balance of the above components in human bodies. Moreover, TCM usually cannot produce immediate effect on patients, therefore, the efficacy of individual component remains largely unknown. This study aims to investigate whether Chinese tocolytic drug components could modulate fertility by affecting the in vitro spheroid (blastocyte surrogate) attachment process by using trophoblastic (JEG-3) and endometrial epithelial (Ishikawa) cells to mimic the embryo-endometrial implantation process. Nine Chinese herbal medicinal compounds (Atractylenolide I(白术内酯), Atractylenolide II(白术内酯II), Atractylenolide III(白术内酯III), Paeoniflorin(芍药苷), Albiflorin(芍药内酯苷), Nuzhenide(女贞子甙), Ecliptasaponin A(旱莲甙A), Wedelolactone(蟛蜞菊内酯) and Columbianadin(二氢欧山芹醇当归酸酯)) which are commonly found in traditional Chinese tocolytic drug formula were selected to study (1) the toxicity of the drugs on trophoblastic (JEG-3) and endometrial epithelial (Ishikawa) cells growth, (2) the effect of three tocolytic drugs (Atractylenolide I, Atractylenolide II and Atractylenolide III) on spheroid attachment, and (3) their effect of the expression of Wingless (Wnt) signaling molecules (Active-β-Catenin, Axin-2, β-catenin, E-cadherin, GSK-3β, and Mucin-1). It was found that the nine compounds, Atractylenolide I, Atractylenolide II, Atractylenolide III, Paeoniflorin, Albiflorin, Nuzhenide, Ecliptasaponin A, Wedelolactone and Columbianadin did not affect cell viability at 25μM, 25μM, 5μM, 0.2μM, 125μM, 125μM, 125μM, 5μM and 25μM, respectively, by cell proliferation assay. However, at these concentrations, the spheroid attachment was not significantly increased by Atractylenolide I, Atractylenolide II and Atractylenolide III. Interestingly, the protein expression of GSK-3β and Active-β-catenin were up-regulated by the three compounds in both cells and JEG-3 cells respectively. The expressions of Axin-2 and E-cadherin were up-regulated by Atractylenolide III in Ishikawa cells and Atractylenolide II in JEG-3 cells. Atractylenolide I and Atractylenolide III increase the Ishikawa cells expression of Active-β-catenin and β-catenin respectively and together suppress the JEG-3 cells Mucin-1 and β-catenin expression. In conclusion, the nine tocolytic compounds have different effect on cell proliferation. Atractylenolide I, Atractylenolide II and Atractylenolide III did not enhance the attachment rate of JEG-3 spheroid onto Ishikawa monolayer. However, they affected Wnt-signaling molecules expression, suggesting that they may modulate endometrial receptivity. Further experiments are needed to study their combined effect on co-culture and expression of Wnt-signaling molecules. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Medical Sciences

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Cryopreservation of gametes is a valuable tool for the fast-growing aquaculture industry in New Zealand. In the present study, research was aimed to improve the cryopreservation of Pacific oyster (Crassostrea gigas) oocytes. For this, two main approaches were used: the modification of an existing published (standard) cryopreservation protocol for oyster oocytes and the modification of the oocytes themselves prior to cryopreservation. The objectives in the chapters of this thesis were: (a) determination of the cryobiological characteristics of oyster oocytes; (b) assessment and reduction of intracellular ice formation (IIF) in oocytes; and (c) modification of the lipid fraction (cholesterol and fatty acids) of oocytes prior to cryopreservation. Knowledge of the membrane permeability parameters in response to concentrations of water and ethylene glycol (EG), the influence of temperature upon these parameters, and the osmotic tolerance limits of oyster oocytes were used to develop computer models that simulated the cellular volume changes that oocytes underwent during EG addition and removal. The models predicted that when one part of EG was added in one step to one part of oocyte suspension and equilibrated for 20 min at 20 �C, similar volume changes in oocytes would be obtained, compared to a more complicated multi-step addition method. This method of addition resulted in similar post-thaw fertilization rates to those obtained by using the multi-step addition method, thus reducing oocyte handling. Cryomicroscopy was used to assess the effect of cooling rates and EG concentration on the temperature at which oocytes underwent IIF. It was found that IIF occurred at higher subzero temperatures when fast cooling rates were used (30 and 5 �C min⁻�) and at EG concentrations ranged between 0 and 15%. At a relatively slower cooling rate of 0.3 �C min⁻� and with 10% EG, which are the conditions employed in the standard cryopreservation protocol, no IIF occurred. The steps of the standard protocol that were more likely to cause oocyte damage were identified by evaluating the fertilization rate of oocytes at each step. Results showed that oocytes were most damaged by cooling them to -35 �C and followed by plunging them in liquid nitrogen. Contrary to what had been observed under the cryomicroscope, transmission electron microscopy (TEM) analysis revealed that all oocytes cryopreserved by the standard protocol contained cytoplasmic ice. In addition, it was also observed that oocytes were at two developmental stages when frozen (prophase and metaphase I). These observations prompted the development of alternative cooling programmes aimed to reduce intracellular ice. The effect of cooling rate, plunge temperature and time held at the plunge temperature were thus evaluated, based on post-thaw fertilization rate of oocytes. Overall, neither the cooling rate nor the holding time had an effect on oocyte fertility. However, the plunge temperature had an effect, where oocytes plunged at -60 �C had lower post-thaw fertilization rates than oocytes plunged at -35 �C. Through the slowing of the cooling rate, lengthening of the holding time and lowering of the plunge temperature, it was possible to reduce the amount of ice in the cytoplasm. However, the reduction of intracellular ice did not improve the post-thaw fertilization rate of the oocytes; on the contrary, post-thaw fertilization decreased notoriously. From these results, it can be suggested that oyster oocytes are more likely to be damaged by exposure to high intra and extracellular solute concentration than IIF during cryopreservation. In an effort to modify the lipid content of oyster oocytes prior to cryopreservation and thus, making them more resistant during cryopreservation, oocytes were incubated in solutions that would add or remove cholesterol or in solutions rich in long chain fatty acids (EPA or DHA). Oocytes incubated in cholesterol-rich solutions showed a positive uptake of fluorescently labelled cholesterol and this effect was dose dependent. Nevertheless, this uptake did not improve the post-thaw fertilization rate nor did it increase the total cholesterol content of the oocytes. When oocytes were incubated in non-conjugated or conjugated EPA or DHA, no increase in the proportion of these fatty acids was identified in the fatty acid profiles of whole oocytes and no improvement of the post-thaw fertilization rate was recorded. Given that there was no uptake of fatty acids from the incubation media by the oocytes, a different approach was taken. This involved the supplementation of lipid-rich diets to the oyster broodstock during gametogenesis (cold-conditioning) and vitellogenesis (warm-conditioning). Despite results showing that lipid content and, indeed, fatty acid profile was altered through the diet, the results also showed that fresh oocytes from broodstock fed during cold-conditioning did not show any improvement in their fertilization rates, nor did they benefit from a lipid-rich diet during warm-conditioning. On the other hand, cryopreserved oocytes did have higher post-thaw fertilisation rates when broodstock were fed during cold-conditioning and, although no effect was found from feeding broodstock with either of the lipid-rich diets during warm-conditioning, trends indicated that a diet consisting of fresh microalgae or the commercial supplement Algamac would yield the highest post-thaw fertilization rates. This thesis has furthered the understanding of some of the factors that determine cryosurvival in oyster oocytes and has demonstrated that both physical and biological issues must be taken into consideration for cryopreservation. Specifically, the results in this thesis helped to modify an empirically developed cryopreservation protocol for Pacific oyster oocytes. In addition, the results also showed strong evidence of the survival of oyster oocytes to intracellular ice and highlighted the importance of supplying the broodstock with lipid-rich food during the periods of gamete formation and maturation in order to obtain oocytes that are more amenable to cryopreservation. These benefits could be of significant practical importance and may be extended for the development or refinement of cryopreservation protocols for other shellfish species of commercial importance to the aquaculture industry of New Zealand.

Thesis (M.S.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains vi, 45 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 40-45).

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, Waite Agricultural Research Institute, 1989. / Includes bibliographical references (leaves 143-165).

Thesis (M.S.)--University of Missouri-Columbia, 2004. / Typescript. Includes bibliographical references (leaves 69-71). Also available on the Internet.