Thesis (Ph. D.)--University of Wisconsin--Madison, 1995. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Traditional methods of oocyte selection for in vitro studies have proven inefficient with respect to achieving a level of predictability for competency. In this study, a novel method of oocyte selection was implemented that identified a relationship between oocyte morphological parameters (as defined by a ratio of a shape factor (SF) to average fluorescence intensity (AFI) and AFI, followed by in vitro fertilization (IVF) and in vitro culture (IVC) using the Well of Well (WOW) method to evaluate oocyte competency. Specifically, we used non-cytotoxic fluorescent molecular probes and multiphoton microscopy to non-invasively characterize spatial localization and functional activity of mitochondria, mitochondrial membrane potential (Δψm), and intracellular calcium activity ([Ca2+]i) using rhodamine 123, JC-1 and Fluo-4, AM, respectively in bovine and canine in vitro matured (IVM) oocytes. Comparison of morphological grading with fluorescence intensity yielded similar trends between all grades of oocytes for both species with no visually obvious, distinct characteristic staining that would permit classification of each oocyte as a specific morphological grade. Our studies confirmed that oocyte mitochondria were homogeneously distributed but primarily localized to the peri- and sub-cortical regions of the oocyte at MII stage for both species. Further, heterogeneously polarized mitochondria were localized to the peri- and sub- cortical regions of the oocyte for both species. In bovine oocytes labeled with Fluo-4, AM, levels of [Ca2+]i were either unremarkable, or very low and limited to the peri-cortical areas, just beneath the oolema. For canine MII stage oocytes, levels of [Ca2+]i were within the same range of AFI as bovine. Ranges of fluorescence intensity compatible for optimal embryo development for bovine and optimal fertilization for canine oocytes were 30-300 and 20-35, and 20-30 and 20-25.5 for rhodamine 123 and Fluo-4, AM, respectively. The optimal range for bovine oocytes imaged with JC-1 was 1.25-2.25 and <6 for canine.

Traditional methods of oocyte selection for in vitro studies have proven inefficient with respect to achieving a level of predictability for competency. In this study, a novel method of oocyte selection was implemented that identified a relationship between oocyte morphological parameters (as defined by a ratio of a shape factor (SF) to average fluorescence intensity (AFI) and AFI, followed by in vitro fertilization (IVF) and in vitro culture (IVC) using the Well of Well (WOW) method to evaluate oocyte competency. Specifically, we used non-cytotoxic fluorescent molecular probes and multiphoton microscopy to non-invasively characterize spatial localization and functional activity of mitochondria, mitochondrial membrane potential (Δψm), and intracellular calcium activity ([Ca2+]i) using rhodamine 123, JC-1 and Fluo-4, AM, respectively in bovine and canine in vitro matured (IVM) oocytes. Comparison of morphological grading with fluorescence intensity yielded similar trends between all grades of oocytes for both species with no visually obvious, distinct characteristic staining that would permit classification of each oocyte as a specific morphological grade. Our studies confirmed that oocyte mitochondria were homogeneously distributed but primarily localized to the peri- and sub-cortical regions of the oocyte at MII stage for both species. Further, heterogeneously polarized mitochondria were localized to the peri- and sub- cortical regions of the oocyte for both species. In bovine oocytes labeled with Fluo-4, AM, levels of [Ca2+]i were either unremarkable, or very low and limited to the peri-cortical areas, just beneath the oolema. For canine MII stage oocytes, levels of [Ca2+]i were within the same range of AFI as bovine. Ranges of fluorescence intensity compatible for optimal embryo development for bovine and optimal fertilization for canine oocytes were 30-300 and 20-35, and 20-30 and 20-25.5 for rhodamine 123 and Fluo-4, AM, respectively. The optimal range for bovine oocytes imaged with JC-1 was 1.25-2.25 and <6 for canine.

The RNA binding protein DAZL (Deleted in Azoospermia) is essential for germ cell survival and subsequent fertility. The transgenic mouse DAZL model has confirmed that knockout (KO) females are infertile as a direct consequence of complete postnatal oocyte ablation. Interestingly, the heterozygous (Het) DAZL females have increased fertility giving rise to significantly more viable offspring, accompanied by significantly reduced plasma FSH and increased inhibin B compared to levels observed in the wildtype (Wt) females. Recent studies to identify putative DAZL mRNA targets suggest that DAZL may have multiple functions and mRNA targets throughout germ cell development. However, how this protein functions within the oocyte and how functional copy number gives rise to increased fertility remains to be fully elucidated. The studies in this thesis sought to identify putative DAZL mRNA targets in addition to molecular mechanisms which may be either affected direct or indirectly as a result of the functional copy number of DAZL (Wt or Het) within the oocyte or follicular unit. Oocytes from Wt and Het were evaluated for their expression of selected oocyte genes and comparative analysis suggests that oocyte gene expression is significantly altered between the genotypes. Genes of interest include Oosp1 and H1foo, both of which are down-regulated in mRNA expression in Het d21 oocytes and d10 ovaries compared to the Wt. Furthermore, an in silico bioinformatics approach was utilised to identify putative DAZL mRNA targets using a consensus DAZL binding sequence. One candidate target, PDCD4, previously identified as a tumour suppressor gene was selected for further investigation. Despite PDCD4 mRNA and protein being highly expressed within the ovary, no difference in mRNA levels between Het and Wt was observed. However, although not ruling out the possibility of being a DAZL target we now have evidence that PDCD4 can function within the steroidogenic cells of the corpus luteum in relation to functional luteolysis. Indirect actions of DAZL upon local regulation and response of follicle growth in culture were evaluated to investigate follicles at the gonadotrophin dependent stage of growth. Individual follicles from Wt and Het d21 mice were cultured in the presence of FSH at 1iu, 0.5iu, 0.1iu and 0.01iu for a six day period. Final follicle size/morphology did not differ between genotypes at 1iu, 0.5iu and 0.1iu of FSH, but by d3 at 0.01iu FSH growth of Wt follicles was significantly (P<0.001) perturbed compared to the Het. Despite no difference in final size between 1iu, 0.5iu, 0.1iu FSH treatments, mRNA analysis of individual follicles demonstrated a significant up-regulation of FSH receptor (P<0.05), aromatase (P<0.05) and inhibin βA (P<0.01) and a significant down-regulation in inhibin βB (P<0.01) expression in the Het follicles compared to the Wt, suggesting an increase in follicle maturity, sensitivity and hence suitability for selection as viable pre-ovulatory follicles. Furthermore, atresia rates from cultured follicles were significantly lower (P<0.05 (1iu, 0.1iu FSH); P<0.01(0.01iu FSH)) in the Het compared to the Wt. These studies provide strong evidence that multiple mechanisms within the oocyte/follicle are directly and indirectly affected as a result of functional copy number of DAZL. Although direct in vivo targets remain to be identified it is clear that DAZL protein potentially targets multiple mRNAs at different stages of development, pre-programming the oocyte to increase the sensitivity of follicle and/or the functioning within a transcription complex regulating development. In conclusion, the beneficial consequences of increased fertility in the Het females is accompanied by a possible acceleration in oocyte and follicle maturation, an increased sensitivity to FSH in vitro with evidence of advanced stages of growth and, a reduction in follicle atresia. These differences support the suggestion that DAZL is having systemic effects on the paracrine communication within the follicle unit between the oocyte and somatic cells altering regulation and subsequent selection, and affecting final ovulation rate and litter size.

Over recent years, the application of assisted reproductive techniques in the treatment of infertility has increased exponentially, yet these methodologies still remain inherently inefficient. It has long been established that the single greatest obstacle to improving the success of these treatments is determining the quality of the oocytes used. However, currently the methods available for oocyte assessment are mainly qualitative, and suffer due to a lack of standardisation. As such, the efficiency of fertility treatments could benefit from the introduction of a rigorous quantitative measure of oocyte quality and maturation. The principal aim of this thesis was to determine the potential of Raman spectroscopy when applied to the field of oocyte biology. Consequently, this thesis addressed three main areas of investigation: I. the intra-oocyte biochemical variation; II. the biochemistry of oocyte maturation; and finally, III. the effect of environment on the mature oocyte in vivo and in vitro. I. To investigate the presence of intra-oocyte biochemical variation, oocytes from various stages of development were analysed using high resolution Raman mapping, in combination with univariate and multivariate analysis. Images revealed variation between the germinal vesicle and ooplasm in immature oocytes, as well as intra-ooplasmic variation in all oocytes. II. The spectral analysis of oocytes derived from pre-antral and in vitro cultured follicles revealed significant variation: It was found that Raman spectroscopy could successfully discriminate between immature and mature oocytes. III. Finally, the spectral analysis of oocytes derived from unstimulated and stimulated ovulation cycles, as well as those derived from in vitro follicle cultures, revealed that although biochemically similar, in vitro matured oocytes demonstrated reduced protein content. Furthermore, greater spectral variation was observed in superovulated oocytes, which was found to describe the corresponding morphological quality. In conclusion, this thesis has demonstrated the effective application of Raman spectroscopy to the study of fixed murine oocytes. Raman mapping experiments have demonstrated this technique for the visualisation of biochemical variation which exists within the oocyte. Furthermore, using Raman spectroscopy, the identification of the biochemical variation resulting from different maturation mechanisms has been achieved, as has the discrimination of immature and mature oocytes. These results indicate that Raman spectroscopy holds promise as a quantitative analysis method in the field of fertility treatment.

"The Aurora Kinase proteins are a family of serine/threonine kinases that have been shown to play fundamental roles in controlling M phase progression in somatic cells. Aurora Kinase A protein is known to be vital for proper spindle assembly and therefore, chromosome segregation. Previous reports have shown that Aurora Kinase B is vital for proper completion of karyokinesis and cytokinesis in somatic cells. The role of Aurora Kinase C in somatic cells has been found to be less clear; however it appears to play an important role in spermatogenesis. Little is known about the role of these Aurora Kinase proteins mouse oocytes during oogenesis, and even less is known about them in embryos during early development. The objective of these studies was to characterize the presence, localization, and function of Aurora Kinase B and Aurora Kinase C protein and mRNA in mouse oocytes and early embryos. Oocytes and embryos were collected from hormone stimulated CF-1 mice and cultured for varying amounts of time. Cumulus denuded oocytes were either fixed for immunofluorescence microscopy studies, lysed for analysis of mRNA levels through the use of reverse transcription PCR (rtPCR) and quantitative rtPCR (q-rtPCR), lysed for protein analysis employing Western blotting, treated with Aurora Kinase protein inhibitor drugs, or microinjected with a siRNA pool targeting Aurora Kinase B. Samples were processed for immunofluorescence analysis using markers of spindle morphology (tubulins), Aurora Kinase B, Aurora Kinase C, and Aurora Kinase B activity (phospho Histone H3). Analysis of relative levels of Aurora Kinase B and Aurora Kinase C mRNA were assessed by rtPCR and q-rtPCR methods. Western blotting was performed on oocytes and early embryos to quantitate Aurora Kinase B and C protein levels. Aurora Kinase inhibitors, Hesperadin and ZM447439, were added to culture medium with mouse oocytes to determine the effects of the loss of Aurora Kinase activity. siRNAs were used to inhibit Aurora Kinase B mRNA in early embryos to ascertain the effect of functional loss of this transcript on embryo development. Marked differences were observed in the localization of Aurora Kinase B when unfertilized oocytes or pre-zygotic genome activation (ZGA) embryos were compared to post-ZGA samples. There was no evidence of Aurora Kinase B protein localized to the mitotic spindle or resultant midbody in oocytes and blastomeres of early embryos. Western blotting results supported this data. Embryos fixed post-ZGA demonstrated Aurora Kinase B localization at midbodies between dividing cells, as was found in mouse embryonic fibroblast control cells. Aurora Kinase C protein was not demonstrable in mouse oocytes, embryos, or control cells using immunocytochemistry or Western techniques. In contrast, Aurora Kinase B and Aurora Kinase C mRNAs were both found to be present in mouse oocytes and early embryos. q-rtPCR data further supported this finding for Aurora Kinase B and revealed that the mRNA level of this transcript is relatively constant until ZGA at which point a decrease relative to the earlier stages was observed. Transcript levels recovered post-ZGA and were comparable to the pre-ZGA levels. Functional inhibition of the Aurora Kinase family through the use of Hesperadin or ZM447439 demonstrated the importance of these proteins for proper microtubule and spindle organization, as these drugs disrupted both karyokinesis and cytokinesis in mouse oocytes and blastomeres of early embryos. Aurora Kinase B targeting siRNA also established a role for Aurora Kinase mRNA in embryos at the 2-cell stage based on the disruption of the cell cycle that was observed in treated embryos. Given earlier reports showing the vital role of the Aurora Kinase proteins in proliferating somatic cells, knowledge of the expression and localization of these proteins in oocytes and early embryos is vital for the understanding of cell cycle control during oogenesis and early embryogenesis. Our data indicate that Aurora Kinase B mRNA may also play a role in early embryogenesis, demonstrating a need for analysis of transcript as well as protein. Our results, as well as outcomes of future experiments suggested by our work, may provide significant insight into cell cycle regulation differences between somatic and embryonic cells. These differences may have a profound impact upon manipulated embryos including those reconstructed through somatic cell nuclear transfer. "

The overall objective of this thesis was to investigate the vitrification of bovine cumulus oocyte complexes (COCs) as a tool for conservation of female genetics. Specifically, the first objective was to compare in vitro maturation rates (i.e. end point Metaphase II (MII) rate) of bovine COCs following vitrification using two different equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two different cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P<0.001). In the vitrified groups, equilibration time in VS1 [TCM-199 + 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% calf serum (CS)] did not affect MII rate (P=0.964); however, MII rate was higher in COCs vitrified on cryotops than in straws (23 vs 9%, P=0.007).<p> The second objective of this thesis was to compare cleavage and subsequent developmental competence of bovine COCs vitrified using different vitrification solutions (fresh vs frozen) and different equilibration times in VS1 (0 vs 5 min). Immature bovine COCs were vitrified on cryotops using vitrification solutions either prepared fresh or frozen/thawed, and two equilibration times in VS1 (0 vs 5 min). Cleavage and blastocyst production rates were higher (P<0.001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocyst rate 31 vs 0.4%). The cleavage rate of COCs vitrified in frozen/thawed solutions with 5 min equilibration was higher (P=0.05) than in other treatment groups. However, the blastocyst rate did not differ (P=0.993) among vitrified groups.<p> We hypothesized that the nuclear stage of bovine COCs at the time of vitrification will affect post-warming in vitro maturation (IVM), cleavage and embryo development. Firstly, a preliminary study was conducted to validate our in vitro maturation system. The COCs were at germinal vesicle (GV, 89%), germinal vesicle breakdown (GVBD, 47%), metaphase I (MI, 90%), and metaphase II (MII, 84%) stages at 0, 6, 12 and 22 h of IVM respectively. In a subsequent study, bovine COCs were vitrified after 0, 6, 12 and 22 h of IVM by loading on cryotops and plunging in liquid nitrogen. Following 1 min in warming solution (TCM-199 + 17% sucrose + 20% CS), COCs were placed in IVM medium to complete 22 h of IVM and nuclear stages were evaluated using lamin A/C-DAPI staining. The nuclear maturation (MII) rates of COCs were 23, 23, 35 and 89% (P<0.001) in the 0, 6, 12 and 22 h IVM groups, respectively. In the final nuclear stage experiment, cleavage and embryo development was determined after vitrification of COCs after 0, 12 and 22 h of IVM. The cleavage and blastocyst rates were higher (P<0.001) in the non-vitrified (control) group than vitrified groups (73 vs 15% and 22 vs 0.3%, respectively). The cleavage rates (14% in 0 h, 17% in 12 h and 14% in 22 h groups; P=0.825) and blastocyst rates (0% in 0 and 22 h and 1% in 12 h groups) did not differ. Results from this study indicated that a greater proportion of COCs progressed to MII if vitrification occurred at MI rather than at GV and GVBD stages. However, the nuclear status of vitrified COCs appeared to have no effect on fertilization or subsequent embryo development.<p> A final set of experiments were designed to determine if the exposure of bovine COCs to cryoprotectant solutions and different warming time intervals affects their ability to become fertilized, cleave and develop into blastocysts. In both experiments, the cleavage and blastocyst rates in the vitrified group were lower (P<0.001) than in the non-vitrified control groups VS1 group, and in the VS1 + vitrification solution 2 (VS2; TCM-199 + 15% EG + 15% DMSO + 20% CS) group. However, the cleavage and blastocyst rates did not differ (P>0.05) in control, VS1 and VS1+VS2 groups. In the second experiment, there was no difference in cleavage rates between 1 and 5 min intervals in the warming solution. In conclusion, cryotop can be used as a preferred cryodevice for vitrification of bovine COCs. Vitrification solutions (VS1 and VS2) and the warming solution can be prepared and stored in the freezer (-20 °C) for convenience. The pre-equilibration of bovine COCs in VS1 had no effect on the nuclear maturation of vitrified/warmed COCs, but 5 min pre-equilibration in VS1 improved cleavage rates. The nuclear stage of COCs at the time of vitrification and cryoprotectant exposure appeared to have no effect on subsequent cleavage. Furthermore, there was no difference in cleavage and blastocyst rates following vitrification and 1 or 5 min warming time intervals. When compared to non-vitrified controls, the vitrification of immature bovine COCs resulted in reduced nuclear maturation, cleavage and embryo development rates. Further studies are needed to elucidate the causes of the poor embryo development in vitrified-warmed bovine COCs at genomic, proteomics and metabolomics levels.

The overall objective of this thesis was to investigate the vitrification of bovine cumulus oocyte complexes (COCs) as a tool for conservation of female genetics. Specifically, the first objective was to compare in vitro maturation rates (i.e. end point Metaphase II (MII) rate) of bovine COCs following vitrification using two different equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two different cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P<0.001). In the vitrified groups, equilibration time in VS1 [TCM-199 + 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% calf serum (CS)] did not affect MII rate (P=0.964); however, MII rate was higher in COCs vitrified on cryotops than in straws (23 vs 9%, P=0.007).<p> The second objective of this thesis was to compare cleavage and subsequent developmental competence of bovine COCs vitrified using different vitrification solutions (fresh vs frozen) and different equilibration times in VS1 (0 vs 5 min). Immature bovine COCs were vitrified on cryotops using vitrification solutions either prepared fresh or frozen/thawed, and two equilibration times in VS1 (0 vs 5 min). Cleavage and blastocyst production rates were higher (P<0.001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocyst rate 31 vs 0.4%). The cleavage rate of COCs vitrified in frozen/thawed solutions with 5 min equilibration was higher (P=0.05) than in other treatment groups. However, the blastocyst rate did not differ (P=0.993) among vitrified groups.<p> We hypothesized that the nuclear stage of bovine COCs at the time of vitrification will affect post-warming in vitro maturation (IVM), cleavage and embryo development. Firstly, a preliminary study was conducted to validate our in vitro maturation system. The COCs were at germinal vesicle (GV, 89%), germinal vesicle breakdown (GVBD, 47%), metaphase I (MI, 90%), and metaphase II (MII, 84%) stages at 0, 6, 12 and 22 h of IVM respectively. In a subsequent study, bovine COCs were vitrified after 0, 6, 12 and 22 h of IVM by loading on cryotops and plunging in liquid nitrogen. Following 1 min in warming solution (TCM-199 + 17% sucrose + 20% CS), COCs were placed in IVM medium to complete 22 h of IVM and nuclear stages were evaluated using lamin A/C-DAPI staining. The nuclear maturation (MII) rates of COCs were 23, 23, 35 and 89% (P<0.001) in the 0, 6, 12 and 22 h IVM groups, respectively. In the final nuclear stage experiment, cleavage and embryo development was determined after vitrification of COCs after 0, 12 and 22 h of IVM. The cleavage and blastocyst rates were higher (P<0.001) in the non-vitrified (control) group than vitrified groups (73 vs 15% and 22 vs 0.3%, respectively). The cleavage rates (14% in 0 h, 17% in 12 h and 14% in 22 h groups; P=0.825) and blastocyst rates (0% in 0 and 22 h and 1% in 12 h groups) did not differ. Results from this study indicated that a greater proportion of COCs progressed to MII if vitrification occurred at MI rather than at GV and GVBD stages. However, the nuclear status of vitrified COCs appeared to have no effect on fertilization or subsequent embryo development.<p> A final set of experiments were designed to determine if the exposure of bovine COCs to cryoprotectant solutions and different warming time intervals affects their ability to become fertilized, cleave and develop into blastocysts. In both experiments, the cleavage and blastocyst rates in the vitrified group were lower (P<0.001) than in the non-vitrified control groups VS1 group, and in the VS1 + vitrification solution 2 (VS2; TCM-199 + 15% EG + 15% DMSO + 20% CS) group. However, the cleavage and blastocyst rates did not differ (P>0.05) in control, VS1 and VS1+VS2 groups. In the second experiment, there was no difference in cleavage rates between 1 and 5 min intervals in the warming solution. In conclusion, cryotop can be used as a preferred cryodevice for vitrification of bovine COCs. Vitrification solutions (VS1 and VS2) and the warming solution can be prepared and stored in the freezer (-20 °C) for convenience. The pre-equilibration of bovine COCs in VS1 had no effect on the nuclear maturation of vitrified/warmed COCs, but 5 min pre-equilibration in VS1 improved cleavage rates. The nuclear stage of COCs at the time of vitrification and cryoprotectant exposure appeared to have no effect on subsequent cleavage. Furthermore, there was no difference in cleavage and blastocyst rates following vitrification and 1 or 5 min warming time intervals. When compared to non-vitrified controls, the vitrification of immature bovine COCs resulted in reduced nuclear maturation, cleavage and embryo development rates. Further studies are needed to elucidate the causes of the poor embryo development in vitrified-warmed bovine COCs at genomic, proteomics and metabolomics levels.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this thesis was to examine whether cumulus cells exhibit a low incidence of apoptosis due to their close association with oocytes and their exposure to OSFs, and to investigate if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). This thesis includes a series of studies designed to examine by various means the nature of the paracrine network of bone morphogenetic proteins (BMPs) and their binding proteins involved in the regulation of cumulus cell apoptosis. OSFs, in particular BMP- 15 and BMP-6, but not growth differentiation factor 9 (GDF-9), reduced apoptosis of cumulus cells by following a gradient from the site of the oocytes. Morever, follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked ~50% of the antiapoptotic actions of oocytes. These results demonstrated that OSFs, particularly BMP-15 and BMP-6, maintain the low incidence of apoptosis by establishing a localized gradient of bone morphogenetic proteins. Results from the present thesis also demonstrated that OSFs enhance oocyte developmental competence during IVM, whether in their native form as an uncharacterized mix of growth factors secreted by the oocyte, throughout the oocyte maturation period, or as exogenous BMP-15 and GDF-9, during the first 9 hour of IVM. Also, OSFs improved embryo quality as evident by increased blastocyst total and trophectoderm cell numbers. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP-15 and GDF-9, respectively, neutralized the stimulatory effects of the exogenous growth factors, and impaired the developmental competence of control cumulus-oocyte complexes (COCs). The work presented in this thesis has provided multiple lines of evidence that OSFregulation of the COC microenvironment is an important determinant of cumulus cell apoptosis and oocyte developmental programming. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1260892 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2006

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this thesis was to examine whether cumulus cells exhibit a low incidence of apoptosis due to their close association with oocytes and their exposure to OSFs, and to investigate if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). This thesis includes a series of studies designed to examine by various means the nature of the paracrine network of bone morphogenetic proteins (BMPs) and their binding proteins involved in the regulation of cumulus cell apoptosis. OSFs, in particular BMP- 15 and BMP-6, but not growth differentiation factor 9 (GDF-9), reduced apoptosis of cumulus cells by following a gradient from the site of the oocytes. Morever, follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked ~50% of the antiapoptotic actions of oocytes. These results demonstrated that OSFs, particularly BMP-15 and BMP-6, maintain the low incidence of apoptosis by establishing a localized gradient of bone morphogenetic proteins. Results from the present thesis also demonstrated that OSFs enhance oocyte developmental competence during IVM, whether in their native form as an uncharacterized mix of growth factors secreted by the oocyte, throughout the oocyte maturation period, or as exogenous BMP-15 and GDF-9, during the first 9 hour of IVM. Also, OSFs improved embryo quality as evident by increased blastocyst total and trophectoderm cell numbers. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP-15 and GDF-9, respectively, neutralized the stimulatory effects of the exogenous growth factors, and impaired the developmental competence of control cumulus-oocyte complexes (COCs). The work presented in this thesis has provided multiple lines of evidence that OSFregulation of the COC microenvironment is an important determinant of cumulus cell apoptosis and oocyte developmental programming. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1260892 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2006