Pre-pubertal pig oocytes possess lower developmental competence than those from adult pigs following in vitro maturation (IVM). Previous studies have demonstrated that exposure of pre-pubertal oocytes to 1 mM dibutyryl cAMP (dbcAMP), a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, for the first 20 h of IVM improves the rate of blastocyst development. Developmental competence of in vitro matured pig oocytes has been reported to increase with increasing follicle size. In this thesis, experiments were carried out using pre-pubertal and adult pig oocytes to investigate the relationship between donor age, intra-oocyte cAMP level and follicle size in terms of oocyte maturation and developmental competence. These experiments demonstrated that, while ovarian, follicular and oocyte morphology are immediately altered with the onset of puberty, pre-pubertal oocytes must be exposed to more than the first oestrous cycle to achieve improved developmental competence in vitro. Later experiments demonstrated that pre-pubertal oocytes accumulate less cAMP during IVM, undergo more rapid meiotic progression and display reduced rates of blastocyst development compared to in vitro matured adult oocytes. Treatment with dbcAMP for 22 h IVM increased the cAMP content of pre-pubertal oocytes, slowed meiotic progression during IVM and improved the rate of blastocyst formation. While the cAMP concentration of pre-pubertal oocytes was increased to levels similar to that of adult oocytes, rates of blastocyst formation remained lower, suggesting that additional factor(s) are required for oocyte maturation. This thesis also examined the follicle size cohorts that make up the 3-8 mm aspiration range on pig ovaries. The surface of pre-pubertal ovaries contained around double the number of 3 mm follicles compared with adult ovaries. Blastocyst development of pre-pubertal oocytes increased with increasing follicle size and was highest using oocytes from 5-8 mm follicles, while adult oocytes from all follicle size cohorts displayed similar high rates of blastocyst formation. The interaction between follicle size and cAMP content in pre-pubertal oocytes was examined next. Cumulus-oocyte complexes (COCs) from 3 mm follicles accumulated less intra-oocyte and inter-COC cAMP and displayed reduced cumulus expansion compared with COCs from 5-8 mm follicles. While dbcAMP treatment increased the cAMP content of oocytes from 3 mm follicles, it had no effect on the cAMP content of the whole COC. These findings suggest that inadequate levels of intra-oocyte cAMP during IVM contribute to the low developmental competence of pre-pubertal oocytes from 3 mm follicles, suggesting that cAMP transfer, production or degradation processes are incomplete. Analysis of steroid content from different follicle size cohorts revealed that the progesterone content of prepubertal follicular fluid (FF) increased with increasing follicle size, yet overall was lower than that of adults. This suggests that differences may exist in the gonadotropinstimulated steroidogenic activity of granulosa cells of pre-pubertal COCs from different follicle sizes. Since progesterone secretion did not differ between pre-pubertal and adult COCs, it appears that the downstream pathway from the granulosa cell response rather than the actual quantity of progesterone is important for subsequent maturation processes. These studies then examined gap junction communication (GJC) within the pre-pubertal COC during IVM to examine whether the positive effects of increasing follicle size and dbcAMP on intra-oocyte cAMP levels relates to improved cAMP transfer between the cumulus cell layer and oocyte. Cumulus cell-oocyte GJC during IVM was maintained for a longer period in pre-pubertal COCs from 3 mm follicles than in those from 5-8 mm follicles. Treatment with dbcAMP had minimal effect on GJC in either COC type, thus the dbcAMP-induced increase in intra-oocyte cAMP levels appears independent of GJC. Differences in GJC during IVM together with the COCs ability to increase intraoocyte cAMP levels during IVM, suggests that differences may exist in the quantity of gonadotropin receptors, which are responsible for cAMP production, within the cumulus layer of COCs from 3 mm compared with 5-8 mm follicles. In conclusion, this thesis has demonstrated that an increase in intra-oocyte cAMP is necessary during maturation for completion and synchronisation of maturation and high developmental competence of the pig oocyte. Comparison of 3, 4 and 5-8 mm follicle sizes in the pre-pubertal pig, as described here, provides an excellent model for further investigation into the role of cAMP and the other factors required for co-ordination of oocyte nuclear and cytoplasmic maturation and subsequent embryo production. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297309 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

No description available.

Widespread evidence indicates that exposure of cell cultures to α particles results in significant biological changes in both the irradiated and non-irradiated bystander cells in the population. The induction of non-targeted biological responses in cell cultures exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation and to radiotherapy. Here, we investigated the mechanisms underlying the induction of stressful effects in confluent normal human fibroblast cultures exposed to low fluences of 1000 MeV/u iron ions (linear energy transfer (LET) ~151 keV/µm), 600 MeV/u silicon ions (LET ~50 keV/µm) or 290 MeV/u carbon ions (LET ~13 keV/µm). We compared the results with those obtained in cell cultures exposed, in parallel, to low fluences of 0.92 MeV/u α particles (LET ~109 keV/µm).Induction of DNA damage, changes in gene expression, protein carbonylation and lipid peroxidation during 24 h after exposure of confluent cultures to mean doses as low as 0.2 cGy of iron or silicon ions strongly supported the propagation of stressful effects from irradiated to bystander cells. At a mean dose of 0.2 cGy, only ~1 and 3 % of the cells would be targeted through the nucleus by an iron or silicon ion, respectively. Within 24 h post-irradiation, immunoblot analyses revealed significant increases in the levels of phospho-TP53 (serine 15), p21Waf1 (also known as CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation. The magnitude of the responses suggested participation of non-targeted cells in the response. Furthermore, when the irradiated cell populations were subcultured in fresh medium shortly after irradiation, greater than expected increases in the levels of these markers were also observed during 24 h. Together, the results imply a rapidly propagated and persistent bystander effect. In situ analyses in confluent cultures showed 53BP1 foci formation, a marker of DNA damage, in more cells than expected based on the fraction of cells traversed through the nucleus by an iron or silicon ion. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure to a mean absorbed dose of 0.2 cGy of 3.7 MeV α particles, but not after 0.2 cGy of 290 MeV/u carbon ions.Analyses in dishes that incorporate a CR-39 solid state nuclear track detector bottom identified the cells irradiated with iron or silicon ions and further supported the participation of bystander cells in the stress response. Mechanistic studies indicated that gap junction intercellular communication, DNA repair, and oxidative metabolism participate in the propagation of the induced effects.We also considered the possible contribution of secondary particles produced along the primary particle tracks to the biological responses. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cells cultures exposed to HZE particles comprise <1 % of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 µm Thus, the latter are unlikely to significantly contribute to the stressful effects in cells not targeted by primary HZE particles.

De nombreuses études ont montré que l'exposition de cultures cellulaires à des particules α conduit à des changements biologiques importants autant dans les cellules irradiées que dans les cellules bystander non-irradiées. L'étude des réponses biologiques non-ciblées dans des cultures cellulaires exposées à de faibles fluences d’ions lourds permet d’estimer les risques pour la santé du rayonnement spatial et de la radiothérapie. Nous avons caractérisé les mécanismes sous-jacents de l'induction d'effets stressants dans des cultures confluentes de fibroblastes normaux humains exposés à de faibles fluences d’ions fer de 1000 MeV/u (transfert d'énergie linéique (TEL) ~151 keV/µm), d’ions silicium de 600 MeV/u (TEL ~50 keV/µm) ou d’ions carbone de 290 MeV/u (TEL ~13 keV/µm). Nous avons comparé ces résultats avec ceux obtenus dans des cultures cellulaires exposées, en parallèle, à de faibles fluences de particules α de 0,92 MeV/u (TEL ~109 keV/µm). L'induction de dommages à l'ADN, les changements dans l'expression des gènes, la carbonylation des protéines et la peroxydation lipidique durant les 24 h suivant l'exposition de cultures confluentes à de faibles doses (0,2 cGy et plus) d’ions fer ou d'ions silicium ont très largement contribué à la propagation d’effets stressants des cellules irradiées aux cellules bystander non-irradiées. Pour une dose moyenne de 0,2 cGy, seules ~1 et 3 % des cellules seraient irradiées dans le noyau par un ion, respectivement, fer ou silicium. Les immunoblots ont révélés des augmentations significatives des niveaux de phospho-TP53 (sérine 15), p21Waf1 (CDKN1A), HDM2, phospho-ERK1/2, de carbonylation des protéines et de peroxydation lipidique dans les 24 h suivant l’exposition. L'ampleur de ces réponses suggère la participation de cellules non ciblées dans les effets observés. De plus, lorsque les populations cellulaires irradiées ont été ré-ensemencées dans un milieu de culture frais peu après l'irradiation, les niveaux de ces marqueurs ont aussi augmentés durant 24 h. Ensemble, ces résultats montrent un effet rapidement propagé et persistant. Des analyses in situ réalisées dans des cultures cellulaires confluentes ont montré que la formation de foyers de la protéine 53BP1, marqueur de dommages à l'ADN, touchait un nombre de cellules plus important que celui auguré par la fraction de cellules traversées dans le noyau par un ion fer ou silicium. Cet effet est exprimé dès 15 min suivant l'exposition, atteint son maximum 1 h après l’exposition puis diminue jusqu’à 24 h. Une tendance similaire s'est produite après exposition à une dose moyenne absorbée de 0,2 cGy de particules α de 3,7 MeV, mais non après 0,2 cGy d’ions carbone de 290 MeV/u.Des analyses utilisant des puits de cultures intégrant une fine épaisseur de CR-39, détecteur solide de traces nucléaires, et permettant ainsi l’identification des cellules irradiées aux ions fer ou silicium, confirment la participation de cellules bystander dans la réponse au stress. Des études mécanistiques ont, de plus, indiqué que les jonctions gap permettant la communication intercellulaire, certaines voies de la réparation de l’ADN, ainsi que le métabolisme oxydatif participent à la propagation des effets non ciblés induit par des radiations de haut TEL. Nous avons également examiné la contribution possible des particules secondaires produites le long des traces d’ions primaires dans les réponses biologiques. Les simulations réalisées avec le code de transport de particules FLUKA ont révélé que la dose due aux produits de fragmentation, autres que les électrons, est inférieure à 1 % de la dose absorbée dans les cultures cellulaires exposées à des ions lourds. De plus, la dose radiale des ions lourds secondaires est limitée à ~10-20 µm autour de l’ion primaire. Ainsi, ces derniers sont peu susceptibles de contribuer de manière significative à la réponse biologique observée dans des cellules non ciblées par des ions lourds primaires / Widespread evidence indicates that exposure of cell cultures to α particles results in significant biological changes in both the irradiated and non-irradiated bystander cells in the population. The induction of non-targeted biological responses in cell cultures exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation and to radiotherapy. Here, we investigated the mechanisms underlying the induction of stressful effects in confluent normal human fibroblast cultures exposed to low fluences of 1000 MeV/u iron ions (linear energy transfer (LET) ~151 keV/µm), 600 MeV/u silicon ions (LET ~50 keV/µm) or 290 MeV/u carbon ions (LET ~13 keV/µm). We compared the results with those obtained in cell cultures exposed, in parallel, to low fluences of 0.92 MeV/u α particles (LET ~109 keV/µm).Induction of DNA damage, changes in gene expression, protein carbonylation and lipid peroxidation during 24 h after exposure of confluent cultures to mean doses as low as 0.2 cGy of iron or silicon ions strongly supported the propagation of stressful effects from irradiated to bystander cells. At a mean dose of 0.2 cGy, only ~1 and 3 % of the cells would be targeted through the nucleus by an iron or silicon ion, respectively. Within 24 h post-irradiation, immunoblot analyses revealed significant increases in the levels of phospho-TP53 (serine 15), p21Waf1 (also known as CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation. The magnitude of the responses suggested participation of non-targeted cells in the response. Furthermore, when the irradiated cell populations were subcultured in fresh medium shortly after irradiation, greater than expected increases in the levels of these markers were also observed during 24 h. Together, the results imply a rapidly propagated and persistent bystander effect. In situ analyses in confluent cultures showed 53BP1 foci formation, a marker of DNA damage, in more cells than expected based on the fraction of cells traversed through the nucleus by an iron or silicon ion. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure to a mean absorbed dose of 0.2 cGy of 3.7 MeV α particles, but not after 0.2 cGy of 290 MeV/u carbon ions.Analyses in dishes that incorporate a CR-39 solid state nuclear track detector bottom identified the cells irradiated with iron or silicon ions and further supported the participation of bystander cells in the stress response. Mechanistic studies indicated that gap junction intercellular communication, DNA repair, and oxidative metabolism participate in the propagation of the induced effects.We also considered the possible contribution of secondary particles produced along the primary particle tracks to the biological responses. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cells cultures exposed to HZE particles comprise <1 % of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 µm Thus, the latter are unlikely to significantly contribute to the stressful effects in cells not targeted by primary HZE particles.