Abstract not available

Abstract not available

ABSTRACT The purpose of this project was to investigate monocarboxylate (i.e. pyruvate and lactate) transport in the preimplantation stage of embryo development. Much progress has been made over the last 15 years towards understanding preimplantation and peri-implantation embryo physiology, including metabolic preferences during this period. It is known that as the cells (blastomeres) of an embryo compact via tight junctions and the embryo differentiates into a blastocyst, a metabolic “switch” occurs to allow the blastocyst to take up glucose at a rapid rate, obtaining energy derived from glycolysis. Glucose transporter molecules have been identified and characterized during this period of development and a paradigm for glucose transport has been described. However, during the early cleavage stages (days 1-3 post-fertilization), the embryo preferentially derives its metabolic energy from the monocarboxylate pyruvate. Evidence for the expression of pyruvate transporter molecules (a family of proton-coupled monocarboxylate co-transporters, MCT) has only been indicated via some kinetic studies on pH homeostasis and PCR analysis for MCT expression, and results have been conflicting (Gibb et al., 1997, Harding et al., 1999, Herubel et al., 2002). This project aimed to clarify discrepancies in reports for mRNA expression of MCT and to enhance the understanding of monocarboxylate transport processes during preimplantation development by pioneering investigations into protein expression for various MCT isoforms. Transport kinetics for monocarboxylate, DL-lactate, were examined by measuring the uptake of radioactive [3H]-DL-lactate from the medium by two-cell embryos and blastocysts. It was discovered that blastocysts demonstrate significantly higher affinity for DL-lactate compared to zygotes (Km 20 + 10 v 87 + 35 mM lactate; p=0.03), which suggested that alterations in the expression of various MCT isoforms might be expected as the embryo developed to a blastocyst. The rate of transport showed a trend towards a decrease from the zygote to blastocyst stages, although this could not be confirmed as significant within the limitations of this experiment. Mouse embryos, both in vivo and in vitro-derived, were collected and pooled at the zygote, two-cell, morula and blastocyst stages of development. RNA purification, reverse-transcription and PCR were used to analyze the expression of the four best-characterized MCT isoforms. MCT1, MCT2 and MCT4 were all found to be expressed in oocytes and mouse embryos from the zygote through to the preimplantation blastocyst. MCT3, an isoform uniquely expressed in the retina, was not detected at any stage in embryos. Since glucose has been implicated in regulatory processes involving glucose transporter expression in mouse embryos (Pantaleon et al., 2005, Pantaleon et al., 2001), mRNA expression was examined in the presence or absence of glucose in the culture media to determine whether the same phenomena applied to MCT. It was discovered that MCT1 and MCT4 isoforms were responsive to glucose-deprivation as evidenced by a reduction in mRNA expression in compacted morula cultured from the zygote stage without glucose. When glucose-deprived embryos were exposed to a brief high concentration of glucose during the 4-cell stage of development and continued in culture without glucose, the expression of mRNA for MCT1 and MCT4 persisted post-compaction, demonstrating that glucose exposure is necessary for the continued expression of these two isoforms in the mouse blastocyst. MCT2 mRNA did not respond to the absence of glucose in this way, and mRNA expression persisted in either the presence or absence of glucose. To follow these analyses of MCT gene transcription during early embryo development, confocal laser scanning immunofluorescence and western blotting were used to identify the expression of MCT proteins at various stages of development. Culture in the presence or absence of glucose was again employed to determine whether the changes seen in mRNA expression were conveyed at the protein level. All three proteins were identified throughout preimplantation development, though their locations were uniquely different. MCT1 was notably absent from plasma membranes at all stages, and was detected diffusely within the cytoplasm. In expanding blastocysts MCT1 tended to concentrate in the cortical cytoplasm of blastomeres and staining was more intense in the polar trophectoderm. In this cytoplasmic location its function is unclear. MCT1 does not appear to be a key transporter of monocarboxylates into and out of the embryo, but it may have a role in shuttling pyruvate and lactate within the cytoplasm to maintain metabolic and redox homeostasis. In embryos cultured without glucose, the immunostaining intensity for MCT1 gradually decreased as morulae degenerated and died. Protein loss occurred from the morula stage onwards, whilst mRNA was already undetectable at this stage. This would indicate that glucose signals which maintain mRNA expression most likely operate at the level of gene activation/transcription with latent effects on protein expression. MCT4 appeared to be located on the plasma membranes of oocytes and 2-cell embryos and nuclear staining was evident throughout preimplantation development, however plasma membrane expression was not apparent in morulae and blastocysts. This is consistent with earlier kinetic evidence of a low affinity lactate transporter (Km 87 + 35 mM lactate) operating at the early preimplantation stages. MCT4 has the lowest affinity for lactate of all the characterized MCT to date. Kinetic data also suggests that a change might occur in MCT protein expression as the embryo progresses to a blastocyst with a higher affinity lactate transporter taking precedence, and the loss of MCT4 from the plasma membrane at these later stages supports this view. Similarly to MCT1, MCT4 mRNA expression was also found to be dependent on glucose exposure during the early preimplantation period, and embryos cultured entirely without glucose demonstrated a loss of MCT4 mRNA expression at the morula stage. MCT4 typically exists as a lactate exporter in glycolytic tissues and it most likely exports lactate from the embryo for pH and redox homeostasis during this period of development. Protein localization studies found MCT2 to be located on the plasma membranes of oocytes, zygotes, 2-cell embryos, and polarized to the surface of the outer blastomeres of morulae and blastocyst trophectodermal cells. Throughout preimplantation development, MCT2 protein co-localized with peroxisomal catalase in peroxisome-sized granules throughout the cells. Known to be a high affinity pyruvate transporter, given its location in embryos it was proposed here that MCT2 most likely imports pyruvate to fuel early embryos, and later works as a bifunctional pyruvate/lactate importer/exporter on the transporting epithelium (trophectoderm) of blastocysts to maintain the pH, redox and metabolic status of the embryo. MCT2 was an enigma to the other MCT. Its expression in the absence of glucose behaved in an opposite way to that of MCT1 and MCT4, with mRNA expression persisting in the absence of glucose. In fact, MCT2 and catalase proteins demonstrated a quantitative increase in embryos lacking glucose, and the increase in staining was noticed as an increase in the density of peroxisome-like structures (or peroxisome proliferation) within the embryo. As such, it was decided to investigate the possibility that peroxisome proliferators (Peroxisome Proliferator Activated Receptors, PPARs) were involved in the control of MCT expression in the same way that they are known to control the expression of catalase and other peroxisomal proteins. At this stage, no MCT isoforms had been identified as being under the control of PPARs, although it was known that their expression was most likely controlled at the level of transcription, with no translational or post-translational controlling elements. PPARα, one of three isoforms (α, γ and β/δ) was selected as a likely candidate given that it controls peroxisomal proliferation and fatty acid β-oxidation processes at the level of transcription in other tissues, and it was known to be upregulated in conditions of starvation and oxidative stress. PPARα mRNA was shown to be expressed in early cleavage preimplantation mouse embryos, but its expression was reduced in morulae and blastocysts. Further, lack of glucose led to persistence of PPARα mRNA expression at the morula stage. PPARα protein was also demonstrated to stain more brightly in early preimplantation embryos compared to later stages. Further experimentation demonstrated that the phenomenon of increased catalase and MCT2 expression in embryos cultured without glucose could be mimicked in the presence of glucose by treating these embryos with the PPARα-selective agonist, WY14,643. The timing and quantitative nature of this upregulation were very similar, suggesting that PPARα was in some way involved in the glucose-deprived upregulation pathway for catalase and MCT2. To further investigate this pathway, oxidative stress was investigated in embryos cultured in the presence and absence of glucose to test whether the generation of reactive oxygen species contributed to the PPARα/MCT2 phenomenon. It was demonstrated that within 2 h of culture in the absence of glucose, hydrogen peroxide levels were significantly elevated in zygotes. Amelioration of increased peroxide generation in glucose-deprived embryos using a non-selective flavoenzyme inhibitor diphenyleneiodonium (DPI) eliminated any increases in PPARα and MCT2 protein expression that were earlier noted in the absence of glucose. To summarize, MCT1, MCT2 and MCT4 mRNA and protein expression were successfully demonstrated in mouse preimplantation embryos and all were confirmed to be in some way regulated by glucose in the culture medium. In the absence of glucose, mRNA expression for MCT1 and MCT4 were reduced to undetectable levels in morulae indicating that their expression was glucose-dependent. Paradoxically, glucose deprivation caused an increase in PPARα, catalase and MCT2 protein expression. PPARα-selective agonism in the presence of glucose induced similar timing and effects on catalase and MCT2 upregulation, implicating PPARα in this pathway. Hydrogen peroxide levels were significantly elevated within 2 h of culture in the absence of glucose. This peroxide elevation could be quenched to control levels by treating these embryos with DPI, and reducing hydrogen peroxide to control levels also eliminated the upregulation of PPARα and MCT2, implicating oxidative stress as an important component in the glucose-deprivation induced upregulation of MCT2. The experimental data presented in this thesis demonstrate that from its very conception, the embryo interacts with, adapts to, and is indeed affected by the external environment in which it develops. Even components like glucose, once considered simply as metabolic substrates, have profound effects on gene transcription and protein expression within the embryo which may impact on later its developmental competence, a reality we need to consider more deeply in light of the implementation of artificial reproductive technologies widely used today in zoology, agriculture and clinically, in humans.

Impaired implantation and placental development have been implicated in several disorders of pregnancy such as unexplained miscarriage, preeclampsia, and intrauterine growth retardation. Insulin-Like Growth Factor (IGF)-II has previously been shown to promote blastocyst development and placental growth and function. We were interested in how IGF-II interacts with other factors throughout blastocyst development, implantation and placentation in the mouse to improve pregnancy outcome. In vitro embryo culture increases the risk of pregnancy complications associated with poor placentation. Recent research has focussed on optimising the culture conditions to more resemble that of the in vivo environment. IGF-II, Urokinase Plasminogen Activator (uPA) and Plasminogen individually have all been shown to be important for embryo development. However, it is likely that a combination of factors is required to counteract the negative effects of in vitro culture. Here we show that IGF-II, uPA and Plasminogen, in combination, significantly improve mouse blastocyst hatching rates and implantation rates on day 8 and doubles the number of mothers that are pregnant after embryo transfer. Following implantation, IGF-II is suggested to play a role in promoting placental development and function. We demonstrate that IGF-II is co-localised with both IGF receptors throughout early pregnancy in trophoblasts and in the developing blood vessels and adjacent stromal cells in the mesometrial decidua. This suggests that IGF-II may play a role in both decidual angiogenesis and placentation. We suggest that perhaps murine trophoblasts secrete molecules such as IGF-II to promote angiogenesis in the decidua early in pregnancy to compensate for their shallow invasion and allow for adequate trophoblast remodelling later in pregnancy. The first trimester human placenta experiences a low oxygen environment. The Hypoxia-Inducible Factors (HIFs) mediate the response to low oxygen, inducing genes such as IGF-II. Currently, the role of oxygen in mouse placentation, the mechanisms by which HIFs promote placentation or their interaction with IGF-II in the placenta is unknown. Here, we demonstrate that the early mouse implantation site is exposed to low oxygen levels similar to those seen in humans and expresses HIF-1 protein. We were interested then in the interaction between IGF-II, oxygen and HIFs in trophoblasts in vitro. Prolonged exposure to low oxygen reduced trophoblast outgrowth, and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. Interestingly, we found that antisense (as) Hif-1 may mediate the response to prolonged hypoxia in murine trophoblasts. Importantly, Hif-1 and Hif-2 were differentially regulated by oxygen and IGF-II in cultured trophoblast cells suggesting a novel interaction between IGF-II and oxygen. In conclusion, it appears that IGF-II is a central growth factor which interacts with other molecules to regulate a wide variety of process in early pregnancy to promote blastocyst development, implantation and placentation. The results outlined in this thesis demonstrate a novel interaction between IGF-II, uPA and Plasminogen in promoting blastocyst development and implantation which may be used to improve pregnancy outcome following ART. In addition, we have also identified a novel interaction between IGF-II, oxygen and the HIF system which may regulate trophoblast function. This has important implications not only for placental research, but also for cancer research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326731 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Impaired implantation and placental development have been implicated in several disorders of pregnancy such as unexplained miscarriage, preeclampsia, and intrauterine growth retardation. Insulin-Like Growth Factor (IGF)-II has previously been shown to promote blastocyst development and placental growth and function. We were interested in how IGF-II interacts with other factors throughout blastocyst development, implantation and placentation in the mouse to improve pregnancy outcome. In vitro embryo culture increases the risk of pregnancy complications associated with poor placentation. Recent research has focussed on optimising the culture conditions to more resemble that of the in vivo environment. IGF-II, Urokinase Plasminogen Activator (uPA) and Plasminogen individually have all been shown to be important for embryo development. However, it is likely that a combination of factors is required to counteract the negative effects of in vitro culture. Here we show that IGF-II, uPA and Plasminogen, in combination, significantly improve mouse blastocyst hatching rates and implantation rates on day 8 and doubles the number of mothers that are pregnant after embryo transfer. Following implantation, IGF-II is suggested to play a role in promoting placental development and function. We demonstrate that IGF-II is co-localised with both IGF receptors throughout early pregnancy in trophoblasts and in the developing blood vessels and adjacent stromal cells in the mesometrial decidua. This suggests that IGF-II may play a role in both decidual angiogenesis and placentation. We suggest that perhaps murine trophoblasts secrete molecules such as IGF-II to promote angiogenesis in the decidua early in pregnancy to compensate for their shallow invasion and allow for adequate trophoblast remodelling later in pregnancy. The first trimester human placenta experiences a low oxygen environment. The Hypoxia-Inducible Factors (HIFs) mediate the response to low oxygen, inducing genes such as IGF-II. Currently, the role of oxygen in mouse placentation, the mechanisms by which HIFs promote placentation or their interaction with IGF-II in the placenta is unknown. Here, we demonstrate that the early mouse implantation site is exposed to low oxygen levels similar to those seen in humans and expresses HIF-1 protein. We were interested then in the interaction between IGF-II, oxygen and HIFs in trophoblasts in vitro. Prolonged exposure to low oxygen reduced trophoblast outgrowth, and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. Interestingly, we found that antisense (as) Hif-1 may mediate the response to prolonged hypoxia in murine trophoblasts. Importantly, Hif-1 and Hif-2 were differentially regulated by oxygen and IGF-II in cultured trophoblast cells suggesting a novel interaction between IGF-II and oxygen. In conclusion, it appears that IGF-II is a central growth factor which interacts with other molecules to regulate a wide variety of process in early pregnancy to promote blastocyst development, implantation and placentation. The results outlined in this thesis demonstrate a novel interaction between IGF-II, uPA and Plasminogen in promoting blastocyst development and implantation which may be used to improve pregnancy outcome following ART. In addition, we have also identified a novel interaction between IGF-II, oxygen and the HIF system which may regulate trophoblast function. This has important implications not only for placental research, but also for cancer research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326731 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

L’oviducte joue un rôle central dans le transport et la préparation des gamètes, la fécondation et le développement précoce. Un dialogue embryo-maternel s’établit pour assurer le succès du développement de l’embryon et de son transport vers le site d’implantation. L’objectif principal de ce travail était de confirmer l’existence d’un dialogue moléculaire et fonctionnel précoce grâce à la coculture d’embryons bovins produits in vitro sur des cellules épithéliales tubaires bovines (BOEC). Nous avons montré que les BOEC ont un effet important sur le développement embryonnaire précoce, particulièrement pendant les 4 premiers jours. Cet effet ce traduit par un clivage accéléré, une modulation des gènes exprimés après l’activation du génome embryonnaire, un taux plus élevé de développement au stade de blastocyste et une adaptation de l’expression génique de ces blastocystes. En retour, les embryons induisent dans les BOEC, des changements d’expression de facteurs impliqués dans la réponse à l’interféron. Une spécificité régionale des profils d’expression a également été observée dans l’oviducte. / The oviduct plays a pivotal role in gametes transport and final capacitation, as well as in fertilization and early embryo development. An embryo-maternal communication takes place to ensure the successful early embryo development and transport towards its implantation site. The principal aim of this research was to confirm the existence of such early embryo-maternal molecular and functional dialogue using bovine oviduct epithelial cells (BOEC) as coculture to support the development of in vitro produced bovine embryos. We showed that BOEC had an important effect on early embryo development, especially during the first 4 days. This effect translates into accelerated cleavage kinetics, modulation of gene expression after embryonic genome activation, increased rate of embryo development to the blastocyst stage and improved gene expression profile. Moreover the embryos are triggering a BOEC response by upregulating genes related to interferon signaling. A regional specificity of gene expression profile in the oviduct has also been detected.

O desenvolvimento de protocolos eficientes para a vitrificação de embriões mamíferos ainda é um desafio para os especialistas em reprodução. Soluções crioprotetoras de baixa toxicidade associadas a técnicas seguras de envase são fatores fundamentais, para proporcionarem uma eficiente identificação e controle sanitário das amostras. Dois experimentos foram realizados para determinar a taxa de sobrevivência de embriões Mus domesticus domesticus envasados em palhetas convencionais (0,25 mL), na presença de uma haste metálica de ouro, empregando soluções crioprotetoras descritas para a vitrificação em microvolume. No experimento 1, avaliou-se a toxicidade da solução de desidratação (SD: PBSm + 10% EG + 10% PROH + 0,5 M sacarose), expondo-se os embriões por diferentes tempos: 1 (T1), 3 (T2) ou 10 min (T3). A toxicidade da solução de vitrificação (SV: PBSm + 20% EG + 20% PROH) foi determinada pela exposição dos embriões durante 25, 60 ou 180 seg, previamente desidratados por 1 ou 3 min. No experimento 2, avaliou-se a utilização do macrovolume (palhetas com a haste de ouro) e microvolume (micropipetas de vidro - GMP) na vitrificação dos blastocistos expostos à SV por 25 seg, previamente desidratados por 1 ou 3 min. Os dados foram analisados pelo teste Qui-Quadrado (P<0,05). No experimento 1, não foi observada diferença estatística entre as taxas de eclosão dos embriões desidratados: T1=68,0% (38/56), T2=72,0% (36/50), T3=71,0% (39/55) e o grupo controle, (74,0% - 48/65). No entanto, houve diferença significativa (P<0,05) na taxa de eclosão em relação ao tempo de exposição dos embriões à SV. Os embriões desidratados por 1 ou 3 min e expostos à SV por 25 seg proporcionaram maiores taxas de re-expansão (79,0% vs 84,0%) e de eclosão (58,0% vs 72,0%), em relação aos tempos de exposição de 60 e 180 seg. No experimento 2, após a vitrificação dos embriões envasados nas palhetas com a haste de ouro, a taxa de eclosão dos blastocistos previamente desidratados por 1 min foi de 16,0% (10/64) e de 4,0% (2/57) quando previamente desidratados por 3 min. Por outro lado, os embriões envasados nas GMP, e previamente desidratados por 3 min, foram os que apresentaram maior taxa de eclosão (60,0% - 52/86). A vitrificação de embriões utilizando soluções crioprototetoras descritas para microvolume não foi eficiente na crioproteção dos blastocistos envasados em palhetas convencionais com a haste de ouro. / The development of efficient vitrification protocols for mammalian embryos still is a challenge for reproductive biologists. Low toxicity cryoprotectant solutions and safe vitrifications procedures that allow sample identification and sanitary control are fundamental factors. Two experiments were conducted to determine the survival rate of vitrified Mus domesticus domesticus embryos loaded into straws containing a metallic piece (manufactured in gold), using cryoprotectant solutions described for microvolume vitrification procedures. In Experiment 1, the toxicity of the dehydratation solution (SD: PBSm +10% EG + 10% PROH + 0,5 M sucrose) was evaluated using three different embryo exposure times: 1 (T1), 3 (T2) or 10 min (T3), in addition as well as the toxicity of the vitrification solution (SV: PBSm + 20% EG + 20% PROH) was also tested upon embryo exposure for 25, 60 or 180 sec, previously dehydration for 1 or 3 min. In Experiment 2, the use of macrovolume (straw with a stem of gold) or microvolume (glass micropipettes – GMP) was evaluated for the vitrification of blastocysts after exposure to SV for 25 sec and previous dehydration for 1 or 3 min. Data were analized by the Chi-square test (P<0,05). In Experiment 1, statistical differences were not observed between hatching rates of dehydrated embryos: T1=68.0% (38/56), T2=72.0% (36/50), T3=71.0% (39/55) and control group embryos, (74.0% - 48/65). However, a significant difference (P <0,05) was observed between hatching rates after embryos exposure to the SV. Embryos dehydrated for 1 or 3 min and exposed for 25 sec to the SV showed higher re-expansion (79.0% vs. 84.0%) and hatching rates (58.0% vs. 72.0%) than embryos exposed to SV for 60 or 180 sec. In Experiment 2, after vitrification of the embryos loaded into straws containing a metallic piece showed a hatching rate of 16.0% (10/64) when previouslly dehydrated for 1 min, and 4.0% (2/57) when for 3 min. On the other hand, embryos loaded into GMP, previouslly dehydrated for 3 min, showed a higher hatching rate, (60.0% - 52/60). Embryo vitrification using a cryoprotectant solution described as suitable for microvolume was not efficient to cryoprotect blastocysts loaded into macrovolume straws containing a metallic piece.

Os objetivos dos experimentos foram primeiro testar a presença da dimetilformamida (DF) nas soluções crioprotetoras e segundo avaliar a viabilidade in vitro pós aquecimento de blastocistos Mus domesticus domesticus vitrificados em microcapilares manufaturados industrialmente. O primeiro experimento testou a capacidade de vitrificação das diferentes soluções de vitrificação, compostas pelas combinações de etileno glicol (EG) ou 1-2 propanediol (PROH) com as diferentes porcentagens de DF. No segundo experimento se testou a toxicidade de 3 soluções de equilíbrio: ES1 (PBSm + 10% PROH + 10% DF + 0.5% PVA), ES2 (PBSm+ 10% EG+ 10% DF + 0.5% PVA), ES3 (PBSm + 10% PROH + 10% EG + 0.5% PVA), através da exposição dos embriões durante 3 períodos de tempo (1, 3 e 10 min). O cultivo in vitro após a exposição às soluções de equilíbrio foi realizado em meio de KSOM durante 72 h. Os resultados de re-expansão foram semelhantes entre as três soluções, quando os embriões foram expostos por 1 ou 3 min. Por outro lado, a exposição à ES3 por 10 min, revelou uma maior taxa de sobrevivência dos embriões, em relação às outras soluções testadas. No terceiro experimento, após expor-se os embriões às soluções de equilíbrio, ES1, ES2 e ES3 por 1 min, a vitrificação foi realizada utilizando-se as seguintes soluções: VS1 (PBSm + 20% PROH + 20% DF + 0.5% PVA); VS2 (PBSm + 20% EG + 20% DF+ 0.5%PVA) and VS3 (PBSm + 20% EG + 20% PROH + 0.5% PVA). Imediatamente após a exposição por 30 s às soluções de vitrificação os embriões foram envasados em micropipetas de vidro (GMPs) e mergulhados em nitrogênio líquido superresfriado. As GMPs foram aquecidas no ar durante 10 s e após os embriões foram expostos durante 5 min ao PBSm + 0,25 M sacarose a 37 °C, finalmente transferidos para gotas de meio KSOM e cultivados in vitro durante 72 horas. As taxas de re-expansão dos embriões após o cultivo in vitro foram as seguintes: ES1/VS1 = 12% (13/108); ES2/VS2 = 38% (46/111) e ES3/VS3 = 84% (89/105). A eclosão dos embriões observada após o cultivo in vitro foi de: ES1/VS1 = 3% (3/108); ES2/VS2 = 17% (19/111) e ES3/VS3 = 70% (73/105). As taxas de sobrevivência revelaram que a presença da dimetilformamida na solução crioprotetora reduz a viabilidade embrionária e que a associação de EG + PROH é eficiente na manutenção da viabilidade embrionária após a vitrificação. O segundo artigo descreve os experimentos de vitrificação, utilizando-se para o envase dos embriões um microcapilar de vidro (GMC), produzido industrialmente (Brand®). Blastocistos murinos após a coleta foram divididos em três grupos: Controle, embriões cultivados in vitro em KSOM por 72 horas; Grupo 1, vitrificados envasados em micropipetas de vidro (GMPs) esticadas manualmente; Grupo 2, vitrificados envasados nas GMCs (Brand® - 5 µL). O procedimento de vitrificação foi o seguinte: primeiro os embriões foram expostos à solução de equilíbrio (PBSm + 10% EG + 10% PROH and 0.5% PVA) por 1 min e após transferidos para a solução de vitrificação (PBSm + 20% EG + 20% PROH + 0.5% PVA) por 30 s, envasados nas GMPS ou GMCs e imediatamente mergulhados em nitrogênio superresfriado. As taxas de sobrevivência dos embriões após o aquecimento e cultivo in vitro, não apresentaram diferenças significativas entre os grupos. O envase dos embriões nas GMCs proporcionou sobreviênvia embrionária similar à observada nos embriões envasados nas GMPs. / The aims of these experiments were first, determine the effect of dimethylformamide (DF) into cryoprotectant solutions and sencondly, evaluated the in vitro viability of blastocyst Mus domesticus domesticus vitrified into glass microcapillaries (Brand®). The first article described the efficiency of DF in association with ethylene glycol (EG) and 1-2 propanediol (PROH) on in vitro viability of vitrified mouse blastocysts. Initially, the differents cryoprotectant solutions were tested on its capacities to induce virification. In the second experiment to determine the cryoprotectant toxicity the embryos were exposed during 1, 3 and 10 min to three different equilibrium solutions ES1 (PBSm + 10% PROH+ 10% DF + 0.5% PVA), ES2 (PBSm+10% EG+ 10% DF + 0.5% PVA), ES3 (PBSm + 10% PROH + 10% EG + 0.5% PVA). After 72 hours of in vitro culture in KSOM medium, re-expansion and hatching rates showed no differences among the tested cryoprotectant solutions for 1 or 3 min exposition interval time. However, for the 10 min exposition interval time, ES3 was more effective to promote embryo survival than the others tested cryoprotectant solutions. In the third experiment, blastocysts were vitrified after been exposed to one of the equilibrium solutions (ES1, ES2 or ES3) during 1 min. After that the embryos were transferred to one of the vitrification solutions (VS1 = PBSm + 20% PROH + 20% DF+ 0.5% PVA; VS2 = PBSm + 20% EG+ 20% DF+ 0.5% PVA and VS3 = PBSm + 20% EG+ 20% PROH+ 0.5% PVA) during 30 s, loaded into glass micro pipettes (GMPs) to be plungged into super-cooled liquid nitrogen. The GMPs were thawed in air during 10 s, transferred into drops of PBSm + 0,25 M sucrose at 37 °C for 5 min, and finally transferred to KSOM medium for in vitro culture. After 72 hours the expansion and hatched rates were evaluated. Results demonstrated a significantly difference between the vitrification solutions, showing better hatching rates the embryos vitrified into the ES3/EV3 solution. Therefore, these data shows that the cryoprotectants solutions containning dimethylformamide have deleterious effects on the developmental competence of vitrified mouse blastocysts, and the highest expansion and hatching rates were obtained when the cryoprotectant solution containning an EG and PROH association. The purpose of the second study was to determine the in vitro expansion and hatching rates of vitrified mouse blastocysts loaded into commercially available glass micro-capillaries (GMC - Brand® 5µL). In the early morning at day 4 of the pregnancy, collected blastocysts were divided in three groups: Control: embryos were in vitro culture during 72 hours into KSOM medium; Group 1, blastocyst vitrified into glass micropipettes (GMP); Group 2, blastocyst vitrified into GMC. The embryos were first exposed to the equilibrium solution (PBSm + 10% EG + 10% PROH + 0.5% PVA) for 1 min and then transferred into the vitrification solution (PBSm + 20% EG + 20% PROH + 0.5% PVA) for 30 sec. Blastocysts were loaded into GMP or GMC and plunged into super-cooled liquid nitrogen. Embryo warming was carried out by plunging the narrowest end of the capillaries into droplets of 0.25 M sucrose mantained at 37°C. After 5 min, embryos in vitro culture into KSOM medium for 72 hours. Blastocyst survival rates did not show significant differences between the groups. The tested manufacturated GMC (Brand®) showed the same efficiency as the GMP to load mouse blastocysts for vitrification.

Uma das maiores dificuldades das terapias de reprodução assistida é a seleção de células germinativas de boa qualidade para posterior fertilização e implantação. Atualmente, a seleção de oócitos se dá basicamente por avaliação morfológica, que não reflete satisfatoriamente a competência oocitária. Assim, a busca por bioindicadores da qualidade oocitária é necessária. O cumulus oophorus forma um conjunto de células somáticas que circundam o oócito no folículo antral,mantendo uma relação íntima com a célula germinativa, com comunicação direta via junções tipo GAP. As células do cumulus oophorus são descartadas após técnicas de fertilização in vitro, o que as torna um material de fácil acesso, livre de conflitos éticos. Uma série de metodologias de análise das células do cumulus foram propostas para identificação de anormalidades no oócito. Porém, nenhuma delas é rotineiramente utilizada na clínica. Os processos celulares das células do cumulus refletem as condições do microambiente folicular, e podem, assim, trazer evidências das condições oocitárias. Nosso grupo analisou as células do cumulus primeiramente por meio de abordagens de bioinformática, que revelaram processos celulares relacionados ao desenvolvimento de embriões até o estágio de blastocisto. Com base nestes resultados, análises de parâmetros bioquímicos e expressão gênica das células do cumulus foram realizadas e permitiram a identificação de possíveis biomarcadores da qualidade do oócito que levam em consideração as características clínicas das pacientes. Estas análises indicaram que expressão do gene PTGS2 e a atividade da enzima Glutationa-S-Transferase como indicadores da formação de blastocistos em pacientes com diferentes variáveis clínicas (backgrounds) analisados. Se confirmados, estes parâmetros poderão ser utilizados como biomarcadores no ambiente clínico, elevando as taxas de sucesso em técnicas de reprodução assistida. / One of the great challenges in assisted reproduction techniques is the selection of appropriate germ cells for fertilization and implantation. Nowadays, oocyte selection occurs basically through morphological evaluation, which does not reflect satisfactorily the oocyte competence. Therefore, the search for bioindicators of oocyte quality is necessary. The cumulus oophorus forms a set of somatic cells that surround the oocyte in the antral follicle, participating in the processes of oocyte maturation and folliculogenesis. These cells maintain an intimate relationship with the germ cell, with direct communication via GAP junctions. Cumulus oophorus cells are discarded in in vitro fertilization techniques, which makes them an easy-access material that can be collected in a free-of-ethicalissues way. A series of cumulus cell analysis methodologies were proposed to identify abnormalities in the oocyte. However, none of them is routinely used in clinical environment. The cellular processes of cumulus cells reflect the conditions of the follicular microenvironment, and may thus bring evidences of oocyte conditions. Our group analyzed cumulus cells in search of predictors of oocyte quality primarily through bioinformatics approaches, which revealed cellular processes related to the development of embryos up to the blastocyst stage. Based on these results, analyzes of biochemical parameters and gene expression of cumulus cells were performed and allowed the identification of possible biomarkers of oocyte quality that takes into consideration patients clinical variables. These analysis indicated PTGS2 expression and Glutathione-S-Transferase activity as indicators of blastocyst formation in all patient profiles (backgrounds) analyzed. If confirmed, these parameters may be used as biomarkers in clinical environment, improving assisted reproduction success rates.

Made available in DSpace on 2014-06-11T19:25:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-30Bitstream added on 2014-06-13T20:53:15Z : No. of bitstreams: 1 nabhan_t_me_botib.pdf: 239064 bytes, checksum: a0903b2fc61ab98911efd89418cc6078 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Existem evidências de que os efeitos deletérios do estresse térmico calórico (ETC) sobre a fertilidade são menos pronunciados em raças tolerantes ao calor devido primariamente às diferenças na capacidade de termorregulação. Experimentos in vitro têm demonstrado que embriões zebuínos (Bos indicus) são mais resistentes ao ETC quando comparados a taurinos (Bos taurus). A fim de melhor compreender as diferenças entre zebuínos e taurinos em relação à resistência ao choque térmico, objetivou-se com o presente trabalho verificar se a resistência ao ETC é resultado da contribuição genética do oócito, do espermatozóide ou de ambos e se o intervalo de tempo entre o abate dos animais e a aspiração do oócito influencia o desenvolvimento do embrião. No experimento 1 oócitos de vacas das raças Nelore e mestiças com fenótipo predominante da raça Holandesa preto e branco (mHPB) foram coletados em frigorífico, maturados e fertilizados com espermatozóide de touros das raças Nelore (N), Angus (An), Brahman (Bra) e Gir (Gir). No experimento 2 oócitos de vacas das raças Nelore e HPB puras foram coletados em frigorífico, aspirados 4 e 6:30 horas após o abate dos animais, maturados e fertilizados com espermatozóide de touros das raças N, Gir e HPB. Em ambos os experimentos, noventa e seis horas pós-inseminação (hpi), os embriões com mais de 16 células foram separados ao acaso em dois grupos: controle e ETC. Os embriões do grupo controle foram cultivados a 39 oC continuamente e do grupo ETC expostos a 41 oC por 12 horas, retornando a seguir para 39 oC. No experimento 1 não foi observado efeito do ETC nas várias raças estudadas, não havendo portanto, redução nas taxas de blastocisto e blastocisto eclodido. As taxas de clivagem e mórula dos embriões mHPB x Gir foram inferiores (p<0,05) as das demais raças. As raças mHPB x N apresentaram taxas de blastocisto... / There are evidences that deleterious effects of heat stress (HS) on fertility are less pronounced in breeds tolerant to high temperatures, due mainly to differences on their thermoregulatory capacity. In vitro experiments have shown that Bos indicus embryos are more resistant to HS when compared to Bos taurus. In order to better understand the differences related to heat shock resistance between Bos indicus and Bos taurus, the main objective of this study was to determine if tolerance to HS is cause by genetic contribution from the oocyte, spermatozoa or both. Additionaly, the influence of the time, between collection of ovaries in the abattoir and oocyte aspiration in the laboratory, on early embryo development was ascertained. In experiment 1, oocytes from Nellore and crossbreed Holstein cows (cHST), were collected in a local abattoir, matured and fertilized using semen from Nellore (N), Angus (An), Brahman (Bra) and Gir (Gir) bulls. In experiment 2, oocytes from Nelore and Holstein (HST) cows were collected in an abattoir and the oocytes were aspired in the laboratory 4 or 6:30 h afterwards, matured and fertilized using semen from N, Gir and HST. In both experiments, 96 h post insemination (hpi), embryos with 16 cells were separated in two groups: control and HS. In the control group the embryos were cultured at 39 oC, whereas in the HS group the embryos were submitted to 41 oC during for 12 h, and then returned to 39 oC. In experiment 1, there was no effect of HS on blastocyst and hatched blastocyst rates in all breeds studied.. The percentage of oocytes that cleaved and reached the morula stage was significantly lower (p<0.05) in cHST x Gir as compared to the other breeds. Additionaly, blastocyst rate was higher in cHST x N than in cHST x An and cHST x Gir (P<0.05). In experiment 2, cleavage, morula and blastocyst rates of Group 4 h were higher (P<0.05) as compared to Group ... (Complete abstract click electronic access below)