Orientador: Marcelo Fábio Nogueira / Banca: Fernanda da Cruz Landim e Alvarenga / Banca: Marcos Roberto Chiaratti / Banca: Flávio Vieira Meirelles / Banca: Lawrence Charles Smith / Resumo: Na literatura, não há descrição de que células trofectodérmicas sejam adequadas para a agregação (sem utilizar a fusão ou a microinjeção) e forma cão de embrião quimérico. O presente trabalho buscou validar um modelo para gerar blastocistos quiméricos, pela agrega ção de tecido trofectodérmico, primeiramente validando-o em camundongos e posteriormente testando a sua eficácia na espécie bovina. Nesta, duas técnicas para a reconstrução de blastocistos foram investigadas, mediante a agregação de um fragmento de trofectoderme (TE) com a massa celular interna (MCI) ou com células tronco induzidas a pluripotência (iPS). Os embriões foram bissectados com lâmina, assistida por micromanipulador, exceto no Experimento 1 e 2 (embriões bovinos) em que a bissecação utilizou microlâmina, por em controlada manualmente. De acordo com o delineamento de cada experimento, os embriões, os fragmentos embrionários e/ou as células iPS, foram depositados em micropoços preenchidos com meio KSOMaa (camundongo) ou SOF (gado bovino). Após 24 horas, a taxa de agregação (TA) das estruturas nos micropoços foi avaliada morfologicamente, mediante a detecção de uma única e coesa massa celular ou pela re-expansão e reorganização do blastocisto. Na validação em camundongos, foram utilizados dois hemiblastocistos ou MCI e TE derivados de embriões produzidos in vivo das linhagens Swiss Webster e C57BL/6/EGFP. Três experimentos foram conduzidos para caracterizar: 1) a eficácia da agregação de embriões em estágios pós-compactação; 2) o efeito do aumento de fragmentos embrionários e a utilização de um agente aglutinante biológico (fitohemaglutinina) na agregação; e 3) a exequibilidade da reconstrução do blastocisto pela aproximação entre MCI e fragmento de TE. Na segunda parte do trabalho, foram utilizados embriões bovinos produzidos in vitro em 5 experimentos: 1) utilização de fitohemaglutinina ... / Abstract: There is no report in the literature that trophectoderm cells can be used for aggregation (except in combination with fusion or microinjection techniques) and develop aggregates into a chimeric embryo. We aimed to validate a model to generate chimeric blatocysts using the trophectodermic tissue for aggregation in mice and replicate the validated technique in bovine embryos. We tested two methods of reconstruction in order to aggregate a trophectoderm fragment with either the inner cell mass (ICM) or the induced pluripotent stem (iPS) cells. Embryos were bisected by a blade assisted by micromanipulator, except in experiment 1 and 2 (bovine embryos) in which embryos were bisected by a manually controlled microblade. According to each experimental design, embryos, embryonic fragments and/or iPS cells were deposited in microwells lled with KSOMaa (mice) or SOF (bovine cattle). After 24 h the aggregation rate (AR) was assessed morphologically by the detection of a single, cohesive cell mass or by a re-expanded and reorganized blastocyst. In mice we used two demi-blastocysts or ICM and TE derived from in vivo produced Swiss Webster and C57BL/6/EGFP embryos. Three approaches aimed to characterize: 1) the e ectiveness of aggregation in post-compaction embryos; 2) the e ect on aggregation of increasing embryonic fragments and the use of the cell agglutination agent, phytohemagglutinin-L; and 3) the feasibility of reconstructing blastocysts by culturing ICM and TE fragments in close contact. In the bovine experiment we used in vitro produced embryos in 5 approaches to assess: 1) the effect of phytohemagglutinin-L in the aggregation of post-compaction demi-embryos; 2) blastocyst reconstruction using manually sectioned structures (ICM and TE); 3) microinjection of TE fragments into morula; 4) blastocyst reconstruction using structures (ICM and TE) sectioned with micromanipulator; and 5) production of chimeric ... / Doutor

<p>About 30 % of all infertile couples suffer from infertility of an unexplained cause. Leukemia inhibitory factor (LIF) is a glycoprotein produced by the endometrium and is an important cytokine in the implantation process. LIF exerts its biological functions through heterodimerization of its two receptors: LIF receptor (LIFR) and gp130. Point mutations in the LIF gene have been associated with female infertility. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the genes of LIFR and gp130 could cause reduced fertility in women. To this end, 115 samples from women diagnosed with unexplained infertility and 191 samples from fertile women were studied. Three SNPs in the gp130 gene and two SNPs in the LIFR gene were analyzed using real-time PCR. One significant difference and a tendency to difference were detected in the gp130 gene for women with unexplained infertility. There were no differences in the LIFR gene variations. In conclusion, polymorphisms in gp130, and thereby disturbances in the LIF pathway, could be one cause for infertility in women diagnosed with unexplained infertility.</p>

It was hypothesized that technical factors associated with in vitro production (IVP) of embryos may influence rate of blastocyst development of oocytes matured at 38.5 or 41.0 C. To test this hypothesis, a retrospective meta-analysis was performed. Simple linear regression was performed to analyze continuous variables and ANOVA for categorical variables. Interactions among factors and maturation temperature on blastocyst development were analyzed using dummy regression for continuous variables, and using a factorial treatment design and ANOVA for categorical variables. Month of collection was the only variable that impacted responsiveness of ova to heat stress. Independent of maturation temperature, variables that explained most variation in blastocyst development included technician, total number of sliced ovaries per collection, ova number placed per well of oocyte maturation media, oocyte collection time, bull ID, sperm concentration added to ova, and ova age at IVF. The proportion of 8 to 16-cell embryos at time of cleavage assessment was the best predictor of blastocyst development. Results of model selection showed that development of ova matured at 38.5 C was associated with size of the collection, while development of ova matured at 41.0 C was mainly associated with ova age at fertilization. When data for ova matured at 38.5 and 41.0 C were combined, the effect of number of PZ per well on blastocyst development became evident. Use of these findings for optimizing efficiency of IVP procedures would effectively reduce experimental costs related to embryo production and increase laboratory productivity.

About 30 % of all infertile couples suffer from infertility of an unexplained cause. Leukemia inhibitory factor (LIF) is a glycoprotein produced by the endometrium and is an important cytokine in the implantation process. LIF exerts its biological functions through heterodimerization of its two receptors: LIF receptor (LIFR) and gp130. Point mutations in the LIF gene have been associated with female infertility. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the genes of LIFR and gp130 could cause reduced fertility in women. To this end, 115 samples from women diagnosed with unexplained infertility and 191 samples from fertile women were studied. Three SNPs in the gp130 gene and two SNPs in the LIFR gene were analyzed using real-time PCR. One significant difference and a tendency to difference were detected in the gp130 gene for women with unexplained infertility. There were no differences in the LIFR gene variations. In conclusion, polymorphisms in gp130, and thereby disturbances in the LIF pathway, could be one cause for infertility in women diagnosed with unexplained infertility.

Epidemiological studies have shown that human adults have a higher chance of developing various metabolic disorders, such as type II diabetes and hypertension, due to maternal under nutrition. The concept that conditions encountered in early development can have far reaching implications for an individual’s adult life is known as the Developmental Origins of Health and Disease (DOHaD) hypothesis. In vitro studies have shown that if the amino acid leucine is not available at a high enough concentration the embryo will not exhibit normal trophectoderm protrusive activity which precedes implantation. The amino acid transport system System B0,+ is the main gateway into these cells for leucine but its expression at the transcription level has never been shown in preimplantation blastocysts. We investigated System B0,+ expression in preimplantation blastocysts from dams fed a control versus a low-protein isocaloric diet (18% and 9% casein, respectively). Using RT-PCR to detect the System B0,+ transcript, ATB0,+, we found that indeed there is expression of this amino acid transporter in the pre-implantation rat blastocyst. Due to the gender-specific nature of many DOHaD phenomena, the blastocysts were sexed using gender specific primers and a nested PCR approach. Quantitative real time PCR (qRT-PCR) results show no significant difference in ATB0,+ expression in blastocysts taken from dams fed either diet (9% n= 56, 18% n = 52; 7 dams from each diet group). Furthermore, separating the data by gender reveals no significant difference in expression. However, while not significant, there does appear to be a trend in the protein restricted blastocysts towards increased transcription of ATB0,+, suggesting System B0,+ may be responding at the transcription level to the diet. This could be part of the predictive adaptive response leading to a reprogrammed phenotype as described by the DOHaD hypothesis. Further work is needed to elucidate System B0,+’s role in developmental programming.

Epidemiological studies have shown that human adults have a higher chance of developing various metabolic disorders, such as type II diabetes and hypertension, due to maternal under nutrition. The concept that conditions encountered in early development can have far reaching implications for an individual’s adult life is known as the Developmental Origins of Health and Disease (DOHaD) hypothesis. In vitro studies have shown that if the amino acid leucine is not available at a high enough concentration the embryo will not exhibit normal trophectoderm protrusive activity which precedes implantation. The amino acid transport system System B0,+ is the main gateway into these cells for leucine but its expression at the transcription level has never been shown in preimplantation blastocysts. We investigated System B0,+ expression in preimplantation blastocysts from dams fed a control versus a low-protein isocaloric diet (18% and 9% casein, respectively). Using RT-PCR to detect the System B0,+ transcript, ATB0,+, we found that indeed there is expression of this amino acid transporter in the pre-implantation rat blastocyst. Due to the gender-specific nature of many DOHaD phenomena, the blastocysts were sexed using gender specific primers and a nested PCR approach. Quantitative real time PCR (qRT-PCR) results show no significant difference in ATB0,+ expression in blastocysts taken from dams fed either diet (9% n= 56, 18% n = 52; 7 dams from each diet group). Furthermore, separating the data by gender reveals no significant difference in expression. However, while not significant, there does appear to be a trend in the protein restricted blastocysts towards increased transcription of ATB0,+, suggesting System B0,+ may be responding at the transcription level to the diet. This could be part of the predictive adaptive response leading to a reprogrammed phenotype as described by the DOHaD hypothesis. Further work is needed to elucidate System B0,+’s role in developmental programming.

It was hypothesized that technical factors associated with in vitro production (IVP) of embryos may influence rate of blastocyst development of oocytes matured at 38.5 or 41.0 C. To test this hypothesis, a retrospective meta-analysis was performed. Simple linear regression was performed to analyze continuous variables and ANOVA for categorical variables. Interactions among factors and maturation temperature on blastocyst development were analyzed using dummy regression for continuous variables, and using a factorial treatment design and ANOVA for categorical variables. Month of collection was the only variable that impacted responsiveness of ova to heat stress. Independent of maturation temperature, variables that explained most variation in blastocyst development included technician, total number of sliced ovaries per collection, ova number placed per well of oocyte maturation media, oocyte collection time, bull ID, sperm concentration added to ova, and ova age at IVF. The proportion of 8 to 16-cell embryos at time of cleavage assessment was the best predictor of blastocyst development. Results of model selection showed that development of ova matured at 38.5 C was associated with size of the collection, while development of ova matured at 41.0 C was mainly associated with ova age at fertilization. When data for ova matured at 38.5 and 41.0 C were combined, the effect of number of PZ per well on blastocyst development became evident. Use of these findings for optimizing efficiency of IVP procedures would effectively reduce experimental costs related to embryo production and increase laboratory productivity.

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. Title from PDF of title page (University of Missouri--Columbia, viewed on April 1, 2010). Vita. Thesis advisor: R. Michaels Roberts. "May 2008" Includes bibliographical references

Made available in DSpace on 2014-06-11T19:32:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-26Bitstream added on 2014-06-13T20:42:44Z : No. of bitstreams: 1 000741947.pdf: 4654580 bytes, checksum: b05e20e66d52ef92df24a9907af04254 (MD5) / Na literatura, não há descrição de que células trofectodérmicas sejam adequadas para a agregação (sem utilizar a fusão ou a microinjeção) e forma cão de embrião quimérico. O presente trabalho buscou validar um modelo para gerar blastocistos quiméricos, pela agrega ção de tecido trofectodérmico, primeiramente validando-o em camundongos e posteriormente testando a sua eficácia na espécie bovina. Nesta, duas técnicas para a reconstrução de blastocistos foram investigadas, mediante a agregação de um fragmento de trofectoderme (TE) com a massa celular interna (MCI) ou com células tronco induzidas a pluripotência (iPS). Os embriões foram bissectados com lâmina, assistida por micromanipulador, exceto no Experimento 1 e 2 (embriões bovinos) em que a bissecação utilizou microlâmina, por em controlada manualmente. De acordo com o delineamento de cada experimento, os embriões, os fragmentos embrionários e/ou as células iPS, foram depositados em micropoços preenchidos com meio KSOMaa (camundongo) ou SOF (gado bovino). Após 24 horas, a taxa de agregação (TA) das estruturas nos micropoços foi avaliada morfologicamente, mediante a detecção de uma única e coesa massa celular ou pela re-expansão e reorganização do blastocisto. Na validação em camundongos, foram utilizados dois hemiblastocistos ou MCI e TE derivados de embriões produzidos in vivo das linhagens Swiss Webster e C57BL/6/EGFP. Três experimentos foram conduzidos para caracterizar: 1) a eficácia da agregação de embriões em estágios pós-compactação; 2) o efeito do aumento de fragmentos embrionários e a utilização de um agente aglutinante biológico (fitohemaglutinina) na agregação; e 3) a exequibilidade da reconstrução do blastocisto pela aproximação entre MCI e fragmento de TE. Na segunda parte do trabalho, foram utilizados embriões bovinos produzidos in vitro em 5 experimentos: 1) utilização de fitohemaglutinina... / There is no report in the literature that trophectoderm cells can be used for aggregation (except in combination with fusion or microinjection techniques) and develop aggregates into a chimeric embryo. We aimed to validate a model to generate chimeric blatocysts using the trophectodermic tissue for aggregation in mice and replicate the validated technique in bovine embryos. We tested two methods of reconstruction in order to aggregate a trophectoderm fragment with either the inner cell mass (ICM) or the induced pluripotent stem (iPS) cells. Embryos were bisected by a blade assisted by micromanipulator, except in experiment 1 and 2 (bovine embryos) in which embryos were bisected by a manually controlled microblade. According to each experimental design, embryos, embryonic fragments and/or iPS cells were deposited in microwells lled with KSOMaa (mice) or SOF (bovine cattle). After 24 h the aggregation rate (AR) was assessed morphologically by the detection of a single, cohesive cell mass or by a re-expanded and reorganized blastocyst. In mice we used two demi-blastocysts or ICM and TE derived from in vivo produced Swiss Webster and C57BL/6/EGFP embryos. Three approaches aimed to characterize: 1) the e ectiveness of aggregation in post-compaction embryos; 2) the e ect on aggregation of increasing embryonic fragments and the use of the cell agglutination agent, phytohemagglutinin-L; and 3) the feasibility of reconstructing blastocysts by culturing ICM and TE fragments in close contact. In the bovine experiment we used in vitro produced embryos in 5 approaches to assess: 1) the effect of phytohemagglutinin-L in the aggregation of post-compaction demi-embryos; 2) blastocyst reconstruction using manually sectioned structures (ICM and TE); 3) microinjection of TE fragments into morula; 4) blastocyst reconstruction using structures (ICM and TE) sectioned with micromanipulator; and 5) production of chimeric ...

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-12-16T11:19:46Z No. of bitstreams: 1 michelemunkpereira.pdf: 1917144 bytes, checksum: 6ea13cf025292a61936d375b8b3710e2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-12-16T11:29:32Z (GMT) No. of bitstreams: 1 michelemunkpereira.pdf: 1917144 bytes, checksum: 6ea13cf025292a61936d375b8b3710e2 (MD5) / Made available in DSpace on 2016-12-16T11:29:33Z (GMT). No. of bitstreams: 1 michelemunkpereira.pdf: 1917144 bytes, checksum: 6ea13cf025292a61936d375b8b3710e2 (MD5) Previous issue date: 2010-03-12 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A maturação in vitro de oócitos é um processo importante para a produção in vitro de embriões bovinos. O objetivo do presente estudo foi investigar o efeito da interação do soro e tensão de oxigênio na maturação in vitro, sobre a maturação nuclear, viabilidade das células do cumulus, abundância de transcritos oocitários, desenvolvimento pré-implantacional e apoptose embrionária. Foram utilizados quatro grupos experimentais: G1 (10% soro de vaca no cio [SVC] em 20% de O2); G2 (0,1% álcool polivinílico [PVA] em 20% de O2), G3 (10% SVC em 5% de O2) e G4 (0.1% PVA em 5% O2). A proporção de oócitos em metáfase II, viabilidade das células do cumulus, taxa de blastocistos e número total de células não foi afetado (P>0,05) quando o soro foi substituído por PVA em 5% de O2, enquanto a taxa de blastocistos e número total de células foi maior (P<0.05) no grupo com soro comparado com PVA, ambos em 20% O2. O índice apoptótico foi menor em blastocistos produzidos a partir de oócitos maturados com PVA em 5% de O2 (G4) em relação aos de outros grupos (G1, G2 e G3), mas não foi encontrada diferença (P>0,05) na taxa de maturação, viabilidade das células do cumulus e de blastocistos. Foram encontradas diferenças (P<0,05) na quantidade de transcritos específicos em oócitos maturados sobre as diferentes condições. Conclui-se que a presença de soro durante a maturação é importante para o desenvolvimento embrionário em tensão de 20% de O2, e a suplementação com PVA em 5% de O2 fornece o melhor ambiente de maturação para os oócitos, resultando em blastocistos com baixo índice apoptótico. / In vitro maturation is an important step for in vitro embryo production. The objective of the present study was to investigate the effect of the interaction of serum and oxygen tension during in vitro maturation on nuclear maturation, viability of cumulus cells, oocyte transcript amount, preimplantation development and blastocyst apoptosis. Four experimental groups were designed: G1 (10% estrus cow serum [ECS] with 20% O2); G2 (0.1% polyvinyl alcohol [PVA] with 20% O2), G3 (10% ECS with 5% O2) e G4 (0.1% PVA with 5% O2). Proportion of metaphase II oocytes, viability of cumulus cells, blastocyst rates and the total cell number were not affected (P>0.05) when the ECS was replaced by PVA under 5% O2, whereas higher (P<0.05) blastocyst rate and total cell number were found with ECS than PVA, both under 20% O2. Apoptosis index were lower in blastocysts from oocytes matured with PVA under 5% O2 (G4) than blastocysts from other groups (G1, G2 and G3), but no differences (P>0.05) were found in nuclear maturation, viability of cumulus cells and blastocyst rate. Differences (P<0.05) in amount of specific transcripts were found in oocytes matured under different conditions. In conclusion, presence of serum during maturation is important for embryo development only when under 20% O2, and maturation with PVA and 5% O2 provides better environment for oocyte, resulting in blastocysts with low apoptosis index.