Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, Aug. 2005. / Title from title screen. Includes bibliographical references.

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Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-23Bitstream added on 2014-06-13T19:45:06Z : No. of bitstreams: 1 carmo_as_dr_jabo.pdf: 750695 bytes, checksum: badd16ed8cd1277688580dd61ee13af8 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Estudo de Associação Genômica (GWAS - Genome Wide Association Study) foi conduzido para identificar regiões cromossômicas relacionadas à capacidade de criotolerância espermática em bovinos da raça Nelore, empregando os fenótipos: diferença entre a motilidade do sêmen fresco e descongelado (DMD) e diferença entre a motilidade do sêmen fresco e pós-teste de termo-resistência (DMT), com o intuito de compreender melhor os fenômenos que regem tal característica. O perfil de SNP (SNP - Single Nucleotide Polymorphism) de cento e vinte e cinco touros foi determinado com o auxílio de micro-arranjo de alta densidade de SNP (BovineHD Illumina®). As estimativas dos valores genéticos dos touros (EBV - Estimated Breeding Value) (Experimento 1) e as médias das medidas fenotípicas (Experimento 2) foram calculadas para as características em questão. As metodologias estatísticas GRAMMAR e EIGENSTRAT foram utilizadas para a realização do teste de associação fenótipo / genótipo após procedimento de controle de qualidade dos dados. A análise de enriquecimento funcional foi realizada com auxílio do software DAVID. Essa análise apontou a família de genes Serpina (inibidores de proteases), reguladores de cAMP e componentes do citoesqueleto celular como o grupo de genes que apresentam maior importância funcional para as características avaliadas. Todas as metodologias testadas demonstraram-se capazes de identificar regiões genômicas associadas aos fenótipos DMD e DMT, destacando-se os cromossomos 1, 16 e 19 / Genome Wide Association Study (GWAS) was performed in order to identify chromosomal regions related to sperm cryotolerance capacity in Nellore cattle for the phenotypes: difference between fresh and post-thaw semen motility (DMD) and difference between fresh and post thermal resistance test semen motility (DMT), aiming to better understand the phenomena governing this characteristic. SNP (Single Nucleotide Polymorphism) profile of one hundred and twenty five Nellore bulls were determined using high density SNP chip (BovineHD Illumina®). Estimated Breeding Values - EBV (Experiment 1) and phenotypic measures means (Experiment 2) were calculated for the evaluated traits. The statistical methodologies GRAMMAR and EIGENSTRAT were used to test the phenotype / genotype association after data quality control. Functional enrichment analysis was performed using DAVID software. These analyses pointed out to Serpine (protease inhibitor), cAMP metabolism control and cytoeskeletal components gene families as the most important functional gene groups for the evaluated traits. All the methods tested shown to be able to identify genomic regions associated with phenotypes DMD and DMT, especially chromosomes 1, 16 and 19

Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-12-14Bitstream added on 2014-06-13T20:06:39Z : No. of bitstreams: 1 dellaqua_cpf_dr_botfmvz.pdf: 1556422 bytes, checksum: 1d36e424398f8b56c6af3fc18521061c (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As biotécnicas de refrigeração e congelação podem causar danos irreversíveis à célula espermática devido ao estresse gerado pela queda de temperatura ou, no caso da criopreservação, pela formação de cristais de gelo intracelulares. Estas alterações resultam na diminuição da longevidade do espermatozóide no trato reprodutor da fêmea e, conseqüentemente, na queda dos índices de fertilidade. Sabendo-se que as alterações das características espermática afetam a viabilidade do sêmen sobre as diferentes formas de preservação seminal e que garanhões de diferentes taxas de fertilidade nem sempre apresentam diferenças significativas nas análises seminais padrões, faz-se necessário uma análise mais específica da células espermática, como a avaliação de vários parametros funcionais em células individuais, o que possivelmente reduziria as incertezas inerentes a previsão da fertilidade na avaliação in vitro do sêmen. Para comprovação desta hipótese três experimentos foram realizados. No experimento 1 o objetivo foi identificar e avaliar as principais modificações ocorridas nas características morfofuncionais do sêmen equino estocado a temperatura ambiente (18-22ºC) por um período de 12 horas. No experimento 2 o objetivo foi verificar a possibilidade de modular estas alterações através de duas temperaturas de refrigeração (5ºC e 15ºC), e, no experimento 3, o objetivo foi avaliar e verificar a relação destas alterações com os índices de fertilidade em sêmen congelado. Para os experimentos 1 e 2, três ejaculados de seis diferentes garanhões foram avaliados quanto a cinética espermática através da análise computadorizada do movimento espermático; para a avaliação morfofuncional foram avaliadas a integridade das membranas plasmática... / Cooling and freezing can cause irreversible damage to sperm cells due to the stress generated by the decrease in temperature or, in the case of cryopreservation, the formation of intracellular ice crystals. These changes result in decreased longevity of sperm in the female reproductive tract and, consequently, a decrease in fertility rates. The changes in sperm characteristics affect the viability of the semen, and these changes vary based on the different preservation techniques used to preserve the semen from different stallions. In addition, fertility rates do not always show significant differences in semen analysis patterns. Therefore, a more specific analysis of sperm cells is necessary. This analysis should include a functional assessment of multiple parameters in individual cells, which might reduce uncertainties in the prediction of fertility based on the in vitro evaluation of semen. To test this hypothesis, three experiments were performed. In experiment 1, the objective was to identify and assess the main characteristic morph functional changes in equine semen that is stored at room temperature (18-22°C) for a period of 12 hours. In experiment 2, the objective was to determine the possibility of modulating these changes using two refrigeration temperatures (5°C and 15°C). In experiment 3, the objective was to evaluate and compare the influence of these changes on fertility rates between semen at various temperatures and in frozen semen. For experiments 1 and 2, the sperm kinetics of three different ejaculates from six stallions were evaluated using a computerized analysis of sperm motility. For morphological and functional assessments, the following parameters were evaluated: plasma and acrosomal membrane integrity, mitochondrial membrane potential, rate of DNA fragmentation, caspase activation and... (Complete abstract click electronic access below)

Male factor infertility is a contributing factor in up to 50% of infertile couples. Increasing numbers of couples undergoing assisted reproductive technology (ART) treatment and reports of a possible decline in male fertility suggest that lifestyle, occupational and environmental exposures might impair semen quality. Sperm DNA contains both DNA strand breaks and base damage that has been associated with poor semen quality but few studies have examined the role of double strand breaks (DSBs), a toxic lesion, or DNA damage such as N7-methyldeoxyguanosine (N7-MedG) arising from alkylating agents that can be toxic and mutagenic. The aim of this research was to examine the relationships between exposures, DNA damage and male fertility. Men were recruited from couples attending for ART treatment and they provided information on lifestyle, occupational and environmental exposures as well as a sperm sample. Semen concentration and motility was determined by standard techniques in the neat sample and the prepared sample that underwent density gradient centrifugation for ART treatment. DSBs were measured in individual sperm cells by the neutral Comet assay and N7-MedG levels in sperm DNA by an immunoslot blot assay. Information on ART outcomes (% fertilisation, % cleavage and clinical pregnancy) was collected and associations between DNA damage, exposures, semen quality and ART outcomes were determined. Expression of individual DNA repair proteins was also examined in individual oocytes. Men in manual work had significantly lower semen volumes and higher % immotile sperm. Exposure to dry cleaning fluids and having a fever were associated with a decrease in sperm number and while non-ionizing radiation was associated with an increase in % immotile sperm, X-ray exposure was correlated to a decrease in % progressively motile sperm. Semen parameters were significantly and negatively correlated with DSBs in neat and prepared sperm, and N7-MedG levels in neat sperm. Density gradient centrifugation improved sperm sample quality and decreased DSBs and N7-MedG levels significantly. Successful fertilization of oocytes was negatively associated with DSB levels in neat and prepared sperm and with N7-MedG levels in neat sperm. Lower DSB levels in men were associated with an increased chance of an achieving clinical pregnancy especially in ICSI couples. N7-MedG levels were significantly correlated with driving a car and exposure to detergent or printing inks and dyestuffs. DSBs were correlated negatively with exercise and positively with eating nuts and almonds or exposure to non-ionizing radiation. DNA repair gene expression in individual oocytes showed significant intra and inter-individual variability. Sperm DNA damage can reduce male fertility, but the causes of such damage remain to be identified. The variable ability of individual oocytes to repair this damage may well affect the chance for a successful pregnancy.

INTRODUCTION Phthalates, a chemical class of plasticizers, are ubiquitous in the environment and recognized as endocrine disrupting compounds (EDCs). Recent data suggest that oxidative stress is a potential mediator of poor male reproductive health associated with phthalate exposure. Mitochondria are implicated in the production of excess oxidative stress and sperm mitochondrial copy number (MtCopy) and deletions (MtDeletion) have been linked with male infertility. However, little is known about the relationship of these mitochondrial biomarkers in sperm with phthalate exposure and oxidative stress. OBJECTIVES To examine associations of urinary phthalate metabolites and isoprostane concentrations on sperm MtCopy and MtDeletions in male partners undergoing assisted reproductive technologies (ART). METHODS A total of (n=97) sperm samples were collected from male partners undergoing ART at Baystate Medical Center, in Springfield, MA from 2014 to 2016 as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Seventeen urinary phthalate metabolites (n=103) were analyzed by the Centers for Disease Control using tandem mass spectrometry. 15-F2t-Isoprostane (n=101) was measured using a competitive enzyme-linked immonsorbent assay in urine of male individuals. A triplex Taqman probe-based qPCR method was developed for relative quantification of genomic DNA, MtCopy and MtDeletions. Multivariable linear or logistic regression was employed to examine associations with age, BMI, batch and current smoking status with each outcome to determine confounders used for adjustment. RESULTS Quartiles of MtCopy and MtDeletion were positively associated with the odds of male infertility (p for trend < .0001 and 0.007, respectively). Urinary metabolite concentrations of MCNP displayed a positive association with MtCopy (β=1.56; p =0.03). Urinary MEHP concentrations were positively associated with MtDeletion in only infertile individuals (n=30) (β = 0.075; p = 0.006). Urinary isoprostane concentration was not associated with MtCopy or MtDeletion, but was associated with seven phthalate metabolite concentrations (MEOHP, MEHHP, MBzP, MHBP, MiBP, and MHiBP). CONCLUSIONS To our knowledge, this is the first study to investigate the relationship between sperm MtCopy and MtDeletion with oxidative stress and phthalates. These results suggest that certain phthalate metabolites may be associated with a known biomarker of systemic oxidative stress. Sperm mitochondrial function as measured by MtCopy and MtDeletion may be considered biomarkers of male infertility, although no relationship was shown between mitochondrial outcomes and oxidative stress. Future research is investigating these relationships with developmental outcomes including embryo quality.

This study was conducted to investigate the requirement for sperm processing in microsurgical subzonal placement of sperm in rabbit oocytes. Fertilization rates with standard in vitro fertilization and microsurgical subzonal sperm placement were found to be similar (56 and 55%) when sperm treated with high-ionic strength Brackett's defined inedium to initiate capacitation were used. Statistically significant reductions in fertilization rates for both standard in vitro fertilization and subzonal placement were noted when twice-washed spermatozoa were used. Initiation of capacitation of spermatozoa results in higher fertilization results even when the zona pellucida is bypassed during fertilization.

論文1ページ目の下部に著作権を表示すること。（© 2016, American Society for Microbiology. ） / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19634号 / 医科博第72号 / 新制||医科||5(附属図書館) / 32670 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 近藤 玄, 教授 篠原 隆司, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Fertility is classified as one of the most limiting aspects plaguing successful mammalian reproduction. In addition, sequential collaborations between a quality spermatozoon and oocyte are essential for successful fertilization, and key in subsequent embryonic development. Male fertility has been consistently declining in mammals. Currently, cost efficient and reliable methods predicting fertility are lacking. As a result, the objectives of this research were to establish molecular markers to predict male fertility by determining sperm cellular phenotype variability among bulls of varying fertility by focusing on the roles of protein markers, TUBB 2C, HSP10, HXK1 and SOD1; establish expression characteristics of ITGB5 protein in sperm, oocytes and early embryos; and finally; determine expression level variability and functions of miRNAs, miR-214 and miR-25 in sperm from bulls of varying fertility. We found that while no significant differences occurred in the expression levels of our proteins in high and low fertility bulls, correlations were discovered between SOD1, HSPE1 and fertility, and subpopulations consisting of intact, partially damaged and completely damaged acrosomes in flow cytometry and microscopy experiments were identified. Results also showed that expression levels of itgb5 were significantly higher in the 2-cell bovine embryos, followed by the 8-16 cell embryos, however, no significant difference in expression levels were noted for the morula and blastocyst stages as compared to the MII oocytes. Phylogenetic analyses confirmed conservation of itgb5 across multiple species, further indicating its potential importance and functional role(s) in fertilization and potential as a marker of fertility. Micro-RNA studies revealed differential expression levels of miR-25 and miR- 214 for all samples analyzed, yet there was no significant difference in expression of the microRNAs when comparing high fertility and low fertility bulls. Also, processes associated with spermatogenesis, such as cellular growth, transduction, translation and transformation which are necessary for fertility, were shown to be targets of miR-214 and miR-25. These results imply that sperm proteins and sperm-bore miRNAs could potentially be targeted as molecular markers of fertility. The findings are important because they illuminate the molecular and cellular underpinnings of gamete quality that influence successful fertilization and early development.

An alarming number of anuran (frog and toad) species are facing the threat of extinction in the wild. In efforts to address this conservation crisis, captive breeding programs are rapidly being established at zoos and research institutions worldwide. However, the captive management of anurans can be challenging, as their reproduction is a tightly regulated hormonal response to environmental stimuli, often unknown or absent in captivity. Consequently, ex-situ breeding efforts tend to be greatly hindered by a paucity of knowledge in anuran reproductive physiology and, for many species on the brink of extinction, time is running out. Assisted reproductive technologies (ARTs), such as exogenous hormone induction of gamete release, artificial fertilization for population augmentation, and cryopreservation for the long-term storage of genetics, have the potential to greatly enhance captive breeding efforts in lieu of natural breeding. Broadly, research aims were to develop assisted reproductive technologies for captive populations of the declining Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas), the critically endangered Mississippi Gopher Frog (Lithobates captio sevosa) and Puerto Rican crested toad (Peltophryne lemur). Specific objectives were to a) trial the efficacy of exogenous hormone treatments on sperm release in male target species by characterizing spermiation response across time; b) investigate methods for increasing sperm longevity through cold-storage and cryopreservation techniques; c) ascertain motility recovery rates and functional capacity of cold-stored and frozen-thawed spermatozoa through artificial fertilization techniques, and; d) apply successfully developed ARTs to determine the feasibility of genetically linking in-situ and ex-situ populations of A. b. boreas, through artificial fertilization of male and female gametes from wild and captive toads, respectively. Research outcomes from this study include the successful development of exogenous hormone protocols, spermiation profiles and sperm cryopreservation techniques for all target species. Additionally, these studies enabled validation of an alternative method for increasing genetic diversity in captive anurans through in-situ-ex-situ gamete linkage. Overall, this research emphasizes the potential value of assisted reproductive technologies as conservation tools for supporting the recovery of endangered frog and toad species worldwide.