Introduction: In recent years, the average age of first reproduction has risen significantly, the mean now standing at around 30 years in many countries. The adverse effects of maternal age on fertility and reproduction have been well documented. However, the influence of paternal age on fertility, reproduction and postnatal health is relatively poorly understood, and 50% of all male infertility cases are classed as idiopathic or unexplained infertility. Methods: The aim of this study was to investigate factors that contribute to male infertility, split into two main parts. The first part focused on analysing data collected from patients who had undergone fertility treatment to assess the influence of different factors on infertility, especially at the genome level. The second part attempted to deal with some of the technical challenges of screening and diagnostic methods to study the genome, with the aim of providing tools that would assist future studies in pinpointing genetic factors responsible for infertility, especially in cases of idiopathic infertility. Results: Based on data from the first part of the study, it was determined that advanced paternal age can affect sperm progressive motility, sperm DNA integrity and the fertilisation rate of in vitro fertilisation (IVF) cycles, as well as the development of embryos. Direct analysis of sperm DNA fragmentation (SDF) and degradation levels revealed an association between elevated SDF and impaired embryo development. Furthermore, a correlation was shown between chromosome aneuploidy and variance in SDF and sperm DNA degradation. Moreover, aneuploidy can influence abnormal sperm morphology and consequently also progressive motility. Also, embryo development rate of IVF cycles on day three, demonstrated a significant decline in cycles where the sperm used for fertilisation had a high aneuploidy rate, which can highlight the reduced developmental capacity of aneuploid embryos. From the lifestyle factors assessed, only alcohol consumption significantly correlated with the sperm DNA damage. Therefore, poor semen quality may highlight damage that has been incurred by the sperm DNA. When the semen quality is suboptimal, the intracytoplasmic sperm injection (ICSI) technique is suggested as a standard strategy to improve the prognosis of ART. However, when the progressive motility is poor, the ICSI approach is not as effective. Based on our findings and in line with other studies, the only sperm parameter that can be affected by paternal age is sperm motility, which could be an indicator of SDF. Therefore, the decline in ICSI fertilisation rate in patients with impaired sperm progressive motility could be due to sperm DNA damage, and even ICSI cannot improve the fertilisation rate considerably. Discussion: The aim of the second part of this project was to establish a robust workflow for whole- genome amplification (WGA) and whole-genome sequencing of single cells to improve the coverage rate and fidelity, with the aim of providing means of detecting any mutation in the genome that might be responsible for reduced embryonic developmental competence. Towards this end, the efficiencies of two different WGA protocols (REPLI-g and TruePrime) were compared. Multiple technical factors required optimisation in order to create a suitable protocol. Our results demonstrated the overall superiority of REPLI-g compared to TruePrime in almost all the assessed parameters. The amplification rate of REPLI-g was much faster than that of TruePrime, and prolonged incubation led to overamplification and an increased duplication rate. However, the TruePrime method has a slower amplification rate and therefore, by increasing the incubation time, it was possible to improve the quality of the data. The modified protocol with reduced volume also had the most promising outcome in terms of the data produced, and could fulfil our expectations by being fast, cost-effective and efficient. Conclusion: In conclusion, the results from the first part of this study confirmed the negative impact of male age on assisted reproductive treatments, which can result in decreased success rates of fertilisation. Other factors such as sperm DNA damage may also contribute to this age effect, suggesting that assessing this parameter prior to fertility treatment, and attempting to mitigate elevated levels of sperm DNA damage, may be of value to older patients. Additionally, overcoming the technical challenges in studying genetic contributory factors in infertility is a promising step toward better understanding of the mutations and variations that are involved in this phenomenon.

Understanding the establishment of polarity and the cell fate specification of epithelial cells is important for developmental biology, regenerative medicine and the study of cancer. In this thesis, models of pre-implantation epithelial development are used to investigate the relationship between these two processes. The trophoblast is an extraembryonic epithelial tissue which contributes to the placenta. Addition of BMP4 to mouse and human embryonic stem (mES) cells grown in culture has been suggested to induce differentiation of cells to the trophoblast lineage. The use of this differentiation method was investigated as a possible model of trophoblast polarisation and cell fate specification. Unfortunately, with the protocol and reagents available this model did not appear to physiologically recapitulate trophoblast development and was not reliable. The primitive endoderm is an epithelium which arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. When mES cells are grown in suspension they form aggregates of differentiating cells known as embryoid bodies. The outermost cell layer of an embryoid body is an epithelial cell type comparable to the primitive endoderm. Embryoid bodies were used here to study the polarisation and cell fate specification of the primitive endoderm. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. The acquisition of epithelial polarity occurred prior to the maximal expression of cell fate markers. Fgfr/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier which normally blocks free diffusion across the epithelial cell layer occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erkmediated polarisation. This data shows that the formation of a polarised primitive endoderm layer in embryoid bodies requires the Fgfr/Erk signalling pathway.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第19026号 / 農博第2104号 / 新制||農||1030 / 31977 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 教授 松井 徹 / 学位規則第4条第1項該当

Diss. Linköping : Linköpings universitet, 2005.

Orientador: José Gonçalves Franco Junior / Banca: Walter Pinto Junior / Banca: Sang Choon Cha / Banca: Marilza Vieira Cunha Rudge / Banca: José Carlos Peraçoli / Resumo: O diagnóstico genético pré-implantacional (PGD) é um procedimento o qual permite que embriões sejam testados perante uma doença genética antes de sua transferência para o útero materno, ou seja, antes... (Resumo completo clicar acesso eletrônico abaixo) / Abstract: Preimplantation genetic diagnosis (PGD) is a procedure that permits embryos to be tested for a possible genetic diseade before being transferred to the maternal uterus, i.e., before the beginning of pregnancy... (Complete abstract click electronic access below) / Doutor

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19026号 / 農博第2104号 / 新制||農||1030(附属図書館) / 学位論文||H27||N4908(農学部図書室) / 31977 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 教授 松井 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Preimplantation is a short yet critical time period for the embryo that includes dramatic changes in gene expression and developmental potential. During early cleavage, a combination of maternal and zygotic factors program the embryonic genome. These dynamic events result in critical differentiation steps including the first lineage specification (ICM and TE), which is required for implantation. The first half of this thesis investigates the role of Smyd3 as a possible maternal-effect gene, which was identified through an mRNA-based expression assay. Using a Smyd3 conditional allele to remove the function of the protein, we were able to confirm successful allelic recombinations but were unable to confirm the absence of the protein. Due to a lack of a phenotype, two conclusions were made: 1) If we did disrupt Smyd3 function, the lack of a phenotype indicates that Smyd3 is not a maternal-effect gene, and is not required for embryonic viability or adult fertility. 2) It is possible that newly-annotated transcripts are responsible for the necessity of Smyd3, and that the conditional allele used did not disrupt the function of Smyd3. The second half of this thesis uses a reverse genetic RNAi screen that identified Wdr74 as being required for the critical first steps of mammalian development. Knockdown of Wdr74 results in embryos that develop normally until the morula stage but fail to form blastocysts or properly specify the inner cell mass and trophectoderm. In Wdr74-deficient embryos, we find activated Trp53-dependent apoptosis as well as a global reduction of RNA polymerase I, II and III transcripts. In Wdr74-deficient embryos, blocking Trp53 function rescues blastocyst formation and lineage differentiation. These results indicate that Wdr74 is required for RNA transcription, processing and/or stability during preimplantation development and is an essential gene in the mouse.

O arsênio inorgânico, um contaminante ambiental, produz uma série de respostas de estresse em células de mamíferos, incluindo o comprometimento da função mitocondrial, acompanhado por inibição do crescimento celular e carcinogênese. Como previamente identificamos efeitos deletérios do comprometimento da função mitocondrial e dos radicais livres do oxigênio na oogênese, investigamos os efeitos do arsenito na meiose, no desenvolvimento embrionário pré-implantação e na apoptose embrionária em camundongos. Camundongas com 6 semanas de idade foram tratadas com baixa (0,16 mg) ou média dose de arsenito (0,32 mg), por meio de 7 injeções intraperitoneais, 1 a cada 2 dias, durante 14 dias. Os controles foram injetados com solvente. A incidência de anomalias meióticas, caracterizadas por anormalidades do fuso celular e/ou mal alinhamento cromossômico, foi significantemente aumentada tanto nos oócitos in vivo ovulados, como nos in vitro maturados, oriundos dos animais tratados com arsenito. Foram detectadas reduções significativas das taxas de clivagem (24 horas de cultivo), de formação de mórula (72 h) e de desenvolvimento para blastocisto (96 h), nos embriões dos grupos tratados com arsenito. Apesar do número total de núcleos não ter diferido significativamente entre os blastocistos dos grupos controle e de tratamento, a percentagem de núcleos apoptóticos foi significantivamente maior nos blastocistos derivados dos animais tratados com a dose média de arsenito. Estes dados sugerem que o arsenito causa aberrações meióticas, que podem contribuir tanto para o comprometimento do desenvolvimento embrionário pré-implantação, como para a apoptose embrionária. / Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including mitochondrial uncoupling accompanied by growth inhibition and carcinogenesis. Because previously we identified detrimental effects of mitochondrial uncoupling, and reactive oxygen species (ROS) on oogenesis, we investigated effects of arsenite on meiosis, early embryo development, and apoptosis in mice. Six-week-old CD-1 mice were treated with either low (0.16mg) or medium (0.32mg) doses of arsenite every two days by 7 intraperitoneal injections for 14 days, and controls were injected with solvent. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment or spreading, was significantly increased in both in vivo and in vitro treated oocytes. Further, we found a significant decrease in cleavage rates at 24h, morula formation at 72h, and development to blastocyst at 96h in treated groups. Although the total number of nuclei in developed blastocysts did not significantly differ between the treated and control groups, the percentage of apoptotic nuclei was significantly increased in blastocysts derived from the medium dose treated group. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis.

Embryonic cells of many species polarise and the cell polarity is often important for the normal developmental progression. In the mouse embryo, the prototype of epithelial cell polarity, namely apico-basal polarisation, become established at the 2.5 days' post-fertilisation, when the embryos are at the 8-cell stage. The formation of apical domain is necessary and sufficient for the first segregation of extra-embryonic and embryonic cell lineages, as well as the following up morphogenetic transitions, such as the blastocyst formation. This study aims to explore the molecular pathways triggering the first cell polarity establishment in the mouse embryo, and to reveal the mechanism that programmes the timing of this event in the mouse embryo. The results showed that cell polarity establishment during the 8-cell stage development can be divided into two major phases: in the first phase actomyosin complex became polarised to the cell-contact free surface; and in the second phase apical proteins recruited to the actomyosin enriched cell-contact free cortex, they further became centralised in the cell-contact free surface, excluding the local actomyosin meshwork, resulting in the formation of actomyosin ring. The activation and assembly of actomyosin meshwork during the first phase, but not its contractility, was essential for apical protein recruitment. Factors responsible for actin cytoskeleton reorganisation included Phospholipase C (PLC) - Protein Kinase C (PKC) pathway components, they directly activated actomyosin in the first phase through the Rho proteins such as RhoA. Further results showed that the apical protein centralisation step required a proximate transcriptional input that was induced by two transcription factors, Tfap2c and Tead4. RNAi and Genetic depletion of these two factors prevented apical protein centralisation and the final apical domain assembly. The protein expression profile indicated that Tfap2c and Tead4 expression, and therefore their activity, were induced by zygotic genome activation. Significantly, overexpression of Tfap2c, Tead4, together with constitutively activated Rho proteins were sufficient to advance the timing of apical domain formation, indicating that the timer of cell polarity establishment at the 8-cell stage is set by the Rho proteins activation, and the zygotic transcriptional accumulation of Tfap2c and Tead4. Together, these results characterised the molecular events during the cell polarity establishment at the 8-cell stage mouse embryo, and identified the timing regulation of this event.

The purpose of this research was to define the effects of various growth-promoting factors on in vitro embryonic development of in vitro matured and in vitro fertilized bovine embryos. The control medium was a chemically defined medium which improves the possibility of closely determining the in vivo conditions the embryo is actually exposed to. The growth-promoting factors tested in this experiment included transferrin, IGF-I (insulin-like growth factor-one), IGF-II (insulin-like growth factor-two), TGF-a (transforming growth factor-alpha) , TGF-B1 (transforming growth factor-beta1) , PDGF (platelet derived growth factor), EGF (epidermal growth factor), NGF (nerve growth factor), and bFGF (basic fibroblast growth factor). Transferrin was included at 10 micrograms/milliliter , while all other factors were utilized at 10 nanograms/milliliter in the control medium. Bovine cumulus-oocytes were retrieved from slaughterhouse ovaries and were matured i n Medium-199 containing 10% feta l bovine serum for 24 hours at 39°C in a 5% C02 atmosphere. Frozen-thawed bull spe r m were s wim-up separated and capacitated in medium containing heparin for 3 hours prior to insemination. Gametes were co- incubated fo r 18 hours and then cumulus cells were stripped from the ova. Ova which did not cleave were removed from culture 36 hours after insemi nati on and were stained for evidence of fertilization. Embryos were cultured in one of the 10 conditions (including control) described above. A total of 150 total oocy.t.es were cultured per treatment for a tota l of 10 days. EGF improved embryo development, while TGF-Bl and TGF-a only slightly improved embryo development compared to the control. All other factors tested did not have a beneficial effect on embryo development in this culture medium. In summary, EGF improved in vitro development of bovine embryos obtained from in vitro maturated and in vitro fertilized bovine oocytes. Other factors which were t est ed did not significantly improve in vitro bovine embryo development. Further experiments are necessary fo r determining the requirements of bovine embryos in vitro.