Chez les animaux, la conformation unique du noyau du spermatozoïde dont la chromatine est organisée avec des protéines chromosomiques spécifiques telles que les protamines le rend totalement inactif. Le remodelage de la chromatine paternelle à la fécondation par des activités d'origine maternelle sont donc des processus essentiels à la formation d'un embryon diploïde, dont les mécanismes restent très mal connus. Lors de ma thèse j'ai essayé de mieux comprendre ces processus par l'étude, chez la drosophile, d'un mutant létal embryonnaire à effet maternel : maternal haploid (mh). Ce mutant affecte l'incorporation des chromosomes paternels à la première division zygotique menant à la formation d'embryons haploïdes gynogénétiques. L'identification du gène de mh comme CG9203 m'ont permis de caractériser sa fonction. Dans les œufs mh, les chromosomes paternels se condensent anormalement et ne parviennent pas à se diviser correctement lors de la première mitose de l'embryon. Récemment, des études sur son orthologue humain, appelé Spartan/DVC1, ont montré qu'il était impliqué dans la synthèse translésionnelle (TLS), un mécanisme de tolérance aux dommages d'ADN. J'ai pu démontrer que dans les cellules somatiques, la fonction de Spartan dans le TLS est conservée chez la drosophile. Cependant, la fonction maternelle de MH ne relève pas du TLS canonique, mais permet de maintenir l'intégrité de l'ADN paternel avant la réplication. Ensemble, mes travaux soulignent la singularité du pronoyau mâle et la complexité que présente le maintien de son intégrité à la fécondation / In animals, sexual reproduction requires the union between two distinct parental gametes: the spermatozoon and the oocyte. The unique nuclear conformation of the sperm, in which the chromatin is organized with sperm-specific chromosomal protein like protamines, abolishes its activity. The paternal chromatin remodeling and the maintenance of its integrity at fertilization by maternal activities are therefore essential processes for zygote formation. However, although their mechanisms are crucial, they remain poorly understood. During my thesis, I tried to better understand the processes involved during de novo paternal chromatin assembly in Drosophila through the study of a maternal embryonic lethal mutation: maternal haploid (mh). The mutant affects the incorporation of paternal chromosomes during the first zygotic division, leading to the development of gynogenetic haploid embryos. The identification of the mh gene as CG9203, and the generation of the null allele mh2 allowed me to characterize its function. In eggs led by mh mutant females, paternal chromosomes abnormally condense and fail to divide leading to the formation of chromatin bridges at the first embryonic division. Recently, its human ortholog Spartan/DVC1, has been described to be involved in translesion synthesis (TLS), a DNA damage tolerance pathway that ensures replication fork progression. Combining genetic and cytological approaches, I demonstrated that the Spartan function in TLS is conserved in Drosophila. However, I discovered that the critical function of MH during the first embryonic division, was not consistent with a canonical TLS. Alternatively, it is specifically required to maintain paternal integrity and to allow its proper replication at the first cycle. The mh phenotype characterization, led me to compare it with others phenotypes induced by the knock-down of replication factors and to study parental chromosome condensation in the zygote. Surprisingly, one of the proteins allowing the establishment of the pre-replication complex is dispensable for the proper paternal chromosome segregation contrarily to the maternal counterpart. Altogether, these works highlight the difference that exists between the two parental pronuclei and the complexity of maintaining their integrity at fertilization

No description available.

No description available.

No description available.

Oocyte development in Xenopus laevis spans six morphologically distinct stages (stage I-VI), and is associated with a decrease in protein O-GlcNAc levels. As a first step in elucidating the role of O-GlcNAc in developing oocytes, initial efforts were focused on isolation and identification of fifteen modified proteins that decrease during oocyte development. Stage I oocytes due to their high amounts of these proteins, were used as starting material for purification. Multiple affinity and specific antibody based purification technique were initially used in an attempt to enrich the O-GlcNAc proteins. Due to the unique properties of the proteins ultimately identified, these techniques were unable to provide sufficient material for sequencing. However, differential centrifugation coupled with 2D-gel electrophoresis was highly successful. The majority of isolated proteins were strongly basic in nature with pIs 8-10. Coomassie stained bands from 2D-analysis were trypsin digested, and peptides were sequenced by mass spectroscopy (Finnigan LCQ). Mass data were interpreted by Bioworks software, and protein sequences were compared to multiple protein databases. Initially, six proteins were identified as Thesaurin a (42Sp50), cytoplasmic mRNA binding protein p54, y-box homolog, Xp 54 (ATP dependent RNA helicase p54), Vg1 RNA binding protein variant A, Zygote arrest 1(Zar1) and Poly (A) binding protein (PABP). Thesaurin a, the main component of 42S particle of previtellogenic oocytes (stages I-III) is involved in tRNA storage and possess low tRNA transfer activity; y-box factor homolog and Xp54 are present in oocyte mRNA storage ribonucleoprotein particles; Vg1 RBP variant A associates mVg1 RNA to microtubules in order to translocate to the vegetal cortex; Zar1 is involved in oocyte-to-embryo transition; and PABP initiates mRNA translation. This study is the first to characterize these oocyte specific proteins as O-GlcNAc modified proteins. Overall, the presence of several O-GlcNAc proteins in oocytes, the reduction in their levels/ O-GlcNAc levels, and the variation in maturation time in the presence of HBP-flux modulators in developing oocyte indicates O-GlcNAc may play important roles in metabolism, cell growth and cell division of X. laevis oocytes. Therefore, identifying the remainder of these proteins and elucidating the O-GlcNAc role in their function is a worthwhile pursuit.

In the general population, one in five couples experiences difficulty in conceiving a child. The role of obesity on women’s fecundity has become a focus of attention in recent years. Successful treatment of infertility through Assisted Reproductive Technology (ART) is also compromised by the presence of obesity, which occurs in 30 % of women seeking treatment. A negative correlation exists between increased body mass index (BMI) and the number of collected oocytes and a lower birth rate after ART. Furthermore, a number of studies have established that weight loss improves natural conception rates in overweight women. Whether weight management can improve success rates in overweight / obese women undergoing in vitro fertilisation (IVF) has not been studied. The purpose of this project was to explore the role of short–term weight loss on potential pregnancy outcomes in overweight / obese women undergoing IVF programme. However, to establish this relationship, we proposed to carry out two studies to assess the following: (I) The feasibility of very low calorie diet (VLCD) during IVF treatment with respect to duration, level of restriction and tolerability of the diet during hormonal down regulation in women (Chapter 2). (II) How energy restriction may affect the quality of an early embryo in diet - induced obese mice with respect to various body weight and caloric intake (Chapter 3). In study (I), women preferred a shorter dietary intervention with greater energy restriction (456 kcal per day) to gradual energy restriction (1200 kcal / day for the first week, and afterward, 456 kcal / day) prior to oocyte transfer. Women were able to comply with the VLCD during IVF treatment and both dietary groups achieved a significant weight loss (mean 6.3 %). In study (II), by using obese mice, the effect of rapid weight loss (mean 12 %) was observed after 5 days of energy restriction. Ovulation rate was greater in the Obese group (HFD) (55.6%) and equal in both Control (CD) and Energy Restricted (HF / ER) (44.4 %) groups. The HF / ER group showed higher fertilisation rate (80 %) than HFD and CD (55% and 45.5%, correspondingly). The blastocyst stage was reached by half of the cultured embryos in both HF / ER and HFD groups and 33 % in the CD group. The quality of embryos that completed blastocyst formation did not differ between groups. However, postfertilisation development in females fed a high fat diet was slower compared to CD and HF / ER groups. In conclusion, this work illustrated a weight management prior conception and use of VLCD during IVF treatment in clinical study needs further investigation with regard to the dietary duration, level of energy restriction and how this combination will influence IVF treatment outcomes. Furthermore, as we were unable to determine the question of how the dietary intervention affects the quality of oocytes and the animal study illustrated a promising result, thus further studies are required. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311579 / Thesis (M.Med.Sc.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

In the general population, one in five couples experiences difficulty in conceiving a child. The role of obesity on women’s fecundity has become a focus of attention in recent years. Successful treatment of infertility through Assisted Reproductive Technology (ART) is also compromised by the presence of obesity, which occurs in 30 % of women seeking treatment. A negative correlation exists between increased body mass index (BMI) and the number of collected oocytes and a lower birth rate after ART. Furthermore, a number of studies have established that weight loss improves natural conception rates in overweight women. Whether weight management can improve success rates in overweight / obese women undergoing in vitro fertilisation (IVF) has not been studied. The purpose of this project was to explore the role of short–term weight loss on potential pregnancy outcomes in overweight / obese women undergoing IVF programme. However, to establish this relationship, we proposed to carry out two studies to assess the following: (I) The feasibility of very low calorie diet (VLCD) during IVF treatment with respect to duration, level of restriction and tolerability of the diet during hormonal down regulation in women (Chapter 2). (II) How energy restriction may affect the quality of an early embryo in diet - induced obese mice with respect to various body weight and caloric intake (Chapter 3). In study (I), women preferred a shorter dietary intervention with greater energy restriction (456 kcal per day) to gradual energy restriction (1200 kcal / day for the first week, and afterward, 456 kcal / day) prior to oocyte transfer. Women were able to comply with the VLCD during IVF treatment and both dietary groups achieved a significant weight loss (mean 6.3 %). In study (II), by using obese mice, the effect of rapid weight loss (mean 12 %) was observed after 5 days of energy restriction. Ovulation rate was greater in the Obese group (HFD) (55.6%) and equal in both Control (CD) and Energy Restricted (HF / ER) (44.4 %) groups. The HF / ER group showed higher fertilisation rate (80 %) than HFD and CD (55% and 45.5%, correspondingly). The blastocyst stage was reached by half of the cultured embryos in both HF / ER and HFD groups and 33 % in the CD group. The quality of embryos that completed blastocyst formation did not differ between groups. However, postfertilisation development in females fed a high fat diet was slower compared to CD and HF / ER groups. In conclusion, this work illustrated a weight management prior conception and use of VLCD during IVF treatment in clinical study needs further investigation with regard to the dietary duration, level of energy restriction and how this combination will influence IVF treatment outcomes. Furthermore, as we were unable to determine the question of how the dietary intervention affects the quality of oocytes and the animal study illustrated a promising result, thus further studies are required. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311579 / Thesis (M.Med.Sc.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

In the general population, one in five couples experiences difficulty in conceiving a child. The role of obesity on women’s fecundity has become a focus of attention in recent years. Successful treatment of infertility through Assisted Reproductive Technology (ART) is also compromised by the presence of obesity, which occurs in 30 % of women seeking treatment. A negative correlation exists between increased body mass index (BMI) and the number of collected oocytes and a lower birth rate after ART. Furthermore, a number of studies have established that weight loss improves natural conception rates in overweight women. Whether weight management can improve success rates in overweight / obese women undergoing in vitro fertilisation (IVF) has not been studied. The purpose of this project was to explore the role of short–term weight loss on potential pregnancy outcomes in overweight / obese women undergoing IVF programme. However, to establish this relationship, we proposed to carry out two studies to assess the following: (I) The feasibility of very low calorie diet (VLCD) during IVF treatment with respect to duration, level of restriction and tolerability of the diet during hormonal down regulation in women (Chapter 2). (II) How energy restriction may affect the quality of an early embryo in diet - induced obese mice with respect to various body weight and caloric intake (Chapter 3). In study (I), women preferred a shorter dietary intervention with greater energy restriction (456 kcal per day) to gradual energy restriction (1200 kcal / day for the first week, and afterward, 456 kcal / day) prior to oocyte transfer. Women were able to comply with the VLCD during IVF treatment and both dietary groups achieved a significant weight loss (mean 6.3 %). In study (II), by using obese mice, the effect of rapid weight loss (mean 12 %) was observed after 5 days of energy restriction. Ovulation rate was greater in the Obese group (HFD) (55.6%) and equal in both Control (CD) and Energy Restricted (HF / ER) (44.4 %) groups. The HF / ER group showed higher fertilisation rate (80 %) than HFD and CD (55% and 45.5%, correspondingly). The blastocyst stage was reached by half of the cultured embryos in both HF / ER and HFD groups and 33 % in the CD group. The quality of embryos that completed blastocyst formation did not differ between groups. However, postfertilisation development in females fed a high fat diet was slower compared to CD and HF / ER groups. In conclusion, this work illustrated a weight management prior conception and use of VLCD during IVF treatment in clinical study needs further investigation with regard to the dietary duration, level of energy restriction and how this combination will influence IVF treatment outcomes. Furthermore, as we were unable to determine the question of how the dietary intervention affects the quality of oocytes and the animal study illustrated a promising result, thus further studies are required. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311579 / Thesis (M.Med.Sc.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

No description available.

Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.