The laws for the inheritance of animal mitochondrial DNA differ from those revealed for nuclear DNA. In contrast to nuclear genes, animal mitochondrial DNA (mtDNA) is predominantly inherited through the maternal line and is typically assumed to be nonrecombining. The absence of both paternal transmission (hereafter: paternal leakage) and heterologous recombination of mtDNA are assumed to be key characteristics of mitochondrial DNA inheritance, which has enabled evolutionary models to be much simpler than those needed for the interpretation of nuclear DNA. However, recent revelations of paternal leakage in the animal kingdom challenge our current knowledge about mtDNA inheritance and the utility of mtDNA as a molecular marker. The occurrence of paternal leakage potentially introduces new haplotypes into populations and therefore impacts on the interpretation of mtDNA analysis. To date, it is unclear whether the documented cases of paternal leakage are exceptions to the general rule or if these events occur more frequently than so far believed. If this event occurred at a measurable frequency, it is vital to implement such data into models of mtDNA evolution to improve the accuracy at which evolutionary relationships and times of divergence are estimated. In this thesis, I aimed to provide an insight into the broader patterns of mtDNA inheritance using chinook salmon as a model organism. I first sought to delimit the frequency of paternal leakage in chinook salmon and further investigated two major mechanisms which are believed to limit paternal leakage: The many-fold dilution of paternal mtDNA by maternal mtDNA upon fertilization and the genetic bottleneck mtDNA is believed to be exposed to during early developmental stages. A screen of roughly 10.000 offspring did not reveal the presence of paternal mtDNA within these samples delimiting the maximum frequency of paternal leakage in this system to 0.18% (power of 0.95) and 0.27% (power of 0.99), suggesting that the occurrence of paternal leakage is most likely an exception to the general rule. To infer the dilution of paternal mtDNA upon fertilization, I employed real-time PCR and determined the mtDNA content of salmon spermatozoa and oocytes to be 5.73 ± 2.28 and 3.15x109 ± 9.98x108 molecules per gamete, respectively. Accordingly, the estimated ratio of paternal to maternal mtDNA in zygotes is 1:7.35x108 ± 4.67x108. This estimate is 3 to 5 orders of magnitude smaller than the ratio revealed for mammals. Consequently, and if the dilution acts as an efficient barrier against the transmission of paternal mtDNA, paternal inheritance of mtDNA per offspring will be much less likely in this system than in mammals. To estimate at what probability the diminutive contribution of paternal mtDNA in zygotes is potentially inherited to offspring, I determined the size of the bottleneck acting on mtDNA during both embryogenesis and oogensis by examining the transmission of mtDNA variants to offspring and oocytes within a pedigree of heteroplasmic individuals. The number of segregating units (mtDNAs) between a mother’s somatic tissue and oocytes was estimated to be 109.3 (median = 109.3; 62.4 < NeOog < 189.6; 95% confidence interval) and from a mother’s soma to offspring’s soma 105.4 (median = 105.4; 70.3 < NeEmb < 153.1; 95% confidence interval). Detected variances in allele frequency among oocytes were not significantly different from those in offspring, strongly suggesting that segregation of mtDNA occurs during oogenesis with its completion before oocyte maturation. However, considering a ratio of roughly 1:7.35x108 for paternal to maternal mtDNA in zygotes and that approximately 109.3 (NeOog) of the mitochondrial genomes present in zygotes are ultimately inherited to offspring, the probability for paternal mtDNA to be transmitted to offspring is in round terms 1.0x10-11/paternal mtDNA molecule. In summary, the results presented in this thesis document the presence of efficient barriers to prohibit the inheritance of minor allele contributions, such as paternal mtDNA, to offspring. These results strongly suggest that paternal leakage is an exception to the general rule. Furthermore, in comparison to studies undertaken in mammals, my results indicate that mechanisms in place to prevent paternal leakage may be unequally efficient among different animal taxa, reflecting differences in life traits, such as gamete morphology, gamete investment and reproductive strategies. Nonetheless, by the means of the dilution effect in zygotes and the genetic bottleneck during oogenesis, the occurrence of paternal leakage might be simply a quantitative phenomenon and cannot be excluded per se. The increasing number of documented cases of paternal leakage clarifies that its occurrence must be considered when applying mtDNA as a genetic marker. Furthermore, for species in which mtDNA inheritance can be confirmed to be purely random, theoretical frequencies of paternal leakage can be inferred and potentially implemented into models of mtDNA evolution.

The laws for the inheritance of animal mitochondrial DNA differ from those revealed for nuclear DNA. In contrast to nuclear genes, animal mitochondrial DNA (mtDNA) is predominantly inherited through the maternal line and is typically assumed to be nonrecombining. The absence of both paternal transmission (hereafter: paternal leakage) and heterologous recombination of mtDNA are assumed to be key characteristics of mitochondrial DNA inheritance, which has enabled evolutionary models to be much simpler than those needed for the interpretation of nuclear DNA. However, recent revelations of paternal leakage in the animal kingdom challenge our current knowledge about mtDNA inheritance and the utility of mtDNA as a molecular marker. The occurrence of paternal leakage potentially introduces new haplotypes into populations and therefore impacts on the interpretation of mtDNA analysis. To date, it is unclear whether the documented cases of paternal leakage are exceptions to the general rule or if these events occur more frequently than so far believed. If this event occurred at a measurable frequency, it is vital to implement such data into models of mtDNA evolution to improve the accuracy at which evolutionary relationships and times of divergence are estimated. In this thesis, I aimed to provide an insight into the broader patterns of mtDNA inheritance using chinook salmon as a model organism. I first sought to delimit the frequency of paternal leakage in chinook salmon and further investigated two major mechanisms which are believed to limit paternal leakage: The many-fold dilution of paternal mtDNA by maternal mtDNA upon fertilization and the genetic bottleneck mtDNA is believed to be exposed to during early developmental stages. A screen of roughly 10.000 offspring did not reveal the presence of paternal mtDNA within these samples delimiting the maximum frequency of paternal leakage in this system to 0.18% (power of 0.95) and 0.27% (power of 0.99), suggesting that the occurrence of paternal leakage is most likely an exception to the general rule. To infer the dilution of paternal mtDNA upon fertilization, I employed real-time PCR and determined the mtDNA content of salmon spermatozoa and oocytes to be 5.73 ± 2.28 and 3.15x109 ± 9.98x108 molecules per gamete, respectively. Accordingly, the estimated ratio of paternal to maternal mtDNA in zygotes is 1:7.35x108 ± 4.67x108. This estimate is 3 to 5 orders of magnitude smaller than the ratio revealed for mammals. Consequently, and if the dilution acts as an efficient barrier against the transmission of paternal mtDNA, paternal inheritance of mtDNA per offspring will be much less likely in this system than in mammals. To estimate at what probability the diminutive contribution of paternal mtDNA in zygotes is potentially inherited to offspring, I determined the size of the bottleneck acting on mtDNA during both embryogenesis and oogensis by examining the transmission of mtDNA variants to offspring and oocytes within a pedigree of heteroplasmic individuals. The number of segregating units (mtDNAs) between a mother’s somatic tissue and oocytes was estimated to be 109.3 (median = 109.3; 62.4 < NeOog < 189.6; 95% confidence interval) and from a mother’s soma to offspring’s soma 105.4 (median = 105.4; 70.3 < NeEmb < 153.1; 95% confidence interval). Detected variances in allele frequency among oocytes were not significantly different from those in offspring, strongly suggesting that segregation of mtDNA occurs during oogenesis with its completion before oocyte maturation. However, considering a ratio of roughly 1:7.35x108 for paternal to maternal mtDNA in zygotes and that approximately 109.3 (NeOog) of the mitochondrial genomes present in zygotes are ultimately inherited to offspring, the probability for paternal mtDNA to be transmitted to offspring is in round terms 1.0x10-11/paternal mtDNA molecule. In summary, the results presented in this thesis document the presence of efficient barriers to prohibit the inheritance of minor allele contributions, such as paternal mtDNA, to offspring. These results strongly suggest that paternal leakage is an exception to the general rule. Furthermore, in comparison to studies undertaken in mammals, my results indicate that mechanisms in place to prevent paternal leakage may be unequally efficient among different animal taxa, reflecting differences in life traits, such as gamete morphology, gamete investment and reproductive strategies. Nonetheless, by the means of the dilution effect in zygotes and the genetic bottleneck during oogenesis, the occurrence of paternal leakage might be simply a quantitative phenomenon and cannot be excluded per se. The increasing number of documented cases of paternal leakage clarifies that its occurrence must be considered when applying mtDNA as a genetic marker. Furthermore, for species in which mtDNA inheritance can be confirmed to be purely random, theoretical frequencies of paternal leakage can be inferred and potentially implemented into models of mtDNA evolution.

A questionnaire based survey was done to assess the views of fertile individuals, infertile individuals, egg donors and recipients towards gamete donation. The survey showed that fertile individuals were significantly less inclined towards the use of donated eggs in research and treatment, compared to infertile individuals. Acceptability of gamete donation was found to be very high in all groups regardless of their fertility, but the majority of individuals, whether fertile or infertile, were opposed to the use of fetal and cadaveric sources of obtaining eggs. The effect of modifications of the freeze-thaw process was investigated in the mouse model. It was seen that slight modifications of the slow freeze protocol affected survival rates and that ultrarapid freezing achieved better survival rates than slow freezing. Human oocyte cryopreservation was performed using a slow freeze-rapid thaw protocol. In total, 34.4% of oocytes survived cryopreservation and these were randomly allocated for fertilisation by conventional <I>in vitro</I> fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Resulting embryos were spread for chromosomal analysis. ICSI significantly improved the rates of normal fertilisation (43.2% versus 2.7%) compared to IVF (P<0.001). A normal diploid karyotype was achieved by ICSI. These studies show that oocyte donation is acceptable to the majority of both fertile and infertile individuals. Further research is required to improve the methods of oocyte cryopreservation. Once the techniques of cryopreservation have been established, ICSI may successfully be applied to enhance subsequent fertilisation rates.

Openness about sperm and egg donation and the regulation of donor anonymity or non-anonymity are new phenomena. How do affected families, clinics, and regulators deal with information about gamete donors and the donation itself? And how does this knowledge management contribute to the creation and enactment of kinship? Addressing these questions in Germany and Britain, this ethnography makes a comparative contribution to the empirical and theoretical analysis of kin-formation and social change. Maren Klotz reveals a contemporary renegotiation of the values of privacy, information-sharing, and connectedness as they relate to the social, clinical, and regulatory management of kinship information. Transparency, not genetics, is the moral imperative, and instead of an unambiguously discernible “geneticization,” her findings on donor non-anonymity and parental openness display a pattern of “transparentization.” This pattern represents a shift in authority over kinship away from the sometimes highhanded reproductive medical profession towards concerned groups, parents-by-donation, and policymakers.

The mechanism of gamete fusion remains largely unknown on molecular level despite its indisputable significance. A few molecules requisite for membrane interaction are known, among them IZUMO1 on sperm and tetraspanin CD9 on egg. A concept of a large multitprotein complex on both membranes forming a fusion machinery is recently emerging. CD9 is expected to play a role in organizing egg membrane order and to interact laterally with other factors. On the other hand, IZUMO1 contains one immunoglobulin (Ig) domain, which is known for its interaction with various molecules and thus is expected to have a binding partner on the egg membrane. Ig domains are known for their involvement in interactions with Fc receptors and Fc receptor-like molecules, which makes these a potential partner for sperm in gamete binding and fusion. Recent experiments identified Fc receptor-like 3 as a candidate binding partner for sperm by one-bead one-compound assay. The aim of presented study was to assess the localization of FCRL3 in mouse eggs, together with its potential co-localization with IZUMO1 on complexes of zona-free eggs with bound sperm. In this study, FCRL3 molecule has been found across the cytoplasm, possibly in the intracytoplasmic membrane compartments of the cell, as well as on the egg membrane. Its presence...

With effect from 1st April 2005, UK law was amended such that gamete donors must now consent to their identity being released to their donor offspring, should they request it after the age of eighteen. This qualitative study investigates the views and experiences of those donating in this new context. Drawing primarily on twenty-four in-depth interviews with donors, supplemented by twenty staff interviews and observation in two fertility clinics, I examine how donors make sense of their role in relation to offspring, recipients and the wider community. I argue that donors make sense of their role as “biological” parents to offspring through creative reference to kinship repertoires, drawing on their own experiences of “doing family.” However, crucially, kinship connections are always qualified in some way to show that they are not quite family to donor offspring, and certainly not their “real” parent. Often this discursive work involved emphasising their relationship to recipients or the wider community (rather than offspring), framing the donation as a gift or a public act. In addition, donors drew on their kinship expertise to dilute, reshape or “re-route” their connection to offspring. Ultimately, this is a thesis about the limiting work involved in “doing kinship.” I demonstrate that donors did this limiting work in highly creative ways, not restricted to forgetting or ignoring connections. Instead, I show that not constructing kinship claims can be as active a process as making them.

The Testis Specific Serine Kinase (Tssk) family of proteins is a large group of kinases that present high level of conservation within paralogs, as well as within species. In addition, in all reported cases as well as in the analysis of expressed sequence tags available in databases, this family of proteins presents a very strict pattern of either testicular or male-gonadal expression. This high level of conservation prompted the postulate that these kinases ought to be important for either testicular function or fertilization. In this work we attempt a biochemical characterization of one family member (Tssk6) in the mouse. We also analyze the male infertility phenotype presented by mice null for Tssk6 revealing its requirement for actin dynamics and the relocalization of proteins necessary for gamete fusion. In this analysis we described Tssk6 as the second protein known to date to be necessary in the sperm for gamete fusion to take place. We also examined a novel member of the Tssk family in the mouse as well as ortholog proteins in two invertebrates (C. elegans and D. melanogaster). Although our understanding of the function, activity and regulation of these kinases remains small, this work constitutes a significant advance towards the understanding of the identity of the Tssk family. The results that follow have far reaching effects that surpass the realm of the Tssk family. They influence the study of sperm biological processes like the changes in sperm cytoskeletal structures and the acrosome reaction. They also influence the field of developmental biology and scientist working in the molecular characterization of the process of gamete fusion and zygote formation. Lastly, the work here presented influences as well evolutionary developmental biology through the study of a highly conserved family of proteins that is essential for reproduction and could play a role in the process of speciation.

<p>Syftet med studien var att beskriva vilka attityder som finns bland recipienter och donatorer angående de riktlinjer som bör efterföljas för ägg- och spermiedonation. Syftet var även att undersöka om det finns skillnader i attityder inom grupperna recipienter kvinnor respektive män och donatorer till huruvida barnet har rätt att få kunskap om sitt ursprung.</p><p>Studien blev en del av ett stort nationellt projekt. Data samlades in med enkäter som berörde de tidigare beskrivna områdena. Enkäterna var utformade med påståenden att ta ställning till som ”instämmer” eller ”instämmer inte”. Antal deltagare var 333 stycken och data samlades in från april 2005 till och med våren 2008.</p><p>Huvudresultatet som framkom i studien var att för donatorernas attityder gällande barnets rätt att få kunskap om sitt ursprung så visade det sig att kvinnorna var i flera påståenden i signifikant högre grad mer positiva än männen. I resultatet angående attityder till riktlinjer som bör efterföljas för könscellsdonation så hade recipienter och donatorer snarlika inställningar, om än inte fullt ut. Gällande för recipienter så var det endast kvinnorna som hade en signifikant skillnad i ett påstående, i övrigt fanns det inga skillnader för de båda grupperna i attityderna till barnets rätt att få kunskap om sitt ursprung.</p> / <p>The purpose of the study was to describe what attitudes there were among recipients and donors about the guiding principles that should comply with oocyte- and sperm donation. The purpose was also to investigate if there were any differences within the groups of recipient women and men and the donor group considering disclosure to the child about their origin.</p><p>The study became a part of a national project. Data were collected with questionnaire that included earlier described aspects. It was formed with statements to take stands towards in form of “agree” or “disagree”. Number of participants were 333 and data were collected from April 2005 until spring 2008.</p><p>The main results found that among donors and the attitudes toward the child’s right to get knowledge about their origin showed that women was significantly more positive in several aspects then the men. The results also showed that considering the guiding principles that should comply with gametes donation the recipients and donors had just about similar attitudes. For the recipients there was only the women who had a significant difference in one of the statements, otherwise there was no difference in either of both groups for the attitudes about disclosure to the child.</p>

Syftet med studien var att beskriva vilka attityder som finns bland recipienter och donatorer angående de riktlinjer som bör efterföljas för ägg- och spermiedonation. Syftet var även att undersöka om det finns skillnader i attityder inom grupperna recipienter kvinnor respektive män och donatorer till huruvida barnet har rätt att få kunskap om sitt ursprung. Studien blev en del av ett stort nationellt projekt. Data samlades in med enkäter som berörde de tidigare beskrivna områdena. Enkäterna var utformade med påståenden att ta ställning till som ”instämmer” eller ”instämmer inte”. Antal deltagare var 333 stycken och data samlades in från april 2005 till och med våren 2008. Huvudresultatet som framkom i studien var att för donatorernas attityder gällande barnets rätt att få kunskap om sitt ursprung så visade det sig att kvinnorna var i flera påståenden i signifikant högre grad mer positiva än männen. I resultatet angående attityder till riktlinjer som bör efterföljas för könscellsdonation så hade recipienter och donatorer snarlika inställningar, om än inte fullt ut. Gällande för recipienter så var det endast kvinnorna som hade en signifikant skillnad i ett påstående, i övrigt fanns det inga skillnader för de båda grupperna i attityderna till barnets rätt att få kunskap om sitt ursprung. / The purpose of the study was to describe what attitudes there were among recipients and donors about the guiding principles that should comply with oocyte- and sperm donation. The purpose was also to investigate if there were any differences within the groups of recipient women and men and the donor group considering disclosure to the child about their origin. The study became a part of a national project. Data were collected with questionnaire that included earlier described aspects. It was formed with statements to take stands towards in form of “agree” or “disagree”. Number of participants were 333 and data were collected from April 2005 until spring 2008. The main results found that among donors and the attitudes toward the child’s right to get knowledge about their origin showed that women was significantly more positive in several aspects then the men. The results also showed that considering the guiding principles that should comply with gametes donation the recipients and donors had just about similar attitudes. For the recipients there was only the women who had a significant difference in one of the statements, otherwise there was no difference in either of both groups for the attitudes about disclosure to the child.

Les gènes impliquées dans la reproduction évoluent rapidement et sont très souvent soumis à sélection positive. L’objectif de ma thèse a été d’étudier l’évolution de certains de ces gènes, potentiellement impliqués dans le phénomène de spéciation via leur implication dans les barrières prézygotiques.Nos résultats montrent que pour le gène SAL1, impliqué dans la reconnaissance phéromonale chez le porc, trois acides aminés sous sélection positive participent à la liaison de la phéromone spécifique porcine. Nous avons également réalisé l’analyse évolutive des gènes prouvés expérimentalement comme ayant un rôle dans l’interaction spermatozoïde-ovocyte lors de la fécondation. Chacune des dix neuf espèces de vertébrés étudiées présente un profil évolutif particulier pour ces gènes, caractérisé par le gain et la perte de gènes, ainsi que la position des acides aminés sous sélection positive. L’évolution divergente de l’ensemble de ces gènes pourrait être impliquée dans la spéciation ou au moins dans le renforcement des barrières d’espèces.Enfin, le serveur web PhyleasProg a été conçu au cours de la thèse. Cet outil permet désormais aux scientifiques, peu aguerris aux méthodes d’analyses phylogénétiques, d’acquérir simplement et rapidement un grand nombre de résultats sur l’histoire évolutive de leurs gènes d’intérêts. / Genes involved in reproduction evolve rapidly and are often under positive selection. The objective of this work was to study the evolution of some of these genes, potentially involved in speciation, through their involvement in prezygotic barriers.Our results show that for the SAL1 gene, involved in pheromonal recognition in pig, three amino acids under positive selection participate in the specific binding of the pig pheromone. We also perform an evolutionary analysis of genes experimentally shown to be involved in the sperm-oocyte interaction during fertilization. Each of the nineteen species studied exhibit a particular pattern of evolution, characterized by gene gains and losses, as well as the position of amino acids under positive selection. The divergent evolution of all these genes could be involved in speciation or at least in the reinforcement of species barriers.Finally, the PhyleasProg web server was designed during the thesis. This tool permits to scientists with no experience in phylogenetic analyses to acquire a large number of results quickly and easily on the evolutionary history of their genes of interest.