The allocation of resources to male or female progeny, or to male or female reproductive function more generally, is one of the most important life history decisions a sexually reproducing individual must ever make. Sex determination is thus a fundamental process, yet the mechanisms which control it are surprisingly diverse. In this thesis, I examine sex determination in the plant species Mercurialis annua L. (Euphorbiaceae). I assess the mechanism of sex determination operating in dioecious and androdioecious populations of M. annua and also investigate the conservation and evolution of sex-determining mechanisms across the annual mercury clade, the lineages of which display exceptional variation in sexual system. First, using crosses, I establish that sex in dioecious M. annua is controlled by a single-locus genetic mechanism, consistent with recent work that identified a single male-linked DNA marker in the species. My search for new sex-linked genes revealed none, however, suggesting that M. annua possesses at most a small non-recombining region around sex-determining loci. Why many dioecious plants lack heteromorphic sex chromosomes is still poorly understood and I consider explanations for this. I extend my investigation by comparing genetic diversity between loci that differ in their linkage to the sex-determining locus. I find a single male-linked marker to possess significantly lower diversity than autosomal loci, but no difference in the diversity of partially sex-linked and non-sex-linked genes. I also assess the conservation of a sex-linked marker among annual mercury lineages and conduct crosses between lineages to examine the conservation of sex determination. My findings indicate a conserved mechanism of single-locus genetic sex determination and I consider the role polyploidisation and hybridisation have played in sexual system evolution and the modification of sex-determining mechanisms in the clade. Finally, I assess the presence of environmental sex determination in androdioecious M. annua, concluding that although male frequency is not influenced by growing density, a degree of sexual lability exists in the lineage.

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No description available.

Gaining a good understanding of marine turtle mating systems is fundamental for their effective conservation, yet there are distinct gaps in our knowledge of their breeding ecology and life history, owing largely to the difficulty in observing these highly mobile animals at sea. Whilst multiple mating by females, or polyandry, has been documented in all marine turtle species, the fitness consequences of this behaviour have not been fully investigated. Furthermore, male mating patterns, operational sex ratios and the number of males contributing to breeding populations are poorly understood, impeding accurate assessments of population viability. In this thesis, I use molecular-based parentage analysis to study, in detail, the genetic mating system of two green turtle (Chelonia mydas) populations. In the focal population in northern Cyprus, I show that, despite exhibiting a strongly female-biased hatchling sex ratio and contrary to our expectations, there are at least 1.3 breeding males to every nesting female. I go on to assess the breeding frequency of male turtles in the population and determine that males do not breed annually at this site, demonstrating that the observed relatively equal sex ratio of breeders is not the result of a few males mating every year, but that the number of breeding males in the population is greater than expected. I show that 24% of nesting females in the population produce clutches with multiple paternity, but do not detect any fitness benefits to polyandrous females, and discuss the potential role of sexual conflict in influencing female mating decisions. Finally, I reveal a high frequency of multiple paternity in green turtle clutches on Ascension Island, one of the largest green turtle rookeries in the world, and discuss possible causes of variation in the level of polyandry among marine turtle populations. The results presented here shed new light on aspects of marine turtle mating systems that are challenging to study, and illustrate the value of molecular data, not only in describing mating patterns, but in elucidating aspects of life history and behaviour that would otherwise be very difficult to ascertain.

All-male fry are preferred to prevent uncontrolled reproduction before harvest in intensive Nile tilapia (Oreochromis niloticus) aquaculture. Males also grow faster than females. An alternative approach to direct hormonal masculinisation of tilapia fry is to produce fry that are genetically male. However, sex determination system in tilapia is fairly complex. Recent developments have resulted in a linkage map and genetic markers that can be used to analyse the sex determination system. To analyse the genetic sex determination mechanism and to develop marker-assisted selection in the Stirling Nile tilapia population, a fully inbred line of clonal females (XX) was verified using test crosses and DNA markers (mostly microsatellites) to use as a standard reference line in sex determination studies. A series of crosses were performed involving this line of females and a range of males. Three groups of crosses were selected (each group consisted of three families) from progeny sex ratio distributions, and designated as type ‘A’ (normal XY males x clonal XX females), type ‘B’ (putative YY males x clonal XX females) and type ‘C’ (unknown groups of males x clonal XX females), for sex linkage study. For type ‘A’, inheritance of DNA markers and phenotypic sex was investigated using screened markers from tilapia linkage group 1 (LG1) to confirm the LG1-associated pattern of inheritance of phenotypic sex and the structure of LG1. Screened markers from LG1, LG3 and LG23 were used to investigate the association of markers with sex in families of type ‘B’ and ‘C’. In addition, a genome-wide scan of markers from the other 21 LGs was performed to investigate any association between markers and sex, in only families of cross type ‘B’. LG1 associated pattern of inheritance of phenotypic sex was confirmed by genotype and QTL analyses in families of cross type ‘A’. Analyses of genotypes in families of type ‘B’ and ‘C’ showed strong association with LG1 markers but no association with LG3 and LG23 markers. Genome wide scan of markers from all other LGs did not show any significant association between any markers and the sex. The allelic inheritance of two tightly linked LG1 markers (UNH995 and UNH104) in families of type ‘B’ and ‘C’ identified polymorphism in the sex determining locus: one of the alleles was associated mostly with male offspring whereas another allele was associated with both progeny (mostly males in type ‘B’ families, and approximately equal numbers in type ‘C’ families). This knowledge was used to identify and separate supermales (‘YY’ males) that should sire higher proportions of male progeny, reared to become sexually mature for use as broodstock. Two of them were crossed with XX females (one clonal and one outbred) to observe the phenotypic expression of the strongest male-associated allele in progeny sex. The observations of 98% male (99 males out of 101 progeny) and 100% male (N=75) from these two crosses respectively, suggest that a marker-assisted selection (MAS) programme for genetically male Nile tilapia production could be practical. This study also suggests that the departures from the sex ratios predicted using a “simple” XX/XY model (i.e., YY x XX should give all-male progeny) were strongly associated with the XX/XY system, due to multiple alleles, rather than being associated with loci in other LGs (e.g., LG3, LG23). This study also tentatively names the allele(s) giving intermediate sex ratios as “ambivalent” and emphasizes that the presence and actions of such allele(s) at the same sex-determining locus could explain departures from predicted sex ratios observed in some earlier studies in Nile tilapia.

Keimzellen entwickeln sich aus den sogenannten Urkeimzellen, die auch als primordiale Keimzellen (PGC) bezeichnet werden. PGC entwickeln sich während der Embryonalentwicklung und wandern dann in die Gonadenanlagen, wo sie sich vermehren und bei getrenntgeschlechtlichen Arten in Spermien in Männchen und Oozyten bei Weibchen differenzieren. Während der Geschlechtsdifferenzierung sind die Gonaden anfällig für den Einfluss externer Faktoren, wie z. B. der Wassertemperatur. Eine Erhöhung der Wassertemperatur von 28°C auf 36°C während der kritischen Phase der Geschlechtsdifferenzierung, vom 10. bis zum 20. Tag nach der Befruchtung kann zu einer Vermännlichung genetisch-weiblicher Tilapien führen. In der ersten Studie führte eine entsprechende Temperaturbehandlung bei genetisch weiblichen Tilapien zu einem Anteil funktioneller Männchen von 37%. Makromorphologisch konnten die Gonaden 90 Tage alter Weibchen aus Kontroll- und Behandlungsgruppen als unreife Ovarien klassifiziert werden. Erste Reifungsprozesse bis hin zur Oogenese begannen 120 Tage nach der Befruchtung. Die Oogenese konnte mikro-morphologisch weiterhin in folgende Stadien eingeteilt werden: Chromatin Nukleolus, Peri-Nukleolus, kortikale Alveolus, Vitellogenese und reife Eizelle. Oozyten in den Phasen des Chromatin Nukleolus und des Peri-Nukleolus, welche auch die primäre Wachstumsphase darstellen, wurden bei weiblichen Fischen aller Altersgruppen festgestellt. Während weiter fortgeschrittenen Oozytenstadien erst ab einem Alter von 120 Tagen festgestellt werden konnten. Oozyten in der Phase des kortikalen Alveolus wurden demnach frühestens am 120. Lebenstag gefunden. In der Vitellogenese befindliche Oozyten traten erst nach 150 Tagen auf. Reife Eizellen wurden ab einem Alter von 180 dpf festgestellt. Es konnten keine signifikanten Unterschiede in der Eizellentwicklungsstadien zwischen Kontroll- und Behandlungstieren festgestellt werden. Das Ziel der zweiten Studie war die Identifikation von Keimbahn-Stammzellen bei Tilapien, die während der Geschlechtsdifferenzierung verschiedenen Aufzuchttemperaturen ausgesetzt waren. Die Identifizierung der Keimbahn-Stammzellen erfolgte anhand von Immunohistochemie mittels Vasa und PCNA (Proliferierendes Cell Nuclear Antigen) Antikörperfärbung. Es wurden zunächst histologische Schnitte von in Paraffin eingebettetem Ovargewebe hergestellt. Keimbahn-Stammzellen wurden im Keimepithel der Ovarien identifiziert, wobei diese in einzelner, isolierter oder in Clusterform vorlagen. Keimbahn-Stammzellen wurden sowohl bei weiblichen Tieren aus Kontroll- als auch Behandlungsgruppen identifiziert. Zusammenfassend, konnten erstmalig Keimbahn-Stammzellen und ihre Lage in den Gonaden genetisch weiblicher Tilapien, aus Kontroll- und Behandlungsgruppen, mittels Vasa und PCNA Antikörperfärbung charakterisiert werden.

Le palmier dattier (Phoenix dactylifera L.) est une espèce fruitière dioïque tropicale qui revêt une importance capitale sur le plan alimentaire, socio-économique et écologique pour les régions arides du globe. Malgré l'intérêt de disposer d'un outil moléculaire pour discriminer les plante s mâles et femelles pour les programmes d'amélioration génétique, aucun marqueur spécifique du sexe n'a été identifié et validé à ce jour. Afin de pouvoir étudier et comprendre le déterminisme sexuel du palmier dattier, nous avons entrepris la description et la caractérisation des processus cellulaires et moléculaires associés à la différenciation des organes sexuels. L'étude histologique du développement reproducteur a montré que le bourgeon floral est d'apparence bisexuelle jusqu'à l'initiation des primordia de l'androcée et du gynécée. Le premier dimorphisme sexuel observé à ce stade correspondant à un gynécée plus large dans les fleurs femelles résulterait d'une activité mitotique plus importante dans les cellules du gynécée fertile par rapport à son équivalent non fonctionnel. Les organes sexuels stériles, staminodes et pistillodes, cessent ensuite leur développement et présentent une différentiation incomplète. Des études d'hybridation in situ de l'expression du gène codant l'histone H4, marqueur de l'activité mitotique, ont montré que le blocage du développement des staminodes et des pistillodes serait dû à un arrêt des divisions cellulaires. Nos investigations de l'intégrité cellulaire par des observations en microscopie électronique à transmission et par coloration de l'ADN confirmeraient que l'avortement des organes stériles ne résulte pas d'un processus de dégradation cellulaire et nucléaire. De plus, l'étude de la méthylation de l'ADN par immunodétection des cytosines méthylées révèle que, par rapport aux organes fertiles, les pistillodes et les staminodes se distinguent par leur niveau plus élevé de méthylation. Ces résultats sont en cohérence avec la réversibilité du blocage de ces organes observés in planta ou in vitro en réponse à une induction hormonale. L'ensemble de ces données montrent que l'unisexualisation des fleurs de palmier dattier est associée à une hyperméthylation globale de l'ADN suivi d'un arrêt des divisions cellulaires dans les organes sexuels stériles. Cette étude a permis d'améliorer nos connaissances sur les mécanismes qui gouvernent la différenciation des organes sexuels et permettra d'ouvrir des perspectives pour l'identification de marqueurs moléculaires du sexe chez le palmier dattier. / The date palm (Phoenix dactylifera L.) is a dioecious tropical fruit crop plant which has vital dietary, socio-economic and ecological importance in arid regions of the world. Despite the interest of developing molecular tools to discriminate male and female plants for the benefit of biodiversity preservation and genetic improvement programs, no sex-specific markers have been identified and validated to date. To study and understand the sex determination of date palm, we undertook to characterise the cellular and molecular processes underlying sex organ differentiation in this plant.A histological study of date palm reproductive development showed that the immature flower is bisexual in appearance until the initiation of the androecium and gynoecium. The first sign of sexual dimorphism is observed at this stage, namely a wider gynoecium in female flowers resulting from greater mitotic activity in the functional gynoecium of female flowers compared to the pistillode of male ones. The sterile sex organs (pistillode and staminodes) were observed to cease their development by progressive loss of cell proliferation and ultimately displayed incomplete differentiation.Cell division patterns and the nuclear integrity of reproductive organs were investigated respectively by RNA in situ hybridization to a histone H4 gene probe and by DNA coloration combined with scanning electron microscopy. The results obtained revealed an absence of cell cycle activity and nuclear degradation in the residual sex organs. In addition, a study of DNA methylation, by immunodetection of methylated cytosines revealed that compared to the fertile reproductive organs, staminodes and pistillodes displayed relatively high levels of global DNA methylation. These results are consistent with the observed reversibility of sterile organ developmental arrest observed in planta or in vitro in response to hormonal induction. Overall, these data demonstrate that the floral unisexuality of date palm is characterized by cell cycle arrest, higher DNA methylation in sterile sexual organs and an absence of cell degeneration rather than a cell death process. This study has improved our understanding of the mechanisms that govern the differentiation of sex organs and forms a useful starting point for research on the identification of molecular markers of sex determination in date palm.Kewords: Date palm - flower - sex determination - cell cycle - DNA methylation

Sexual development begins with the process by which the bipotential gonads of the embryonic urogenital ridge develop into either testes or ovaries. In the mouse, sex determination occurs at around 11.5 dpc and depends on the presence or absence of the Y chromosome and the associated activity of the testis-determining gene, Sry, in supporting cell precursors. The mutually antagonistic male and female developmental pathways are regulated by many cellular and molecular processes, disruption of which can lead to disorders of sex development (DSDs). However, many of the molecular mechanisms regulating the differentiation of the two gonads are still unknown. The boygirl (byg) mutant was identified in an ENU-based forward genetic screen for embryos with gonadal abnormalities. On the C57BL/6J background, XY byg/byg homozygotes exhibited complete embryonic gonadal sex reversal. The defective gene in byg, Map3k4, is a component of the mitogen-activated protein (MAP) kinase signalling pathway and provides the first evidence for a function of this pathway in sex determination. This thesis describes experiments aimed at investigating the cellular and molecular basis of the sex reversal phenotype associated with the XY Map3k^4byg/byg mutant. Cellular characterisations revealed a defect in male-specific proliferation at 11.5 dpc, which was attributed to a defect in Sry up-regulation. Elucidation of the downstream kinases activated by MAP3K4 during sex determination was attempted, with particular focus on identifying a role for p38α MAP kinase (MAPK). Using a conditional knockout approach, the function of p38α in Steroidogenic factor-1 (Sf1)-positive somatic cells was assessed. However, specific inactivation in these cells did not affect gonad development. Conditional inactivation of Map3k4 itself in these Sf1¬-positive cells also did not disrupt gonad development, suggesting that this pathway is either initiated in a different cell lineage or at an earlier stage than deletion driven by Sf1-Cre can disrupt. Conditional inactivation of p38α in the Müllerian duct mesenchyme and ovarian granulosa cells using Amhr2-Cre did reveal a function for p38α in female fertility, but did not disrupt embryonic sexual development. Gene knockdown in organ culture was attempted to determine a role for multiple p38 MAPKs in all cell types of the gonad. Therefore, this thesis details further characterisations of a novel signalling pathway important for the expression of Sry, focussing on the role of the p38 MAPKs.

Objetivo: Avaliar os resultados da genitoplastia masculinizante, com a técnica de Denis Browne, realizada em um grande grupo de pacientes com distúrbios do desenvolvimento sexual (DDS) tratados em um único hospital de referência. Pacientes e Métodos: Avaliamos 65 pacientes (57 com DDS 46,XY e 8 com DDS 46,XX) com hipospádia proximal e genitália ambígua. Os resultados cosméticos e sintomas urinários foram avaliados objetivamente e os pacientes responderam a um questionário sobre sintomas urinários, atividade sexual e satisfação pessoal após o tratamento cirúrgico. A idade dos pacientes na primeira cirurgia foi em média de 9 ± 10 anos e o segundo tempo cirúrgico foi realizado em média 14,5 ± 16,3 meses após a primeira cirurgia. O seguimento destes pacientes foi em média de 15,1 ± 10 anos e a idade dos pacientes na avaliação final foi em média de 25,9 ± 14,1 anos. Resultados: O aspecto cosmético foi considerado bom em 44%, regular em 53% e ruim em 3% dos pacientes. Houve diferença estatisticamente significante na média do tamanho peniano antes do tratamento entre os grupos com deficiência de 5-RD2 e com DDS de etiologia indeterminada (p<0,05). A média do tamanho peniano na avaliação final dos pacientes póspúberes foi de 7,8 ±2,4 cm, variando de 4 to 12 cm correspondendo a -4,4 ± 1,3 DP (-6,5 a -1,5 DP). Houve diferença estatisticamente significante no tamanho peniano entre os grupos com deficiência na produção de testosterona e com deficiência de 5-RD2 e entre os grupos com DDS de etiologia indeterminada e deficiência de 5-RD2 (p<0,05). O grupo com deficiência de 5-RD2 apresentou o menor tamanho peniano na avaliação final (-5,4±1 DP). As complicações mais freqüentes foram a fistula uretral encontradas em 50% dos pacientes seguida de estenose, presente em 20% dos pacientes. O sintoma urinário mais freqüente foi a perda urinária pós miccional. A atividade sexual foi referida por 86% dos pacientes adultos sendo definida como adequada em 60%, satisfatória em 29% e insatisfatória em 11% dos pacientes. Em relação ao resultado cirúrgico, 84% dos pacientes referiram estar satisfeitos porem 11% estavam insatisfeitos com o tamanho peniano e 5% com a presença de estenose uretral. Conclusão: A maioria dos pacientes com DDS submetidos a genitoplastia masculinizante pela técnica de Denis Browne mostrou-se satisfeita com os resultados cirúrgicos. Entretanto, queixas sobre o tamanho peniano, atividade sexual e micção indicam que novas abordagens devem ser desenvolvidas para melhor resultado morfológico e funcional dos pacientes com distúrbio do desenvolvimento sexual. / Purpose: To evaluate the results of masculinizing genitoplasty with the Denis Browne technique performed in a large cohort of patients with disorders of sex development (DSD) treated at a single tertiary centre. Patients and Methods: We evaluated 65 patients (57 with 46,XY DSD and 8 with 46,XX DSD) with proximal hypospadias and genital ambiguity. Cosmetic results and the urinary stream were evaluated objectively, and the patients responded questionnaires regarding satisfaction with the surgical results, as well as urinary and sexual symptoms. The age at first surgery was 9±10 years and the second stage was performed after 14.5±16.3 months. The mean followup was 15.1±10 years and the average patients age at the last examination was 25.9±14.1 years. Results: Cosmetic results were considered good in 44%, regular in 53% and poor in 3% of the cases. The comparison of the mean penile length among 46,XY DSD groups identified a significant statistically difference between 5-RD2 deficiency and undetermined DSD groups at diagnosis (p<0.05). The mean penile length at last clinical evaluation in post-pubertal patients was 7.8 ±2.4 cm, ranging from 4 to 12 cm corresponding to -4,4 ± 1,3 SD (-6.5 to -1.5 SD) and there was a significant statistically difference in the mean penile length amongst testosterone production deficiency and undetermined DSD groups with 5-RD2 deficiency group (p<0.05). The 5RD2 deficiency group presented the smallest penile length at the last evaluation (-5.4±1 SD). The most common complications were urethral fistula (50%) and stenosis (20%) and the most frequent urinary symptom was dribbling after micturition. Sexual activity was reported by 86% of adult patients and was adequate in 60%, satisfactory in 29% and unsatisfactory in 11% of them. Overall, 84% referred satisfaction with surgical results, but 11% complained about penile length and 5% about urethral stenosis. Conclusion: Most of the DSD patients were satisfied with the longterm results of masculinizing genitoplasty using Denis Browne technique, although specific complaints about small penis size, sexual activity and urinary symptoms were frequent. New approaches should be developed to achieve full satisfaction of DSD patients in adulthood.

Orientador: Silvia Helena Venturoli Perri / Banca: José Fernando Garcia / Banca: José Antônio Visintin / Resumo: O crescente avanço da produção in vitro de embriões bovinos intensificou a utilização de outras biotecnologias da reprodução tais como a micro-manipulação embrionária e o diagnóstico genético pré-implantacional, sendo a identificação do sexo embrionário utilizada na rotina comercial de laboratórios de produção in vitro. O objetivo deste trabalho foi avaliar as interações de diferentes fatores sobre a taxa de mortalidade embrionária e a proporção do sexo de embriões bovinos submetidos ao processo de sexagem. Foi realizado levantamento no banco de dados da Transfix - Transplante de Embriões Ltda, Patrocínio Paulista / Brasil, referente a 4.650 embriões produzidos in vitro e sexados entre 2005 e 2007. Os embriões foram submetidos à micro-manipulação pela técnica de micro-aspiração, e as biópsias à reação em cadeia pela polimerase (PCR). Somente as fêmeas foram transferidas para receptoras previamente sincronizadas. O diagnóstico de gestação e a determinação do sexo fetal foram realizados por ultra-sonografia. As variáveis foram classificadas de acordo com o sexo dos embriões (macho, fêmea e indeterminado), cinco laboratórios (A, B, C, D e E), seis raças bovinas (Nelore, Brahman, Girolando, Simental, Holandês e Jersey), estágio embrionário (MO, BI, BL, BX e BE), qualidade embrionária (1, 2 e 3) e qualidade da biópsia ("dentro do padrão" e "fora do padrão"). As análises estatísticas foram realizadas pelos testes 2 de associação, 2 de aderência para proporção 1:1 e pela análise de regressão logística com o método de Hosmer-Lameshow utilizando o procedimento logístico (PROC LOGISTIC) programa computacional SAS. A PCR apresentou eficiência de 93,3%, acurácia de 93,2% e taxa de machos e fêmeas de 52,9% e 47,1%, respectivamente. A taxa de mortalidade dos embriões... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Crescent progress of in vitro bovine embryo production has improved the use of other reproductive biotechnologies, as embryo micromanipulation and preimplantation genetic diagnosis, being embryo sexing used in commercial routine of in vitro embryo production laboratories. The present study aimed to evaluate the interactions among different factors on the mortality rate and sex ratio of in vitro produced bovine embryos. A survey was performed in the Transfix - Transplante de Embriões Ltda, Patrocínio Paulista / Brazil data base, referring to 4.650 in vitro produced bovine embryos sexed during years 2005/2007. Embryos were submited to the biopsy by the microaspiration technique, and biopsies to the polymerase chain reaction (PCR). Only female embryos were transferred to synchronized recipients. Pregnancy diagnosis and fetal sex determination were carried out by ultrasound. The variables were classified according embryo sex (male, female and indeterminate), five laboratories (A, B, C, D and E), six bovine breeds (Nellore, Brahman, Girolando, Simmental, Holstein and Jersey), embryo stage (MO, EB, BL, XB and HB), embryo quality (1, 2, and 3) and biopsy quality ("standard" and "non standard"). The statistical analysis was carried out by association 2 test, 2 for 1:1 ratio and logistic regression analysis with Hosmer- Lameshow method using logistic procedure (PROC LOGISTIC) of SAS package. The PCR showed 93.3% efficiency, 93.2% accuracy and male and female ratio of 52.9% and 47.1%, respectively. Mortality rate of biopsied embryos was 10.3% and pregnancy rate was 31.7%. Although no significant differences were observed between male and female ratio, indeterminate embryos possess greater possibility to die after micromanipulation. For quality 2 and 3 embryo mortality rate after biopsy was 3.19 and 11.37 fold higher, respectively, than for quality 1 embryo. For those whose biopsy... (Complete abstract click electronic access below) / Mestre