Evidence of feminising effects, including additional or enlarged female organs, have been reported in the (sub)-tropical freshwater prosobranch Marisa cornuarietis exposed to vertebrate estrogen receptor agonists. The primary symptom is an increase in the number of eggs laid, but this is only observed when exposure occurs at relatively low temperatures. This research project exposed temperate freshwater prosobranchs and a pulmonate to 17β-oestradiol (10-200 ngL-l, nominal) in an outdoor mesocosm subject to natural seasons to determine whether similar effects occur in European native temperate freshwater gastropods. Laboratory exposures to 17β-oestradiol (1-100 ngL-l, nominal) and Bisphenol-A (0.2-20 ngL-l, nominal) were also carried out over a range of different temperatures and photoperiods to simulate natural seasons. In the mesocosm exposures, significant increases in reproduction were measured in Viviparus viviparus, Bithynia tentaculata and, if the mortality rate was not significantly increased, Planorbarius corneus. It was observed that increases only occurred after the onset of autumn. In the laboratory, the oviposition rate in P. corneus was constant at 20oC with a 16h photoperiod, but declined significantly at 15oC with a 12h photoperiod, except at 100 ngL-l 17β-oestradiol, when the rate remained constant. There were no similar effects from Bisphenol-A exposure. Small increases in reproduction were observed in all the prosobranch exposures to both compounds, but the interpretation of the data was confounded by several factors (test chemical degradation, high mortality rates and parasitized organisms) and there were no significant differences. In conclusion, there were indications that all of the assessed species were capable of increased reproduction, and in P. corneus this appears to be a perpetuation of summer oviposition rates in autumnal conditions. The consequences of this effect in annual semelparous pulmonates may not be detrimental at the population level, but the long-term fitness of iteroparous prosobranchs that practice ‘restrained reproduction’ in early breeding seasons may be adversely affected.

Características morfológicas, histomorfométricas, ultraestruturais e tridimensionais do ducto deferente de codorna doméstica (Coturnix coturnix) da variedade italiana foram estudadas e descritas. Para tanto, foram utilizadas 30 codornas sexualmente maduras, procedentes de um criatório da Fazenda Edgardia de Botucatu, Estado de São Paulo, Brasil. Os ductos deferentes foram coletados, observados e documentados ao longo do ano nas estações de outono, inverno, primavera e verão, e estudados por métodos de macro-mesoscopia, microscopia de luz, microscopias eletrônicas de transmissão (MET) e de varredura (MEV). Cada ducto deferente foi reduzido em três segmentos: proximal ou adepididimal; médio ou justa-renal, e distal ou adcloacal. Os estudos ultraestruturais e tridimensionais se concentraram no segmento médio do ducto. Diferenças marcantes na morfologia foram observadas, incluindo características estruturais e histomorfométricas, sobre os valores médios de altura epitelial, diâmetros tubular e luminal dos ductos deferentes, mensurados durante as quatro estações do ano, sendo mais marcantes as observações feitas no outono, a fase quiescente, e na primavera, a fase ativa, do ciclo testicular anual. Nas fases intermediárias do ciclo, compreendendo os meses de inverno (fase recrudescente), e de verão (fase regressiva), os reflexos da cinética testicular sobre a estrutura do ducto deferente se fizeram notar principalmente nas características subcelulares das células principais do epitélio de revestimento. As observações de MEV confirmaram a variabilidade morfológica do ducto deferente nas estações marcantes do ano (outono e primavera) em termos das fases do ciclo testicular anual. Estes estudos permitiram concluir que esta espécie de ave mostra um padrão circanual no ciclo reprodutivo, influenciando as características morfológicas dos ductos excretores do testículo / The morphological, histomorphometric, ultrastructural and three-dimensional features of the vas deferens of quail (Coturnix coturnix) of the Italian variety were studied and described. For this study were utilized 30 adult sexually active quails taken from the experimental Edgardia Farm of the UNESP, Botucatu, São Paulo, Brazil. The vas deferens was collected, observed and documented along the year in the autumn, winter, spring and summer seasons, and studied by macro-mesoscopy, light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Each vas deferens was reduced in three segments: proximal (adepididymal); middle (just the median lobe of the kidney); and distal (adcloacal). The ultrastructural and three-dimensional studies were concentred in the middle segment of the duct. It was verified marked morphologic differences in the vas deferens, including structural and histomorphometric features concerning the middle values of the epithelial height, tubular and luminal diameters measured during the four seasons of the year. The observations made in the autumn the quiescent phase and in the spring the active phase of the annual testicular cycle were more evident. In the intermediaries phases of the cycle, i.e., the winter (recrudescent phase) and summer (regressive phase), the influence of the testicular kinetic on the vas deferens structure was noted mainly in the subcellular characteristics of principal cells of the epithelium lining. The SEM observations confirmed the morphologic variability of the vas deferens in the main seasons of the year (autumn and spring) concerning to the annual testicular cycle phases. These studies allowed to conclude that this avian species showed a circunannual pattern of the reproductive cycle, influencing the morphological features of the excurrent ducts of the testis

Características morfológicas, histomorfométricas, ultraestruturais e tridimensionais do ducto deferente de codorna doméstica (Coturnix coturnix) da variedade italiana foram estudadas e descritas. Para tanto, foram utilizadas 30 codornas sexualmente maduras, procedentes de um criatório da Fazenda Edgardia de Botucatu, Estado de São Paulo, Brasil. Os ductos deferentes foram coletados, observados e documentados ao longo do ano nas estações de outono, inverno, primavera e verão, e estudados por métodos de macro-mesoscopia, microscopia de luz, microscopias eletrônicas de transmissão (MET) e de varredura (MEV). Cada ducto deferente foi reduzido em três segmentos: proximal ou adepididimal; médio ou justa-renal, e distal ou adcloacal. Os estudos ultraestruturais e tridimensionais se concentraram no segmento médio do ducto. Diferenças marcantes na morfologia foram observadas, incluindo características estruturais e histomorfométricas, sobre os valores médios de altura epitelial, diâmetros tubular e luminal dos ductos deferentes, mensurados durante as quatro estações do ano, sendo mais marcantes as observações feitas no outono, a fase quiescente, e na primavera, a fase ativa, do ciclo testicular anual. Nas fases intermediárias do ciclo, compreendendo os meses de inverno (fase recrudescente), e de verão (fase regressiva), os reflexos da cinética testicular sobre a estrutura do ducto deferente se fizeram notar principalmente nas características subcelulares das células principais do epitélio de revestimento. As observações de MEV confirmaram a variabilidade morfológica do ducto deferente nas estações marcantes do ano (outono e primavera) em termos das fases do ciclo testicular anual. Estes estudos permitiram concluir que esta espécie de ave mostra um padrão circanual no ciclo reprodutivo, influenciando as características morfológicas dos ductos excretores do testículo / The morphological, histomorphometric, ultrastructural and three-dimensional features of the vas deferens of quail (Coturnix coturnix) of the Italian variety were studied and described. For this study were utilized 30 adult sexually active quails taken from the experimental Edgardia Farm of the UNESP, Botucatu, São Paulo, Brazil. The vas deferens was collected, observed and documented along the year in the autumn, winter, spring and summer seasons, and studied by macro-mesoscopy, light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Each vas deferens was reduced in three segments: proximal (adepididymal); middle (just the median lobe of the kidney); and distal (adcloacal). The ultrastructural and three-dimensional studies were concentred in the middle segment of the duct. It was verified marked morphologic differences in the vas deferens, including structural and histomorphometric features concerning the middle values of the epithelial height, tubular and luminal diameters measured during the four seasons of the year. The observations made in the autumn the quiescent phase and in the spring the active phase of the annual testicular cycle were more evident. In the intermediaries phases of the cycle, i.e., the winter (recrudescent phase) and summer (regressive phase), the influence of the testicular kinetic on the vas deferens structure was noted mainly in the subcellular characteristics of principal cells of the epithelium lining. The SEM observations confirmed the morphologic variability of the vas deferens in the main seasons of the year (autumn and spring) concerning to the annual testicular cycle phases. These studies allowed to conclude that this avian species showed a circunannual pattern of the reproductive cycle, influencing the morphological features of the excurrent ducts of the testis

La première ovulation induite par effet mâle chez la brebis anovulatoire en anoestrus saisonnier est souvent accompagnée d’un développement de cycles courts qui peuvent être contrecarrés par un traitement progestatif préalable. Nos travaux ont montré que la réponse au mâle est plus rapide en fin d’anoestrus en raison d’une sélection à J0 de follicules dont le stade de développement est plus avancé. En parallèle, ils apportent de nouvelles connaissances sur le mécanisme d’action par lequel la progestérone parvient à supprimer les cycles courts induits par effet mâle, en modulant certaines caractéristiques de la dynamique de croissance folliculaire et en améliorant l’activité stéroïdogénique des follicules préovulatoires. Cependant, d’autres études sont nécessaires pour étudier l’impact de ces effets sur la qualité ovocytaire et la fertilité de ces premières ovulations induites par effet mâle sur un plus grand nombre d’animaux. / The first ovulation induced by male effect in anestrous ewes during the non-breeding season usually result in the development of short cycles that can be avoided by progesterone priming. Our results show that ewes ovulate earlier in June due to selection at J0 of follicles in a more advanced stage of development. However, these observations seem to be unrelated to the luteal outcome after male effect. In parallel, we demonstrate the positive effect of progesterone on the completion of follicular growth and the improvement of the ability of these follicles to respond properly to LH surge and to synthesis ovarian steroids. However, further studies are required to understand whether these changes have functional consequences on the quality of oocytes induced by the male effect and in fertility after male effect on a large number of animals.

Many previous studies in evolutionary genetics have been based on few model organisms that can be reared at ease in the laboratory. In contrast, genetic studies of non-model, natural populations are desirable as they provide a wider range of adaptive phenotypes throughout evolutionary timescales and allow a more realistic understanding of how natural selection drives adaptive evolution. This thesis represents an example of how modern genomic tools can be effectively used to study adaptation in natural populations. Atlantic herring is one of the world’s most numerous fish having multiple populations with phenotypic differences adapted to strikingly different environments. Our study demonstrated insignificant level of genetic drift in herring that resulted in minute genetic differences in the majority of the genome among these populations. In contrast, a small percentage of the loci showed striking genetic differentiation that were potentially under natural selection. We identified loci associated with adaptation to the Baltic Sea and with seasonal reproduction (spring- and autumn-spawning) and demonstrated that ecological adaptation in Atlantic herring is highly polygenic but controlled by a finite number of loci. The study of Darwin’s finches constitutes a breakthrough in characterizing their evolution. We identified two loci, ALX1 and HMGA2, which most likely are the two most prominent loci that contributed to beak diversification and thereby to expanded food utilization. These loci have played a key role in adaptive evolution of Darwin’s finches. Our study also demonstrated that interspecies gene flow played a significant role in the radiation of Darwin’s finches and some species have a mixed ancestry. This thesis also explored the genetic basis for the remarkable phenotypic differences between three male morphs in the ruff. Identification of two different versions of a 4.5 MB inversion in Satellites and Faeders that occurred about 4 million years ago revealed clues about the genetic foundation of male mating strategies in ruff. We highlighted two genes in the inverted region; HSD17B2 that affects metabolism of testosterone and MC1R that has a key role in regulating pigmentation, as the major loci associated with this adaptation.

Studying genetic diversity in natural and domestic populations is of major importance in evolutionary biology. The recent advent of next generation sequencing (NGS) technologies has dramatically changed the scope of these studies, enabling researchers to study genetic diversity in a whole-genome context. This thesis details examples of studies using NGS data to: (i) characterize evolutionary forces shaping the genome of the Atlantic herring, (ii) detect the genetic basis of speciation and domestication in the rabbit, and, (iii) identify mutations associated with skeletal atavism in Shetland ponies. The Atlantic herring (Clupea harengus) is the most abundant teleost species inhabiting the North Atlantic. Herring has seasonal reproduction and is adapted to a wide range of salinity (3-35‰) throughout the Baltic Sea and Atlantic Ocean. By using NGS data and whole-genome screening of 20 populations, we revealed the underlying genetic architecture for both adaptive features. Our results demonstrated that differentiated genomic regions have evolved by natural selection and genetic drift has played a subordinate role. The European rabbit (Oryctolagus cuniculus) is native to the Iberian Peninsula, where two rabbit subspecies with partial reproductive isolation have evolved. We performed whole genome sequencing to characterize regions of reduced introgression. Our results suggest key role of gene regulation in triggering genetic incompatibilities in the early stages of reproductive isolation. Moreover, we studied gene expression in testis and found misregulation of many genes in backcross progenies that often show impaired male fertility. We also scanned whole genome of wild and domestic populations and identified differentiated regions that were enriched for non-coding conserved elements. Our results indicated that selection has acted on standing genetic variation, particularly targeting genes expressed in the central nervous system. This finding is consistent with the tame behavior present in domestic rabbits, which allows them to survive and reproduce under the stressful non-natural rearing conditions provided by humans. In Shetland ponies, abnormally developed ulnae and fibulae characterize a skeletal deformity known as skeletal atavism. To explore the genetic basis of this disease, we scanned the genome using whole genome resequencing data. We identified two partially overlapping large deletions in the pseudoautosomal region (PAR) of the sex chromosomes that remove the entire coding sequence of the SHOX gene and part of CRLF2 gene. Based on this finding, we developed a diagnostic test that can be used as a tool to eradicate this inherited disease in horses.

Le RFRP est une neuropeptide impliqué dans la régulation de l’axe reproducteur, mais ses effets varient en fonction du sexe et des espèces. Le but de cette étude était de décrire en détails l’organisation du système RFRP et de caractériser son rôle dans le contrôle circadien et saisonnier de l’axe reproducteur de hamsters femelles. Les résultats montrent que le système RFRP est régulé par la photopériode et que son niveau d’expression est plus élevé chez les femelles que chez les mâles. Cela se traduit par des actions spécifiques sur l’axe gonadotrope femelle. En effet, L’activité des neurones à RFRP est diminuée au moment du pic pré-ovulatoire de LH et des injections centrales de RFRP-3 dans l’heure qui précède le pic de LH induisent une diminution de l’amplitude de la sécrétion de LH, démontrant une implication du RFRP dans la régulation circadienne du pic pré-ovulatoire de LH. Par ailleurs, des infusions chroniques de RFRP-3 chez des hamsters femelles sexuellement inactifs sont capables de réactiver le fonctionnement de l‘axe reproducteur, ce qui montre que le RFRP a un également un rôle régulateur essentiel dans le contrôle saisonnier de la reproduction. / RFRP neurons regulate the reproductive axis, however, their effects depend on species and sex. Here, we aimed at providing a neuroanatomical description of the RFRP system in the Syrian hamster and at investigating the role of RFRP in the daily and seasonal control of female reproduction. We show that besides being regulated by annual changes in photoperiod, the RFRP system is more strongly expressed in females than in males. In line with this, we unveil that RFRP has multiple roles in regulating female reproduction. RFRP neuronal activity is specifically reduced at the time of the pre-ovulatory LH surge and central RFRP-3 administration prior to the surge decreases LH peak levels, altogether pointing towards a daily down-regulation of the inhibitory RFRP signal necessary for proper generation of the LH surge. Moreover, chronic RFRP-3 infusion in sexually inactive females, with endogenous low RFRP expression, completely reactivates the reproductive axis. Taken together, we demonstrate that RFRP is a key component in the seasonal control of reproduction while at the same time specifically regulating cyclic events controlling reproductive activity in females.