Dissertations / Theses on the topic 'Sex determination in animals'

Thesis (Ph.D.) - University of Glasgow, 2008.
Ph.D. thesis submitted to the Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Much of what we know about temperature-dependent sex determination (TSD) in reptiles stems from constant temperature incubation studies in the laboratory. In recent years, as TSD studies moved into the field it became evident that TSD was much more complex than previously thought. The present study attempted to reveal the complexity of TSD, as it relates to other features of the species' biology and physical characteristics tractable only in the field, such as fluctuations in incubation temperature and reproductive life history. To this end I studied the ecology of the turtle Carettochelys insculpta, a TSD species inhabiting the wet-dry tropics of northern Australia from 1996 to 1998. I tested hypotheses associated with movements, activity, behaviour, reproduction, nest site choice, nest temperatures, embryonic survival, embryonic aestivation, hatch-ling sex ratios, and emergence in the species. Each of these was also considered in the context of the influence of the wet-dry tropics. Compared to other turtles inhabiting lotic habitats, C. insculpta occupied considerably larger home ranges, covering up to 10 km of river. Of previously published factors influencing home range size, low productivity of the (micro) habitat may best explain the extensive home ranges in C. insculpta. Patchiness and low nutrient value of the chief food (aquatic vegetation) of C. insculpta may force turtles to cover large expanses of river to acquire sufficient energy for growth and reproduction. Females were more active, moved farther, and occupied larger home ranges than males. Home ranges of females comprised 1-4 activity centres, many of which were associated with thermal springs. I suggest that females may exhibit increased activity and movements relative to males because of sexual inequality in parental investment, where food is particularly limiting (e.g., in species with biennial reproduction). Biennial reproduction in the population allowed the examination of the influence of reproductive condition on home range size, movements, and activity. Reproductive condition did not influence home range or activity, but gravid turtles moved father between successive sightings than non-gravid females. Individual data corroborate these findings, with females moving farther between successive sightings while gravid compared to while spent. Contrary to previous reports, turtles did not appear to move into estuarine areas or lowland flood plains during the wet season, but moved into the riparian forest and possibly into wetlands adjacent to the main channel in the vicinity of their dry season home ranges. During the study I documented the turtles' use of small, localized thermal springs discharging from the river bottom. Dataloggers attached to the carapace to monitor ambient water temperatures recorded the frequency and duration of thermal spring use by individuals. Turtles used the thermal springs frequently during the winter (4-6 months) when river temperatures were lower than that of the thermal springs (8 = 29 � 0.52� C). Turtles often utilized thermal springs for several consecutive hours, leaving the springs only to surface for air. Thermal springs may be derived from ground water (which maintains a temperature equivalent to the annual mean air temperature), rather than from a specific geothermal heat source. Nine of 19 radio-telemetered adult females were seen to use thermal springs, of which seven were gravid and two non-gravid. Thus, gravid turtles may seek thermal springs more than non-gravid turtles. Frequency, duration, and timing of usage collectively suggest active thermoregulation as the primary function of thermal spring use. Utilization of thermal springs probably permits turtles to be more active in cooler months, which may enhance growth rates and accumulation of energy for reproduction. Onset of nesting along river stretches with thermal springs preceded nesting in a stretch not known to have thermal springs by 24 days. Thus, I speculate that by warming themselves on thermal springs in the months prior to nesting, turtles may have accelerated follicular development and nested earlier. Female C. insculpta matured at ca. 6 kg body mass (38.0 cm carapace length, 30.5 cm plastron length). Turtles produced egg sizes and clutch sizes similar to that of other turtle species of similar size. Turtles reproduced every second year, but produced two clutches in each breeding year, ca. 40 days apart. Thus, it appeared that females were energy limited, possibly due to the low available energy content of the dry season diet (aquatic vegetation). Life history theory predicts that if some costly behaviour is associated with reproduction, skipping years could reduce that cost and allow savings to be directed into future reproduction. The present study revealed no obvious accessory behaviour in the population. Within years, clutch mass did not differ between early (first) and late (second) clutches. However, earlier clutches tended to have more and smaller eggs per clutch but than later clutches, a new finding for turtles that has been demonstrated in lizards and other animals. Because the study spanned both years with 'big' and 'small' wet seasons, I was able to examine how the magnitude of the wet season influenced reproductive characteristics. Following big wet seasons turtles produced larger, heavier, and more eggs per clutch than they did after small wet seasons. Relationships among body size, egg size, and clutch size were evident after two big wet seasons but not apparent after two small wet seasons. Collectively, annual variation in reproductive characteristics and current life history theory suggest that a big wet season is a plentiful time for the turtles. I investigated beach selection of nesting pig-nosed turtles (Carettochelys insculpta) along a 63 km stretch of river in 1997 and 1998. I used three classes of beaches to examine beach choice: beaches with nests, beaches with only crawls, andbeaches without nests or crawls. Across these beach types I compared aspect, solar exposure, temperature, substrate moisture, height, water depth at approach, and the height of cohesive sand. I located 82 nesting beaches with 221 nests, and identified 171 potential nesting beaches based on previously published criteria. Beaches with nests had a greater substrate moisture content and corresponding higher cohesive sand line (hereafter CSL) than beaches without nests. Beaches with nests also had a higher CSL than beaches with only crawls. Apparently, turtles could not excavate a nest chamber above the CSL due to loose substrate consistency causing sand to fall in on itself. Turtles could only nest at low elevations below the CSL on beaches with lower substrate moisture. Turtles apparently avoided nesting on these beaches due to the higher probability of nest flooding, as corroborated by a concurrent study. Beach temperatures increased with a seasonal increase in air temperatures, and were influenced by aspect and total angle of solar exposure. Temperatures did not differ among beaches with nests, beaches with only crawls, and beaches without crawls or nests. Therefore, there was no indication that turtles were manipulating offspring sex through choice of nesting beach. However, turtles may be manipulating sex by nesting in areas with particular thermal characteristics within beaches. Two related aspects of hatchling emergence were studied. Using emergence phenology data, nest temperatures, historical weather data, and a developmental model, I tested the hypothesis that delayed hatching occurred in C. insculpta, and that such a delay would allow hatchlings to time their emergence to match the onset of the wet season. Hatchling C. insculpta emerged, on average, 17 days later than dates predicted from a developmental model. Combined with observations of hatchlings remaining in eggs until emergence, these results confirmed delayed hatching in nature. This delay was synchronized with initial river rises associated with the onsetof wet season rains, and is consistent with published criteria for embryonic aestivation. On a diel scale, I generated predictions of two potentially competing models for nocturnal emergence in hatchling turtles, based on the knowledge that air temperatures decrease with season during the emergence period. A test of those predictions for C. insculpta produced ambiguous results. However, further analysis indicated that C. insculpta, and probably other nocturnally emerging turtle species, respond to a decline in diel temperature rather than an absolute temperature. The former would ensure nocturnal emergence, while the latter is experienced during the day as well as at night. Nocturnal emergence may be associated with nesting in open microhabitats. The 'decision' of when and where to nest can influence both offspring survival and hatchling sex ratios in animals with temperature-dependent sex determination (TSD). Knowledge of how these maternal attributes influence the incubation environment is an important first step in hypothesizing why TSD evolved in a particular species. 1 studied the influence of nest site choice and timing of nesting on embryonic survival and hatchling sex ratios. Predation and flooding were the major sources of embryonic mortality. Embryonic survival was influenced by both lay date and nest site choice: In one year when nesting began later, nests laid later and at lower elevations were destroyed by early wet season river rises. In other years early nesting precluded flood mortality. However, turtles did not nest at the highest available elevations. I hypothesized that turtles were unable to nest at higher elevations because the sand was dry and not cohesive. A field experiment demonstrated that turtles were constrained to nest at lower elevations where they could construct a nest chamber. A mathematical model predicting hatchling sex from fluctuating temperatures was applied to temperature data from 102 natural nests. Resultsconfirmed a type la pattern of TSD, whereby males are produced from cooler temperatures and females from warmer temperatures. The principal determinant of hatchling sex was lay date. Clutches laid earlier in the season produced mainly males, while later clutches yielded mostly females, due to seasonal ramping of air and sand temperatures. However, nest site choice also exerted an influence on hatchling sex. Female-producing clutches were deposited at higher elevations than male-producing clutches. The onset of nesting was not influenced by water temperatures, but may have been related to the magnitude of the previous wet season(s). Turtles nested earlier after two 'big' wet seasons and later following two 'small' wet seasons. This pattern indicates that the wet season is a plentiful time for the turtles. Adaptive 'differential fitness' models for the evolution of TSD have recently been reviewed and clarified. The differential fitness model that best fits C. insculpta is the 'timematching' model, whereby one sex benefits more than the other from early hatching. Male C. insculpta hatched 2-3 weeks earlier then females, on average. Benefit to early hatching males and, therefore, the ultimate selective mechanism (e.g., growth, time to mature) is unknown. Obtaining such data will likely prove difficult in such a long-lived species. A recent adaptive explanation for the evolution and maintenance of temperaturedependent sex determination (TSD) in reptiles rests upon the assumption that mothers can predict or manipulate offspring sex. I postulated that four physiological and behavioural criteria must be met in order for this assumption to be valid: (1) a strong correlation must exist between substrate temperatures during nest site choice and nest temperatures during the period of development when sex is determined in the egg (thermosensitive period = TSP). (2) Assuming that (1) is possible, mothers would need to be capable of correcting for temporal factors obscuring the predictable thermalcharacteristics of nest sites. This could be accomplished in two ways. By contracting nesting times mothers could assess the relative temperatures of alternate nest sites with some accuracy. A protracted distribution of nesting times could greatly reduce a mother's ability to distinguish between, for example, a cooler nest site at a warmer time and a warmer nest site at a cooler time. Alternatively, mothers would need to be able to incorporate temporal changes in nest site temperatures. (3) Sufficient variation in thermal profiles among nest sites, relative to the breadth of temperatures producing both sexes (pivotal temperatures), would be necessary. For example, if most nests produced both sexes, then depth of the eggs would be the deciding factor determining sex, leaving little opportunity for nest site choice to produce one sex or the other. (4) Mothers would need access to nest sites spanning a range of thermal profiles in order to produce either offspring sex. To this end, home range size relative to the number and location of nesting beaches should be important. I tested these four predictions in Carettochelys insculpta, a beach nesting turtle with TSD, using three years of field data on nest site choice, nesting times, thermal characteristics of nests, hatchling sex ratios, and movements of nesting turtles. A strong positive correlation existed between assessable substrate temperatures at nest site choice and mean daily TSP temperatures in all three years. However, the proportion of explained variation was highly variable among years, and low in 1998. Accordingly, the proportion of nests in which substrate temperatures at nest site choice predicted offspring sex correctly was low in 1998 (48- 62 %, depending on treatment of the data). Nesting times were normally distributed, and combined with diel changes in nest site temperatures greatly reduce a turtle's ability to distinguish between sites that would produce different sexes. Considerable among-clutch variation in thermal profiles to produce variable sex ratios existed, agreeing with other studies on turtles. Radiotelemetry indicated that home rangesencompassed several nesting beaches with differing thermal profiles, indicating scope for producing the desired sex. However, the seasonal increase in air temperatures resulted in an overriding effect of mostly males being produced in early (first) clutches and mainly females being produced in late (second) clutches. Collectively, the results suggest that C. insculpta mothers would find it difficult to predict, and therefore, manipulate hatchling sex, supporting the conventional notion that TSD mothers have little or no control over offspring sex.

The mechanism of sex determination and the development control of the life-cycle of the gastro-intestinal parasite, Strongyloides ratti was investigated using a combination of genetic and parasitological techniques. Parasitological analysis investigated the effects of intra- and extra-host factors on the development of the free-living phase of the life cycle. An initial analysis of the distribution of infective stages among host faecal pellets showed that they were significantly overdispersed and well described by the negative binomial distribution. This overidspersion was found to occur over a wide range of infection intensities and to increase significantly during infection. Further investigation, coupled with an artificial selection experiment, suggested the existence of two discrete developmental switches; an intra-host sex determination switch and an extra-host free-living female/directly developing iL3 development conversion. Analysis of the effects of host immunity on these developmental switches clarified the way in which the composition of the free-living phase varies over the course of an infection. Increasing host immunity results in a greater proportion of female larvae developing into free-living females rather than into directly developing iL3s. Further evidence suggested that the increase in proportion of female larvae that develop into free-living females with increased host immunity is due to an increased temperature sensitivity of the free-living female/directly developing iL3 developmental conversion. Increasing host immunity also alters the sex ratio, resulting in a greater proportion of larvae developing into free-living males. In addition, increased parasitic female age appears to increase the proportion of larvae that develop into free-living males, but does not increase the proportion of female larvae that develop into free-living females. In conclusion, this thesis has used a combined parasitological and genetic approach to investigate the mechanism of sex determination of S. ratti. This understanding now allows a rational view of the S. ratti life-cycle to be presented. These findings are discussed in relation to previous studies of Strongyloides spp. and further questions raised by this work are discussed.

Effective reproduction is essential for species survival. Sexual reproduction depends upon functional gonads and reproductive ducts. Zebrafish (Danio rerio) is a popular model organism, but the genetic basis of zebrafish sex determination, gonad development, and reproductive tract development are not fully understood, and understanding this basis could inform about the evolutionary conservation of these genes and the use of zebrafish to investigate and treat reproductive diseases. In chapter I, I give a overview of sex determination systems, gonad development, and reproductive duct development in mammals and fish, and ask how sex is determined and how reproductive ducts develop in zebrafish. In chapter II, I used genome wide association studies (GWAS) to investigate if the genetic basis of sex determination in a variety of zebrafish strains -- two ‘wild-type’ strains cultured for about 30 years in the lab, and four ‘natural’ strains, wild-type strains isolated directly or recently from nature in India -- and identified a sex-associated region on zebrafish chromosome 4 in natural zebrafish strains that was lacking in the lab strains. In chapter III, I investigated whether or not wnt4a is important for zebrafish ovarian development, and found that wnt4a is expressed in the early bipotential gonad and that loss of wnt4a results in male-biased sex ratios, indicating that wnt4a is important for zebrafish ovarian development. In chapter IV, I investigated whether or not wnt4a is important for male reproductive duct development, and found that loss of wnt4a slows the formation of male reproductive ducts and prevents the male fused ducti deferens from connecting to the genital orifice in zebrafish males. Results further showed that wnt4a is expressed in tissue around the site where this connection should occur before and after the connection is formed, revealing a novel wnt4a phenotype in zebrafish that hasn’t been seen thus far, and indicating that wnt4a is necessary for proper male reproductive duct development in zebrafish. This dissertation contains both published and unpublished co-authored material.

Orientador: Rafael Henrique Nóbrega
Resumo: A decisão sobre se uma gônada bipotencial se desenvolverá em um testículo ou em um ovário é considerado um estágio crítico na diferenciação sexual dos vertebrados. A administração de esteróides exógenos durante este período pode afetar essa plasticidade, promovendo a diferenciação sexual na direção feminina ou masculina. Dessa forma, o objetivo desta tese foi avaliar os efeitos do tratamento de 17β-estradiol no desenvolvimento de Astyanax altiparanae (lambari), através de análises histológicas e de análises de expressão genica de possíveis genes envolvidos em vias masculinas e femininas. Para isso, larvas com gônadas indiferenciadas foram alimentadas com Artemia contendo diferentes concentrações de estradiol durante 28 dias, desde o 1 dia pós-eclosão (dpe) até o período que precede a diferenciação gonadal. Nossos resultados mostraram que o E2 modificou o fenotípo e a relação sexual histológica e, surpreendentemente, induziu intersexo com com a presença de óvulos nos testículos nas concentrações de 2 e 6 mg de E2/kg de alimento. Esses dados são uma evidência clara de que o tratamento utilizado não foi suficiente para induzir a reversão completa do sexo em A. altiparanae. Em termos de expressão gênica, o tratamento com E2 (6 mg/kg de alimento) produziu uma notável plasticidade gonadal entre machos e fêmeas aos 90 dias após a eclosão (dph). Os machos, denominados “machos resistentes ao estradiol”, superexpressaram os genes masculinos, como dmrt1, sox9 e amh. Dessa forma, nó... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The decision whether a bipotential gonad will become a testis or ovary is considered a critical stage in vertebrate sex determination. Administration of exogenous steroids can affect this plasticity by skewing the sex gonadal differentiation towards a male or female. The aim of this study is to evaluate the effects of 17β-estradiol (E2) diet on Astyanax altiparanae (lambari) development, focusing on the gonadal development and gene expression analysis of possible candidate genes involved in either male or female pathways. Larvae with undifferentiated gonads were fed with steroid diet containing different concentrations of E2 during 28 days, from the mouth opening until a period that precedes the gonadal differentiation. Animals were sampled at 90 days post-hatching (dhp) for histology and gene expression analysis. Our results showed that E2 disrupted both phenotypic and histological sex ratios, and surprisingly, induced intersex with testis-ova in the concentrations of 2 and 6 mg E2/Kg food. This data is a clear evidence that the treatment used was not enough to induce complete sex reversal in A. altiparanae. However, in terms of gene expression, E2 (6mg/Kg food) induced a remarkable gonadal plasticity between males and females at 90 dph. The males, named as E2 resistant males, overexpressed the male-biased genes, such as dmrt1, sox9 and amh. We suggested that these males were able to resist the E2-induced feminization by the expression of genes related to testis differentiat... (Complete abstract click electronic access below)
Doutor

La molécule de l’éthylène (C2H4) est le régulateur principal du sexe chez les cucurbites. Essentiellement, l’éthylène est connu pour son rôle promoteur dans le développement des carpelles et un rôle inhibiteur des étamines dans les fleurs du melon. L’interaction entre les biosynthétique gènes de l’éthylène (CmACS7, CmACS11, et CmACO3) et le facteur de transcription CmWIP1 détermine différentes formes du sexe chez le melon. Le rôle de ces gènes est bien étudié chez le melon. Cependant, le mécanisme qui contrôle l’initiation et la coordination de formation des étamines et des carpelles dans la fleur reste ambigu. En reposant sur l’importance de l’éthylène dans l’expression du sexe chez le melon, j’ai focalisé sur l’identification des gènes impliqués dans la voie signalisation éthylène-sexe. Au cours de la thèse, le criblage des mutants altérés dans la réponse à l’exogène éthylène nous facilitait d’identifier des nouveaux gènes impliqués dans la détermination du sexe chez la famille de Cucurbitacée. Pendant ma thèse j’ai isolé plus de 10 mutants insensibles à l’éthylène de différentes populations du melon. Deux mutants ont été isolés de deux populations monoïques indépendantes. Ces deux mutants provoquent une transition partielle et complète au melon andromonoïque dans la génération M2, respectivement. Un de ces deux mutants a été identifié et caractérisé. Deux autres mutants gsn106 et vat233 ont été criblés de deux populations andromonoïque, provoquent une transition complète et partielle à androïque melon, respectivement. En utilisant le séquençage à haute débit et les analyses génétiques j’ai essayé de cloner et caractériser ces gènes mutants. Par ailleurs, des autres mutants insensibles à l’éthylène sont en cours d’être phénotypes pour le phénotype du sexe. L’isolation et caractérisation des nouveaux gènes impliqués dans le déterminisme du sexe nous aidera pour mettre en place un model clair explant comment le sexe est contrôlé chez les plantes
Ethylene (C2H4) is an important phytohormone in plants and the main sex regulator in the family Cucurbitaceae. As known, the ethylene promotes the carpel development and inhibits the stamens in the melon flower (Cucumis melo L.). The interplay of the biosynthesis genes (CmACS7, CmACS11, et CmACO3) and the transcription factor CmWIP1 generates different sexual forms in melon. The role of these genes in the sex expression is well studied. However, the mechanism that controls the initiation and coordination of stamen and carpel development in the flower remains ambiguous. Based on the importance of the ethylene in the sex determination, I aimed to isolate novel genes involved in the pathway ethylene-sex in the melon (Cucumis melo L.). For this purpose, I used the response to exogenous ethylene in the etiolated seedlings (known as the triple response phenotype) to isolate ethylene-insensitive mutants. During my thesis I isolated more than 10 ethylene-insensitive mutants from six EMS-mutagenised melon populations. Some of these mutants induced changes in the sex expression of the melon. . Two mutants were isolated from two independent monoecious populations (female and male flowers on the same plant) and induced a partial and a complete sexual transition to the andromonoecious melon in the second generation M2, respectively. One of them was cloned and characterized using Omics tools. Two other mutants (gsn106) and (vat233) screened from two independent andromonoecious melon (bisexual and male flowers on the same plant) populations, induced complete and partial sexual transitions into androecy (only male flowers), respectively. Using Next-Generation Sequencing (NGS) and the genetic analysis, we are trying to clone and characterise these mutants (gsn106) and (vat233). In the same way, we continue to observe others promising ethylene-insensitive mutants (vat306, vat175, and vat230) for the sex phenotype. The isolation and characterisation of novel genes involved in the sex determination will permit to provide a new and clear model explains of the sex determination mechanism in plants

Chez les mammifères, la détermination sexuelle résulte dans le développement d’un individu mâle ou femelle. Le développement correct de deux sexes dimorphiques dotés d’organes reproductifs fonctionnels est primordial pour la survie des espèces. La détermination sexuelle chez les mammifères a une base génétique et les deux sexes sont génétiquement distincts dés la fertilisation. Les embryons féminins possèdent deux chromosomes X et les masculins un chromosome X et un Y. L’expression de SRY déclenche le développement testiculaire en assurant une expression stable et prolongée du gène SOX9 dans les cellules somatiques de la gonade masculine. Dans les gonades féminines, l’expression de SOX9 est activement réprimée et permet le développement ovarien. Ces événements sont placés en haut de la cascade de détermination sexuelle mais, même si beaucoup de facteurs ont été identifiés, beaucoup restent encore ignorés ou mal connus. Pour identifier de nouveaux facteurs génétiques qui contrôlent la détermination sexuelle chez l’Homme j’ai étudié plusieurs individus ayant des troubles de détermination sexuelle (DSD) - troubles congénitaux où le développement sexuel chromosomique, gonadique ou anatomique est atypique. Pendant ma thèse, mon but a été de mieux comprendre le rôle de trois gènes – NR5A1, GATA4 et FOG2 – dans le développement et fonction des gonades. Mutations dans NR5A1 sont associées à une insuffisance ovarienne précoce et à une infertilité masculine. Mutations dans GATA4 et FOG2 sont associées à des cas de 46,XY DSD avec ou sans phénotype cardiaque. Mes résultats ont démontré le rôle de ces gènes dans la cascade de détermination sexuelle chez l’Homme

Aedes aegypti is one of the most important mosquito vectors of human disease, transmitting dengue, Chikungunya and yellow fever viruses. New control methods are much needed for this species and an ability to skew sex ratios toward males would be very useful for a number of strategies. In this study, male-specific chromosomal regions were searched for in Ae. aegypti using novel approaches such as microdissection of chromosomes. The sex determination pathway of Ae. aegypti was also explored, using Drosophila melanogaster as a model, to find candidate genes that could be used to induce male biased sex ratios. The transformer-2 (tra-2) gene is necessary for sexual differentiation of females in D. melanogaster, but its role remains unknown in mosquitoes. A homolog of tra-2 was identified in Ae. aegypti. Rapid Amplification of cDNA ends (RACE) experiments were conducted to characterize this gene in Ae. aegypti. The Ae. aegypti tra-2 gene, as for D. melanogaster, was highly variable in transcription due to alternative splicing and alternative polyadenylation, with 9 different variants identified. RNA interference (RNAi) was then used to determine if knockdown of all variants of Ae. aegypti tra-2 can be achieved and if it would cause gender switching in individuals, initially by means of direct injection of double stranded RNA, and then progressing to germline transformation. A construct designed to produce tra-2 dsRNA was injected into Ae. aegypti embryos and integrated into the Ae. aegypti genome. The transgenic population showed up to 100% male bias in single pair crosses. The male bias effect could still be seen in the heterozygous population. The results suggest that knockdown of tra-2 could provide a useful tool for sex ratio distortion as part of the development of novel control methods for Ae. aegypti.

In this work, the evolution of sex determination gene networks is inves tigated using a modelling approach. Recent evidence indicates that an in crease in the complexity of interactions has played an important role in gene network evolution. Sex determination mechanisms offer a good model for studying gene network evolution because, among other reasons, they evolve rapidly. In chapter 2, the potential for evolutionary change of the existing Drosophila sex determination gene network is considered. With the aid of a synchronous logical model, theoretical concepts such as a network-specific form of mutation are defined, as well as a notion of functional equivalence between networks. Applying this theoretical framework to the sex deter mination mechanism, it is found that sex determination networks generally exist within large sets of functionally equivalent networks all of which satisfy the sex determination task. These large sets are in turn composed of sub sets which are mutationally related, suggesting a high degree of flexibility is available without compromising the core functionality. The technique for finding functional equivalence between networks suggests a general method for gene network reconstruction, which is explored in chapter 3. Lastly, in chapters 4 and 5, a hierarchical model is presented which integrates popu lation genetics techniques with network dynamics. This model consists of a core population genetics simulation within which parameters such as the sex and fitness of the genotype are calculated from the corresponding network dynamics. The model is used to investigate the early evolution of sex deter mination networks. Following from a hypothesis proposed by Wilkins (1995), the assumption is made that sex determination networks have evolved in a retrograde manner from bottom to top. Starting from the simplest possible ancestral system, based on a single locus, we explore the way in which more complex systems, involving two or three loci, could have evolved.

Many reptiles display temperature-dependent sex determination (TSD), in which the primary sex is determined by incubation temperatures rather than sex chromosomes. However, temperature is not the only factor that play critical roles in sex determination in the species with TSD. Previous studies in the snapping turtle, a species with TSD, showed that dihydrotestosterone (DHT) induces ovary development at temperatures that normally produce males or mixed sex ratios. In addition, the feminizing effect of DHT was found to be associated with increased expression of the ovary-determining gene Foxl2, suggesting a potential androgen-Foxl2 regulatory mechanism. This dissertation aims to clarify the molecular mechanisms underlying TSD in several aspects. First, determine the role of androgen in TSD; second, identify novel thermosensitive genes involved in TSD and lastly, reconstruct gene regulatory networks underlying sex determination.

To test the hypothetical androgen-Foxl2 interaction, I cloned the proximal promoter (1.6 kb) and coding sequence for snapping turtle Foxl2 (tFoxl2) in frame with mCherry, a red fluorescent protein. The tFoxl2-mCherry fusion plasmid or mCherry plasmid were stably transfected into mouse KK1 granulosa cells. Although expression of tFoxl2-mCherry was not affected by androgen treatment in KK1 cells, androgen inhibited expression of the endogenous mouse Foxl2 gene, suggesting the androgen-Foxl2 interaction does exist but it differs between species. We also found tFoxl2-mCherry potentiated low dose DHT effects on aromatase expression, which has not been reported in any other studies.

To identify novel sex-determining genes in TSD, I first de novo assembled and annotated the transcriptome of the snapping turtle using next-generation sequencing (NGS) and then performed RNA-seq analyses on the newly assembled reference transcriptome. With the differential gene expression analyses, I identified 293 thermosensitive genes. Among these genes, I find AEBP2, JARID2, and KDM6B of particular interest because these genes could influence expression of many other genes via epigenetic modifications.

To further investigate the molecular mechanisms underlying sex determination, I reconstructed gene regulatory networks using an entropy based network reconstructing algorithm—ARACNE with public microarray experiments in mouse gonads. The subsequent hub gene analyses revealed the basic molecular pathways underlying gonadal development and the master regulator analyses identified 110 candidate sex-determining genes including both known sex-determining genes and novel candidate genes.

My findings demonstrate that androgens can influence expression of key ovarian genes but further studies are needed to understand the androgen signaling in TSD. Furthermore, my study provides a first description of the snapping turtle transcriptome and the effects of temperature on transcriptome-wide patterns of gene expression during the TSP. In addition, hub genes and master regulators identified for mammalian gonad determination will guide the direction of future studies in the field of sex determination. However, additional studies are needed to validate the computational findings.

Abstract Mammalian female sex development was considered a default developmental pathway. However, the deletion of the Wnt4 gene, a member of the Wnt family of secreted signals, was shown to reverse the sex of XX female mouse embryo and caused exhibition of certain male characteristics. This indicated that the female sexual development cannot be default but depends on active signaling and cell-cell interaction. The aim of the current study was to reveal the functional role of the Wnt4 gene in the control of sex determination, ovariogenesis and female sex duct formation. This study demonstrates that testosterone is produced by the ovary of Wnt4-deficient female embryos. The inhibition of androgen action by an antiandrogen, flutamide, during gestation leads to complete degeneration of the Wolffian ducts in 80% of the Wnt4 mutant females. This suggests that testosterone is the possible mediator of the masculinization phenotype in Wnt4-deficient females. Wnt4 is expressed by ovarian somatic cells, which are vital for the control of female germline development. This work has shown that Wnt4 is the factor maintaining germ cell cysts, cell-cell interaction and early follicular gene expression. In addition, the findings indicate a critical role for Wnt4/5a signaling in meiosis. Our research has proven that Wnt4 has roles during postnatal ovary development as its defective signaling leads to premature ovarian failure associated with diminished Amh levels, defective basement membrane and cell polarization. The Mullerian duct, the anlagen of oviduct, uterus and upper part of vagina, does not form in Wnt4-deficient females. This study indicates that Wnt4 is needed for migration initiation and maintenance during Mullerian duct formation prenatally. During the postnatal uterine differentiation Wnt4 is essential for endometrial gland formation. The present study provides new evidence for Wnt4 function during embryonic and adult female sexual differentiation
Tiivistelmä Nisäkkäiden naaraspuolista kehitystä pidettiin aiemmin sukupuolisen erilaistumiskehityksen oletusarvona. Signaloivien proteiinien Wnt-perheeseen kuuluvan Wnt4-geenin puutteen todettiin kuitenkin johtavan XX naarasalkion sukupuolen kääntymisen koiraaksi sekä aiheuttavan tiettyjä koiraille ominaisia piirteitä. Tämä osoitti, ettei naaraspuolinen kehitys ole oletusarvo, vaan se riippuu aktiivisesta signaloinnista ja solujen välisestä interaktiosta. Tämän väitöstutkimuksen tarkoitus oli selvittää Wnt4-geenin roolia sukupuolen määräytymisessä, munasarjojen kehittymisessä sekä naaraan sukupuolitiehyitten muodostumisessa. Tutkimuksessa osoitettiin, että munasarjat tuottavat testosteronia niillä naaraspuolisilla alkioilla, joilta puuttuu Wnt4-geeni. 80 prosentilla naaraista, joilla on Wnt4-geenin puute, androgeenivaikutuksen esto raskauden aikana annettavalla antiandrogeenilla, flutamidilla, estää sukupuolen vaihtumisen fenotyypin. Tämä viittaa siihen, että testosteroni toimii mahdollisena koiraan fenotyypin välittäjänä naarailla, joilta puuttuu Wnt4-geeni. Wnt4 ilmentyy munasarjojen somaattisissa soluissa, jotka ovat tärkeitä naaraspuolisen ituradan kehityksen säätelyn kannalta. Väitöstutkimus osoittaa, että Wnt4 on itusoluryppäitä, solujen välistä interaktiota sekä varhaista follikkeligeeni-ilmentymistä ylläpitävä tekijä. Tulokset osoittavat myös, että Wnt4/5a -signaloinnilla on tärkeä rooli meioosissa. Tutkimus osoittaa lisäksi, että Wnt4 vaikuttaa munasarjojen kehitykseen myös syntymän jälkeen. Puutteellinen signalointi alentaa Anti-Müllerian hormonin tasoa, heikentää tyvikalvoa ja vähentää solujen polarisaatiota, joka johtaa ennenaikaiseen munasarjojen toiminnan hiipumiseen. Müllerin tiehyet, joista myöhemmin kehittyvät munanjohtimet, kohtu ja vaginan yläosa, jäävät kokonaan muodostumatta naarailla, joilta puuttuu Wnt4-geeni. Tulokset viittaavat siihen, että Wnt4 on tarpeen alkioaikaiseen Müllerin tiehyen muodostavien solujen liikkeellelähtöön ja ylläpitoon. Wnt4:llä on myös keskeinen rooli kohturauhasten muodostumisessa sukukypsyyden saavuttamisen aikana ja sen jälkeen

The accurate determination of sex is the first analytic task of physical anthropologists in the study of human skeletal remains. Correct sex determinations are essential because most other skeletal analyses rely on this to be accurately determined. Although morphological features of the skull and pelvis have been traditionally used for sex determination, certain metric analyses have also been useful. In the absence of the most commonly used bones, metric measurements obtained from the tarsals have the ability to provide accurate information regarding sex. Metric sex assessments are based on the levels of sexual dimorphism between males and females. This thesis evaluates the presence of sexual dimorphism in the tarsals and their dimensions of length, width, and height and assesses which bones and dimensions are most useful for sex determination using a modern skeletal sample (n = 160) comprised of individuals of known sex and age. Summary statistics were calculated for males and females separately to assess the sexual variation for each measure. Pearsonâs Correlation Coefficient was used to establish correlations between sex and each measurement. Logistic regression analysis was used to determine the levels of sexual dimorphism in the bones and dimensions for the sample. Accuracy of sex estimations was compared between the left and right sides for each bone to determine if one side was more sexually dimorphic than the other. Logistic regression coefficients from the most accurate bones and dimensions were generated from the sample, which can be used to develop equations to accurately sex other samples of similar Euro-American ancestry. The most sexually dimorphic tarsals were the talus and first cuneiform. Tarsals from the right side were slightly more sexually dimorphic than those from the left. Length and height dimensions were more variable than breadth dimensions. Correct classifications for individual variables were as high as 87.8 %, with the highest overall percentage of correct classification of 93.5% obtained from a combination of all height measurements from the right tarsals for the total sample. This has implications for sexing skeletal remains from archaeological samples when other more commonly used bones are absent or poorly preserved.

Abstract There are a remarkable variety of sex determination systems among different animal taxa. In most animals, sex is determined chromosomally. Although in an increasing number of animals sex determination has been found to be influenced primarily by the environment. Species with genotypic sex determination (GSD) have their sex determined at the time of fertilization, by genetic factors alone and those with environmental sex determination (ESD) have their sex determined by environmental factors that act after fertilization. Temperature-dependent Sex Determination (TSD), whereby the sex of the developing embryos depends on the temperature at which they develop is widespread in oviparous reptiles and occurs in all crocodilians, marine turtles and tuatara examined to date and is common in many freshwater turtles and lizards. SECTION ONE Temperature-dependent sex determination (TSD) was never expected to occur in viviparous reptiles, as thermoregulation by pregnant females would result in relatively stable gestation temperatures. Temperature-dependent sex determination and viviparity goes against all the basic assumptions that TSD occurs in oviparous reptiles where temperatures within a nest vary widely. However, skewed sex ratios as a result of incubation temperature indicated the possibility of TSD in the viviparous lizard Eulamprus tympanum. In my first experiments I show the first recorded case of a viviparous reptile with TSD. The developing embryos of the viviparous skink E. tympanum are subject to TSD, with gestation temperature having a highly significant effect on sex and warmer temperatures giving rise to male offspring (Chapter 1). Sex is fully determined at the time of birth and can be differentiated histologically into testes or ovaries (Chapter 2). The morphology and histological characteristics of the gonads of neonatal E. tympanum resulting from the treatment temperatures described in chapter 1 illustrate that sex in E. tympanum is easily distinguished at the time of birth and corresponds with the presence or absence of hemipenes. Males are histologically characterised by an elongated gonad consisting of seminiferous tubules with either no cortical epithelium or, if present at all, in a very thin band. If they are present, M�llerian ducts, showing signs of degeneration, are attached to the kidney by a shortened mesosalpinx. Females are histologically characterised by an irregularly shaped gonad consisting of a thick cortical epithelium that occasionally contains oocytes. The M�llerian ducts are obvious structures attached to the kidney by a fibrous mesosalpinx. The presence or absence of hemipenes is a reliable technique for determining sex in newborn E. tympanum. Sex determination is easiest to perform on neonates within the first few days of birth as hemipenes become increasingly difficult to evert as neonates age, however, with practice they are easily identified without full eversion. SECTION TWO The thermal biology of E. tympanum in the field is restricted by both the thermal properties of their habitat (Chapter 3) and behavioural modifications when faced with a predation threat (Chapter 4). The available temperatures in the field suggest that TSD is biologically relevant in the species and not just a laboratory artefact; E. tympanum can attain mean selected temperatures achieved in the laboratory but the proportion of time at the temperature is restricted. Females actively thermoregulate in the field, although they are restricted in their efficiency of thermoregulation by environmental constraints, for example, microhabitat structure, weather conditions, predator avoidance and social ranking. The highly territorial nature and high densities of E. tympanum present in Kanangra Boyd National Park potentially force less dominant individuals into less favourable habitats that are significantly cooler. An important point is that gravid females in more favourable habitats in the period encompassing the middle third of development (the assumed sex determining period) are selecting higher temperatures, with lower variance and have greater thermoregulatory efficiency than during the rest of pregnancy, therefore, thermoregulating more precisely during this thermosensitive period (Chapter 3). Chemosensory cues provide important information on the risk of predation. Hence, chemoreception is a common mechanism used by many species to detect the presence of, and subsequently respond to, a potential predator. The perceived risk of predation may force retreat to sub-optimal conditions, forcing a trade-off between the risk of predation and the ability to acquire resources. The basking regime maintained by gravid female E. tympanum, can directly alter sex ratios of offspring produced through temperature-dependent sex determination (Chapter 1). The avoidance of predator scents may restrict basking ability and in turn alter the sex of offspring produced. I measured responsiveness to chemical cues using tongue flicks as an indicator of chemical discrimination in females of different reproductive condition. I then measured activity and basking behaviour of gravid and non-gravid females in experimental enclosures in the presence of various chemical stimuli to determine if basking opportunity is compromised by the presence of a predator scent. Females respond differently depending upon reproductive condition, with gravid females responding most significantly to a predator scent. Activity, basking frequency, and time spent in the open (basking duration) are significantly reduced in gravid females in the presence of a predator stimulus. Under laboratory conditions, gravid females modify their behaviour and forego the opportunity to bask when there is a perceived predation risk (Chapter 4). SECTION THREE As female viviparous reptiles can regulate the temperature of the embryo by maternal temperature selection (Chapter 1), the occurrence of TSD in E. tympanum opens the possibility for females to select the sex of offspring. Reproducing females may benefit by facultatively adjusting their investment into sons over daughters or vice versa, in response to population wide shifts in adult sex ratios. Female E. tympanum, can manipulate the sex of their offspring in response to sex imbalances in the population using temperature-dependent sex determination (Chapter 5). When adult males are scarce, females produce male-biased litters and when adult males are common, females produce female-biased litters. The cues used by a female to assess the adult population are not known, but presumably depends upon the female�s experience throughout the breeding season and is the subject of further investigation (Chapter 6). The maternal manipulation of offspring sex ratio in E. tympanum suggests a selective advantage of temperature-dependent sex determination. Any facultative sex ratio response needs to recognise the scarcity of one sex in order to overproduce that sex in the next generation; offspring sex ratio will vary inversely with adult sex ratio. Maternal sex allocation in E. tympanum is linked with population (or adult) sex ratio (Chapter 5), and one of the mechanisms by which females recognise an imbalance may be linked to visual recognition of males (Chapter 6). Females maintained throughout pregnancy without any male stimulus produce entirely male offspring (Chapter 5). In contrast females exposed to male stimulus produce both sexes (Chapter 5). Females respond differently to varying degrees of male stimulus and visual recognition of males in a population may be more important than chemoreception. In the absence of visual cues, females produce more male offspring, even when chemosensory cues are present (Chapter 6). The study system presented here offers many advantages over oviparous species with TSD, due to E. tympanum being relatively short lived and fast maturing. Thus, the fitness consequences over multiple generations as a result of gestation can be investigated. Viviparity allows maternal control of embryonic temperature during gestation and a means of maternal sex allocation. Until now the maternal side of TSD and sex allocation has been where the mother deposits her eggs and the allocation of sex steroid hormones at oviposition, both of which have been difficult to study. The work presented and the study system itself should inspire great interest in TSD and viviparous reptiles.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007.
Additional advisors: Asim Bej, Gene Hines, Douglas Watson, Douglas Weigent. Description based on contents viewed Oct. 2, 2008; title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--University of Sydney, 2004.
Title from title screen (viewed 5 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Biological Sciences, Faculty of Science. Degree awarded 2004; thesis submitted 2003. Appendices contains published articles co-authored by Robert. Includes bibliographical references. Also available in print form.

Interactions among living organisms fall along the mutualism-parasitism continuum; where mutualistic interactions benefit one or both organisms and parasitic interactions harm them. Mutualisms are a particularly interesting form of interaction as their evolutionary stability is constantly at risk of destabilisation by cheaters which take greater advantage of their partners than other symbionts in the population. This has resulted in organisms involved in mutualistic interactions have evolved many mechanisms to prevent destabilisation by cheaters. Insects are involved in mutualistic interactions with a myriad of organisms, in particular micro-organisms. Some of the most well documented insect–micro–organism interactions are those of the fungus–farming insects; Attine ants, termites and ambrosia beetles, and the obligate mutualistic fungi they cultivate. These mutualisms have remained stable over millions of years. Another, less well studied, apparently stable, obligate insect-fungus mutualism is the interaction between Sirex woodwasps and Amylostereum fungi. In this review we examine the evolution of mutualisms from initial interaction, through to maintenance of a stable obligate interaction, and explore the mechanisms that act to stabilise them. We explore the evolutionary and ecological factors necessary for the maintenance of the Sirex–Amylostereum mutualism in the context of work that has been done on the evolution of other more extensively studied insect-fungus mutualisms.
Dissertation (MSc)--University of Pretoria, 2014.
Genetics
MSc
Unrestricted

Thesis (Ph. D.)--Georgia State University, 2007.
Title from file title page. Mattew S Grober, committee chair; Kim Wallen, Charles Derby, Laura Carruth, Tim Bartness, committee members. Electronic text (107 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Jan. 31, 2008. Includes bibliographical references. (p. 94-107)

Sry on the Y-chromosome triggers the fetal gonad to begin differentiation into testis in mammals. Mutation or absence of Sry results in development of ovaries and the female phenotype. However, XY sex reversal in the presence of wild-type Sry exists in mice and man. One such example is the B6-YTIR mouse, whose autosomes and X-chromosome are of the C57BL6/J mouse (Mus musculus molossinus) whereas the Y-chromosome is from a mouse originating in Tirano, Italy (Mus musculus domesticus). B6-YTIR mice develop only ovaries or ovotestes in fetal life. The objective of my thesis was to identify the mechanism of sex reversal in the B6-YTIR mouse. The results indicate that onset of Sry transcription in B6-YTIR gonads is comparable to control B6 XY gonads. On the other hand, onset of Mis, 17alpha-HA, 3beta-HSD (testicular cell products), p450arom as well as inactivation of Sry transcription are delayed or absent in the sex reversed gonads. It has been suggested that low levels of Sry transcription may account for aberrant testis differentiation in B6-YTIR mice. We observed relatively low levels of Sry transcripts not only in B6-YTIR but also in B6 mice. However, levels in normal B6-YSJL mice were significantly greater. On the SJLB6F1 background, where no sex reversal occurs, Sry transcript levels of the TIR allele increased while those of B6 and SJL alleles remained the same as in the B6 background. Thus, low levels of Sry transcript from the B6 allele are sufficient whereas the levels from TIR and SJL alleles (both DOM type) appear to be critical for testis determination. We then compared the levels of endogenous Sry proteins. A combination of immunoprecipitation and immunoblotting succeeded in detecting a protein band whose expression profile and molecular size are consistent with those of the predicted Sry. Sry protein levels in B6-Y TIR gonads were roughly two fold greater than in B6 XY gonads. We hypothesize that the Sry protein of the TIR/SJL alleles is less efficient

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.

This study set out to develop molecular biological techniques for the study of sex determination in the tilapias, and, in particular, in Oreochromis niloticus (Linnaeus), Lake Manazala strain. Two heterospecific probes, pDP1007 (supplied by Dr. D. Page) which gives a Y-chromosome specific hybridization pattern in humans, and pUGD0600 (supplied by Dr. S. Mizuno) which is W chromosome specific in chickens, were tested for sex specificity in Oreochromis niloticus. Results from hybridizations of these probes to Southern blotted digests of O.niloticus DNA are reported. Both probes hybridized to O.niloticus genomic DNA but neither gave sex specific patterns. Four short repeat oligonucleotide sequences were synthesized. Two sequences were labelled both non-radioactively, with dioxygenin and with ³²P and were used to probe Southern blots of O.niloticus DNA and dried agarose gels containing O.niloticus DNA. The probes (GATA)₄(GACA)₁ and (GATA)₃(GACA)₂ both gave fingerprint like patterns but no sex specific banding patterns were observed. The implications of these results for the organization of repetitive sequences in the O.niloticus genome are discussed. A partial DNA library, enriched for male specific sequences was created by subtractive hybridization using the Phenol Emulsion Reassociation Technique (PERT). Thirteen recombinant colonies containing inserts were isolated. Inserts from the library were screened for sex specificity. Two inserts were partially sequenced and PCR primers for them were constructed which were used to amplify the sequences in genomic DNA. Both sequences were successfully amplified from genomic DNA but neither was male specific. The amplified sequences were restricted with a number of restriction enzymes to identify sex specific restriction fragment length polymorphisms (RFLPs) but, again, none were observed. Sequences were also tested for similarity to sequences in the EMBL/GENBANK nucleotide databases using the suite of programs available on SEQNET (Daresbury laboratory). Future approaches to the isolation of sex specific DNA sequences in the Tilapias are discussed in detail.

The PhD research studied two aspects in tilapia, firstly the analysis of sex determination in Nile tilapia (evidence of complex sex-determining systems) and secondly the genetic management of the tilapia species, using different genomic analysis approaches. This research started with the development of two techniques: minimally invasive DNA sampling from fish mucus, which was found to be suitable for standard genotyping and double-digest restriction-site associated DNA sequencing – ddRADseq; and pre-extraction pooling of tissue samples for ddRADseq (BSA-ddRADseq), which was found to be suitable for identifying a locus linked to a trait of interest (sex in this case). The first molecular evidence concerning the sex determination in genetically improved farmed tilapia (GIFT) was described using BSA-ddRADseq. Given the multiple stock origin of GIFT, surprisingly only a single locus (in linkage group 23) was found to be associated with the phenotypic sex across the population. The first evidence of LG23 influence on phenotypic sex in the Stirling population of Nile tilapia was also found. Different combinations of estrogen hormones and high temperature were tested for feminising Nile tilapia: a combined treatment of estrogen hormone and high temperature was found to be more efficient in feminising Nile tilapia than the estrogen alone. A set of species-diagnostic SNP markers were tested which were found to be suitable to distinguish pure species (O. niloticus, O. mossambicus and O. aureus), and these were used to analyse species contribution to GIFT and a selected tilapia hybrid strain. The results of the current research added novel information to our understanding of sex determination in Nile tilapia, which will be helpful in the development of marker-assisted selection in GIFT and other Nile tilapia strains towards the production of all male offspring. The methods developed also have broader applicability in genetic and genomics research.