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In order to combine previous observations from different sources on sex determination, and to identify sex chromosomes including the major sex determination locus in Nile tilapia, physical and genetic maps based on sex-linked markers and genes (such as sex-linked AFLPs, microsatellites, ovarian aromatase and DMO genes) were integrated and anchored. An accurate physical map using FISH techniques on mitotic cells was developed based on a previous map and 23 tilapia BAC clones previously assigned to linkage groups (LGs) 1, 3, 6, 7, 10 and 12; and on meiotic cells, 2 BAC clones containing the SLAM OniY227 and the dmrt4 gene were mapped. The six linkage groups were then assigned to different chromosomes, but surprisingly, the putative sex LG1 was located to a small submetacentric chromosome and not to the larger subtelocentric chromosome 1, where LG3 was assigned instead. The other LGs were assigned to different chromosomes and oriented with respect to the centromeres. A detailed comparison of the physical distribution of markers on chromosome 1 with respect to LG3 revealed a suppression in recombination in the subtelomeric region of the q arm between the marker GM354 (0 cM) and clcn5 (29 cM) and an abrupt increment of recombination between clcn5 (29 cM) and GM128 (77 cM) close to the centromere (Flpter=0.2). The unpairing region (20% of the total length) observed on the larger bivalents of XY fish during early pachytene in meiotic cells has been confirmed by DAPI staining and FISH to be at the terminal part of the q arm, opposite to the centromere. Comparison with six other tilapia species (2n=44) revealed a well conserved karyological distribution of the suspected LGs associated with sex determination (1 and 3). Besides, in O. karongae (2n=38) it was shown by SATA and UNH995/UNH104 marker hybridisation that LG1 has been re-arranged into the subtelomeric chromosome 2 as a result of a telomere-telomere fusion. A pool of 15 tilapia BAC clones previously localised on chromosome 1 and containing sex-linked AFLPs, dmrt1, dmrt4 and several SINEs were screened for new microsatellites; BACs were digested with SAU3AI and TC, GT, ATCT and CTGT probes radio-labelled with 32P. The high abundance of repetitive sequences in the BACs used led to only one useful polymorphic and co-dominant marker being obtained, associated to a BAC clone containing a copy of the dmrt1 gene on chromosome 1 (Flpter=0.85). Four linkage maps were constructed from an XY male, XY neofemale, XX neomale and XX female, mapping 4 and 8 markers on LG1 and LG3 (including the dmrt1 associated microsatellite) respectively. A specific sex-determination locus was identified on LG1 clearly linked with UNH995. However there appeared to be different allelic strengths for this sex determination locus, as shown by different sex ratios associated with different UNH995 genotypes. Additionally, one of the two XX fish mapped, showed the location of the recessive black blotching trait on LG3 (chromosome 1) between the markers GM128 and GM526, close to the centromere (Flpter=0.14). The results presented suggest a nascent Y chromosome in early stage of differentiation in Nile tilapia and with a functional master gene on LG1 close to the marker UNH995 (Flpter=0.67) located on the q arm of a small submetacentric chromosome. The potential influences of the autosomal LG3 (chromosome 1) in sex differentiation are also discussed.

Thèse--Université de Strasbourg. / "Index bibliographique": p. 483-497.

Inaug.-diss.--Bonn. / Lebenslauf. "Literaturangabe": p. [28]-29.

Inaug.-diss.--Göttingen. / Lebenslauf. "Verzeichnis der benutzten literatur": p. 26-27.

By controlling the inheritance of sex, the sex determination mechanism constrains sex allocation strategies and sex ratio adaptation; however, sex ratio selection also influences the evolution of sex determination mechanisms. Much of the sex determination literature focuses on how sex determination mechanisms transition between genetic and environmental factors (i.e. GSD vs. ESD), and if genetic sex factors are involved, how many (e.g. chromosomal vs polygenic systems). The study of sex allocation largely focuses on deviations in sex ratio from a theoretically 1:1 evolutionarily stable strategy, such as when sex ratios reflect ‘cost’ differences between the sexes. Tigriopus californicus is a tidepool copepod with polygenic sex determination, and shows wide variability in sex ratios in the field and lab that cannot be explained by genetic and stochastic processes alone, which suggests that an environmental variable might influence sex ratio. Females and their offspring were fed diets of different nutritional quality in a crossed design, and the sex ratio of each clutch was recorded for up to 8 clutches from a given female: this design allowed the influence of female diet vs. that of her offspring to be distinguished. The clutch sex ratio changed over the laying order according to the offspring’s diet, which is evidence for environmental sex determination in this species. Sex ratio also showed the influence of maternal diet, consistent with sex allocation theory. While dietary carotenoids showed no association with sex ratio or clutch size, long chain polyunsaturated fatty acids (particularly EPA and DHA) were implicated as the agent of sex ratio effect, providing a direction for future studies. The situation of T. californicus at the intersection of major themes in sex evolution makes this system an ideal model for selection studies. / Graduate / 2018-09-13

Chum salmon embryonal discs were smeared and the cells stained with acetocarmine (60° C.) for chromosome content. Thirty-eight chromosomes (granular, sigmoid, and j-shaped) were counted, but the number was not considered absolute. The presence of sex chromosomes could not be established. Chromosomes in cells of differentiating gonads showed such excessive clumping that a count could not be made. Sex differentiation was followed in chum salmon. Sections from alevins (three weeks old) showed the gonads to be indifferent. Isolated nests of germ cells appeared which eventually formed a continuous cord (five weeks). At nine weeks the primordial ovary developed oocytes and in the succeeding week formed an endovarial canal. At this time the testis was first recognized (ten weeks). It was a small compact organ containing spermatogonia. Up to the final examination (fifteen weeks) the ovary showed no new structures excepting a prominent vena commites anteriad, while the testis retained its primitive features. The sex of pink and chum salmon fry migrants were shown by dissection. Ultraviolet light and temperature treatment of developing chum, pink, and sockeye embryos did not show that these factors would influence the sex ratio. Temperature data were decidedly inconclusive. An equal distribution of the sexes in pink and chum fry and sockeye smolts was found in data compiled from migrant runs at Port John, B. C. It is suggested that precocious male sockeye mature in the sea. The sex ratios at stages in the life history of the pink salmon are presented to show that there had occurred a natural sea mortality of 40.2 percent. / Science, Faculty of / Zoology, Department of / Graduate

Könsbestämning hos vertebrater är ett komplext område som har intresserat forskare under en lång tid, trots detta är det i dagens forskningsläge inte helt självklart vad det är som avgör kön. Syftet med denna litteraturstudie är att identifiera och sammanställa kunskap om avgörande könsbestämningsmekanismer utifrån det aktuella forskningsläget. I samband med detta kommer arbetet undersöka hur exempel från könsbestämningssystem hos vertebrater kan användas och tillämpas i biologiundervisning på gymnasiet. Arbetet kom fram till att det är flera gener och proteiner som deltar i könsbestämningen, samt att många av dessa konserverats genom evolutionen. Dock betyder inte detta att alla grupper har samma könsbestämningsmekanismer utan det fanns en stor variation mellan vertebratgrupper. Jämförelser av könsbestämningssystemen visade på nya evolutionära samband mellan genetisk och miljöbetingad könsbestämning. Avslutningsvis konstateras möjliga tillämpningar i biologiundervisningen på gymnasiet. Exempel från olika könsbestämningssystem hos vertebrater kan eventuellt användas för att ge elever nya naturvetenskapliga perspektiv på könsbestämning och utveckla biologilärarens arbete med sexualitet, samtycke och relationer. / Sex-determination in vertebrates is a complex area of research that has interested researchers for a long time, still to this day it is not entirely clear what determines sex. The purpose of this literature review is to identify and summarize knowledge about crucial sex-determining mechanisms based on the current state of research. Regarding this review it will investigate how examples from the sex determination system in vertebrates can be used and applied in biology education at high school. The work concludes that there are several genes and proteins that are a part of the sex-determination, and that many of these have been conserved during evolution. However, this does not mean that all the vertebrates have the same sex-determining systems; rather there is a large variance within the vertebrates. Comparison of the sex-determining systems revealed new evolutionary relationships between the genetical- and the environmental sex-determining system. Finally, possible applications in biology education in high school were discussed. Examples from different sex-determining systems in vertebrates could potentially be used to provide students a new scientific perspective of sex-determination and develop the teacher’s work with sexuality, consent and relationships.

<p>A unique feature of the fetal gonad is its ability to form two distinct organs, the testis and the ovary, from a single bipotential primordium. The outcome of this decision, which is made by a population of somatic cells known as the bipotential supporting cell precursors, determines whether an embryo will develop as a phenotypic male or female. Though several molecular pathways have been shown to be required for female fate determination in vertebrates, the intricacies of ovarian morphogenesis are not well understood. A key event in ovarian development occurs around birth, when meiotic germ cells and somatic granulosa cells organize into primordial follicles, the structures that generate mature oocytes for ovulation in adult females. We investigated the embryonic origins and proliferative properties of granulosa cells in the fetal mouse ovary and found that the precursors emerge from the ovarian surface epithelium and then enter mitotic arrest in a specification process that extends from the bipotential stage to the end of the postnatal follicle assembly period. Maintenance of cell cycle arrest in granulosa cell precursors appears to be regulated by Wnt signaling. The first granulosa cells to be specified were exclusively incorporated into the subset of follicles that begin to grow immediately upon assembly. We show that this first group of granulosa progenitors derives from the supporting cell precursors present in the bipotential gonad. Interestingly, both XX and XY supporting cell precursors were mitotically arrested towards the end of the bipotential period, indicating that adoption of supporting cell fate might be regulated by the cell cycle. We also show that antagonism of Notch signaling may be required for these precursor cells to exit the cell cycle and differentiate.</p><p>In Witschi's classic model of vertebrate gonad development, the cortex and medulla of the undifferentiated gonad expand and differentiate in a mutually exclusive manner to yield the mature ovary and testis (Witschi 1951). Estrogen acts on both the cortex and medulla to promote female fate determination and ovary development in non-mammalian vertebrates. However, the downstream receptors and targets through which estrogen exerts its effects on the gonad have not yet been elucidated. We selected the red-eared slider turtle Trachemys scripta as a model with which to address this question. We first characterized the cellular composition of the turtle gonad before and after sex determination, identifying four populations of somatic cells distinguishable by their location within the gonad as well as the complement of transcription factors expressed. This information was then applied to an investigation of estrogen signaling pathways in the turtle ovary. We show that i) estrogen likely acts through its canonical receptors rather than a non-canonical pathway involving ERK signaling; ii) early exposure to estrogen resulted in the premature downregulation of a testis-specific gene, SOX9, in the medulla; iii) less estrogen is needed to promote ovarian differentiation in the cortex of the gonad than to repress testicular differentiation of the medulla, consistent with the localized production of estrogen in the medulla; and iv) estrogen's repressive effect on SOX9 expression may be mediated by Wnt signaling. </p><p>Our findings add complexity to the standard model of how the male and female supporting cell lineages are established in mice, reveal evolutionary conservation between mice and turtles in the timing of granulosa cell specification relative to sex determination., and refine our understanding of how estrogen acts to promote ovarian development in non-mammalian species.</p> / Dissertation

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.