Dissertations / Theses on the topic 'Gonad development'

Links between declining human male fertility (decreased sperm counts, increased incidence of both testicular cancer and genital abnormalities) and the increasing prevalence of endocrine disrupting chemicals (EDCs) in the environment have been reported. We aim to characterise the key developmental processes occurring during human fetal gonad development. Human fetal testis development was characterised by a transient increase in interstitial area proliferation between 13-19 weeks which was accompanied by an increase in steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes. Androgen receptor was expressed by the peritubular myoid cells which had a high bcl-2:bax ratio, indicative of cell survival. Estrogen receptors ( and ) were localised to distinct cell populations. In the ovine gonad similar developmental processes occurred, and comparison with human ovarian development demonstrated interesting parallels. After optimisation, the explant culture system revealed that exposure to the insecticidal EDC dieldrin, at low (<1 ppb) doses reduced LH-stimulated testosterone output in the human fetal testis. This was accompanied by dose-specific changes to the testis proteome, alterations in bcl-2:bax ratios in favour of apoptosis and a down-regulation in StAR expression relative to the LH-treated controls. In conclusion, the processes of proliferation apoptosis, steroidogenesis and steroid action are crucial during fetal gonad development. We demonstrated that in utero exposure to dieldrin may cause reproductive dysfunction in adult life due to reduced steroidogenesis in the fetal gonad; mediated through a down-regulation in StAR expression and alterations in the regulation of gonadal apoptosis.

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

In mammals, the timing of sex determination and a small number of the genes involved in gonadal development have been established. However, other so far unidentified genes are clearly involved in gonadal development, in avian and other species. Chick gonadal development was investigated by studying the morphology of the developing gonads and by attempting to gain a better understanding of the genes involved in the initial stages of this developmental process. The first approach was a histological analysis of chick gonadal development over the period from the indifferent gonad stage to the initial stages of ovary and testis differentiation. Secondly, we analyzed the expression of chick homologues to genes involved in mammalian gonadal development, in an attempt to compare gene regulation and timing of gonadal development between mammals and avians. Finally, we used the technique of differential display to both ascertain the feasibility of this approach in the identification of transcripts in a complex developmental system and to isolate novel genes involved in chick gonadal development. As a result of this study we were able to predict that the sex determination event in chicks occurred earlier in embryogenesis than previously documented. We also established that genes involved in mammalian gonadal development were expressed with similar expression profiles in the developing chick gonads. Finally, twelve candidate clones were isolated by differential display, five of which represented novel sequences. Expression profiles, during chick gonadal development and differentiation, were analyzed by Northern analysis and whole mount in situ hybridization to confirm differential expression.

Wt1 is a complex gene that encodes a protein whose best described function is to act as a transcription factor. Wt1 plays a crucial role in several organ developments, including kidneys, liver, heart or gonads. Moreover, Wt1 is implicated in the etiology of a set of syndromes and diseases ranging from cancer to disorders of sex development (DSD). Additionally, Wt1 has been described to play a role in adult homeostasis. In this thesis, I focused on Wt1 role in development, specifically in gonad and heart morphogenesis. For this purpose, I used two previously described Cre mouse models to generate two different Wt1KOs. The heart is the first organ being formed and it is primarily composed of three layers: the endocardium, the myocardium and the epicardium. The epicardium is a mesothelial layer of cells covering the vertebrate heart surface. Epicardial cells give rise to epicardial derived cells (EPDCs), progenitors of crucial cell types in heart development like vascular smooth muscle cells and cardiac fibroblasts. Moreover, the epicardium contributes to heart morphogenesis by secreting paracrine factors. After myocardial infarction, the epicardium embryonic genetic signature is reactivated. Thus, understanding the mechanisms behind epicardial development constitutes a topic of general interest. The epicardial genetic program has been previously characterized and the Wt1 role in the regulation of this program has been established. One of the pathways modulated during epicardium development is the BMP4 pathway. The results of this thesis demonstrate that WT1 directly regulates Bmp4 expression. Additionally, exposed results demonstrate how the BMP4 pathway is crucial for epicardial cell maturation, through the regulation of cell morphology, from a cuboidal to a squamous cell shape, epicardial proliferation, and transcriptomic changes. Finally, the data shown in this work indicated that changes and mechanisms described for epicardium may be a phenomenon extrapolated to other mesotheliums like the lung mesothelium. To better study Wt1 role in the development of other organs, we have characterized the recombination activity of a Wt1Cre mouse model using two different reporter mouse models, the R26RmTmG and the R26RtdRFP. Results demonstrated that Wt1Cre is efficiently activated in hearts and gonads but not in other organs generated from the genital ridge, like the kidney or the adrenal gland. Taking advantage of this, the Wt1Cre mouse model was used to generate a new Wt1KO: the Wt1Cre; Wt1Loxp/GFP. Wt1Cre; Wt1Loxp/GFP mice show a partial embryonic lethality, potentially caused by defects in heart development, but some of them reach adulthood, becoming an ideal model to investigate Wt1 role from embryonic to adult stages. Therefore, we decided to study Wt1role in gonad development in Wt1Cre; Wt1Loxp/GFP mice. Sex development is a complex and coordinated process that starts with the differentiation of the bipotential gonads. WT1 mutations or haploinsufficiency have been reported in different syndromes and conditions linked to DSD. The study of WT1 role in embryonic development and the impact on adult sex development has been hampered by the complete gonadal agenesis or embryonic lethality presented by other Wt1KO mouse models. The results presented demonstrate a sharp reduction in Wt1 levels in Wt1Cre; Wt1Loxp/GFP gonads since the bipotential stage. This causes, in the adult, the formation of small, atrophic gonads, a hermaphroditism of the genital tract and external ambiguous genitalia. Besides, the data reported in this thesis demonstrates that Wt1Cre; Wt1Loxp/GFP mice present an impaired gonad embryonic development due to the lack of differentiation of the main cell lineages responsible for gonad development and function: supportive cells, steroidogenic cells and primordial germ cells (PGCs). Finally, the observed sub-lethality in Wt1Cre; Wt1Loxp/GFP mice is explained by the observation of a severe heart developmental impairment in a portion of Wt1Cre; Wt1Loxp/GFP embryos, whereas others show no apparent heart defects at embryonic stages. Equally, an histological study performed on Wt1Cre; Wt1Loxp/GFP mice also described changes in the spleen and brown adipose tissue. In summary, our results indicate the importance of morphological changes epicardial cells undergo during development, a process regulated by Wt1 modulation of the BMP4 pathway. In addition, the generation and characterization of the Wt1Cre; Wt1Loxp/GFP mouse renders an informative and useful tool to study Wt1 role beyond embryonic stages and provide a more accurate frame for the understanding of results previously obtained when using the Wt1Cre mouse model.

The study proposed to describe sexual development in pelagic stage loggerhead sea turtles Caretta caretta and compare this to hatchlings and adults. It is meant as an ontogenic approach, in order to understand reproductive development and population composition and their dynamics in the pelagic environment. The study focused on the pelagic loggerheads that are found in the waters offshore Madeira Island (Portugal) in the North-eastern Atlantic and use it as a developmental habitat. The innovating character of this work relied on the lack of any description regarding the gonad ontogenesis and reproductive development for the pelagic stage in any of the 7 existing sea turtle species, all of them in danger of extinction. Three methods were used to diagnose the sex of each juvenile individual and asses the level of reproductive development: (1) laparoscopy, (2) gonad biopsy and (3) the assessment of two sex steroids circulating levels, namely testosterone and estradiol. In order to cover all life stages and compare data obtained for the juvenile stage, hatchlings and nesting female adults were sampled at the nearest nesting rookery at Boa Vista Island in the Cape Verde Archipelago. Gonads from dead hatchlings were collected for gonad histology and blood was collected from nesting females for sex steroids assessment. Laparoscopies revealed to be a valid sexing method for the juvenile stage, since gonads are morphologically differentiated at these size classes. Moreover, laparoscopy was validated using gonad histology. Gonad histology of juveniles showed that gonads are already completely differentiated into ovaries or testes at the size classes examined, but development seems to be quiescent. Males present already developed seminiferous tubules with spermatogonia lining the interior of the seminiferous tubule. Female gonads present oocytes at different development stages, but only oocytes up to stage III were observed. The maximum oocyte diameter in each individual correlated with body size, suggesting that reproductive development is an on-going process in juvenile females. The circulating levels of both testosterone and estradiol in juveniles of both sexes were very low and consistently lower than the ones observed in the nesting females from Boa Vista Island. No bimodal distribution was found for any of the sex steroids analysed and thus circulating hormone levels were not a reliable tool for sexing juvenile individuals with a non-invasive technique. The ratio testosterone:estradiol did not show a bimodal distribution either. The levels of testosterone correlated with sea surface temperature. The fact that temperatures observed during this study were below 24ºC might have hindered a differential testosterone pattern between juvenile males and females. Sex ratios for this population were generated according to laparoscopy results and compared among years and size classes. An overall sex ratio of 2 females for each male was found, but they varied among size classes but not among years. Possible causes for the sex ratios observed are discussed. This study is a contribution to our knowledge on the pelagic stage of loggerhead turtles, namely on the population structure regarding sex ratio, which is a vital tool for implementing conservation strategies.
Orientadores: Thomas Dellinger and Adelino Canário

Entomopathogenic Steinernema nematodes (Nematoda: Steinernematidae) have a mutualistic relationship with Xenorhabdus bacteria (Gamma-Proteobacteria Enterobacteriaceae). The two partners form an insecticidal alliance that is successful in killing a wide range of insects. A few studies have shown that Steinernema IJs have an enhanced virulence and reproductive fitness when they associate with their cognate symbionts. However, there are unanswered questions regarding the physiological interactions that govern and perpetuate the interactions between different nematode developmental stages and their bacterial partners. In this study, we evaluated gonad development and maturation time of first-generation adults of S. carpocapsae and S. feltiae adults when reared under four bacterial scenarios: a) cognate symbiotic, b) non-cognate symbiotic bacterial strain, c) non-cognate symbiotic bacterial species and d) non-symbiotic bacteria (Serratia proteamaculans). For comparative purposes, we also considered adult nematodes reared in vivo in Galleria mellonella larvae to assess nematode development under natural conditions. Furthermore, in this study we also measured production of nematode pheromones (ascarosides), which play a key role in mating and reproduction. For this purpose, we considered in vitro rearing methods (with cognate and non-cognate Xenorhabdus symbionts) to qualitatively and quantitatively characterize ascarosides produced by first-generation adults. Our data showed that for both Steinernema spp. tested, time to adult maturation and gonad development was tightly dependent on the bacterial conditions under which juveniles were reared. However, contrasting results were observed when assessing total body length and gonad size. S. feltiae males and females size (body length and width) and respective gonad length were smaller when reared with a non-cognate symbiotic species. Additionally, non-symbiotic bacteria did not sustain S. feltiae maturation to adult stages. Contrarily, S. carpocapsae juveniles developed to adults when reared with any of the bacterial conditions tested, including with non-symbiotic Serratia proteamaculans. Additionally, S. carpocapsae adults, unlike S. feltiae, did not exhibit enhanced body and gonad size when reared with their cognate symbiont. In fact, S. carpocapsae males and females had larger gonad lengths when reared with a non-cognate symbiotic strain, XnAna (X. nematophila associated with S. anatoliense). S. carpocapsae males and females had significantly underdeveloped gonads when reared with non-symbiotic bacteria. In both Steinernema spp., sex ratio was not impacted by the bacterial condition. However, sex ratio (female:male) S. carpocapsae, decreased from 2:1 to 1:1 when reared with non-symbiotic bacteria. The body and gonad sizes of Steinernema spp. reared in vitro with their cognate symbiont were significantly smaller than those grown in vivo. Ascaroside production in either Steinernema spp. was not significantly impacted by the rearing conditions. In S. carpocapsae, a significant increase in glucoside-1 was observed when the nematodes were reared with cognate or non-cognate bacteria. No detectable quantities of asc-C11 were produced by S. feltiae nematodes when reared with a non-cognate symbiotic bacterial species. We conclude that bacterial symbionts influenced maturation and development of first-generation adults’ in both Steinernema spp. tested in this study. However, response to the bacterial symbionts was species specific. Additionally, this study showed that Xenorhabdus as a food source plays an important role in the type and amount of ascarosides produced by Steinernema spp.

In the mouse embryo, the first definitive haematopoietic stem cells (HSCs), capable of repopulating adult irradiated mice, emerge at mid-gestation by embryonic day E11. At this stage, the aorta-gonad-mesonephros (AGM) region is able to initiate and expand HSCs. Recently, it has been shown that the development of HSC in the AGM region results from the maturation of haematopoietic precursors called pre-HSCs. Mounting evidence points at an endothelial origin for these cells, the haematogenic endothelium. Analysis of VEGFs mutants, a critical pathway for endothelial developement, suggested that it also plays a role during early haematopoiesis. The main receptor of the pathway, FLK-1 (also known as VEGRR2 or KDR), is expressed in early hematopoietic and endothelial cells in the mouse embryo. Knock-out mutants for Flk-1 showed a decrease of endothelial and intra-embryonic haematopoietic progenitors. Although Flk-1 has been identified as an essential gene for HSC emergence, its exact point of action in HSC development remains unknown. In this thesis, I investigated the role of FLK-1 signalling in haematopoietic development and defined precise stages and cell types during HSC emergence in which FLK-1 is critically involved. by using a reporter line and antibody staining, I demonstrated that FLK-1 is expressed in the pre-HSCs/HSC lineage. Germ-line Flk-1 knockout results in embryonic lethality at around E9.0, before HSC emergence, mainly due to defects in vasculogenesis. Since arterial specification precedes HSC formation, it has never been elucidated whether the haematopoietic defects found in the knockouts are a secondary effect of the loss of vasculature or it FLK-1 is directly involved in haematopoietic specification. Therefore, to determine the role of the receptor in HSC development, I used a conditional inducible mutagenesis approach that allowed the deletion of Flk-1 precisely when pre-HSCs mature into HSCs at E10.5 and E11.5. My data showed that Flk-1 deletion at these stages affects both endothelial and haematopoietic progenitors, as well as HSCs. This suggests that the VEGF pathway is not only essential in early stages of haematopoietic development, as previously demonstrated, but it may be also involved in the maturation of pre HSC into HSCs at later stages.

Abalone, Haliotis midae, farmers in South Africa that feed formulated diets reported a periodic drop in abalone growth during periods of increased gonad development. A large drop in abalone biomass was noticed after presumed spawning events. This study was aimed to determine the effect of diet and sex-sorting on gonad development in abalone. Experiments were conducted on a commercial abalone farm from July 2012 to the end of June 2013. Isonitrogenous and isoenergetic diets were formulated with two protein sources. A fishmeal and soybean meal (S-diet) diet and a fishmeal only (F-diet) diet were fed to abalone (50 - 70 g abalone⁻¹) over 12 months. Weight and length gain, gonad bulk index (GBI), visceral index (%) and meat mass index (%) were determined monthly and seasonally. A histological study on the female gonads was conducted. This study also included an experiment to test the effect of sex-sorting (70 - 80 g abalone⁻¹) on growth and body composition with treatments including males (M), females (F) and equal numbers of males and females (MF). Weight gain and length gain were faster in S-diet-fed abalone (RM-ANOVA, F ₍₁, ₁₆₎ = 7.77, p = 0.01; F ₍₁, ₆₉₎ = 49.9, p < 0.001, respectively). Gonad development was significantly affected by the inclusion of soybean meal with S-diet-fed abalone showing higher GBI-values than F-diet-fed abalone (RM-ANOVA, F ₍₁, ₃₃)= 16.22, p = 0.0003). Male abalone had higher GBI-values than females (RM-ANOVA, F ₍₁, ₃₃₎ = 39.87, p < 0.0001). There was no significant difference in average feed conversion ratio (FCR) between diets over time (RM-ANOVA, F ₍₁, ₂₁₎ = 0.008, p = 0.97). However, average FCR-values were significantly highest between November 2012 and March 2013, the presumed spawning season. The visceral mass (gut and gonad) as a proportion of whole mass (visceral index, %) was significantly higher in abalone fed the S-diet (RM-ANOVA; F ₍₁, ₆₉₎ = 68.06, p < 0.0001). There was no difference in meat mass index (%) between diets for both male and female abalone (RM-ANOVA; F ₍₇, ₂₄₈₎ = 0.80, p = 0.60; F ₍₇, ₂₄₁₎ = 1.7, p = 0.11,respectively). Meat mass index significantly decreased from September 2012 to February 2013 coinciding with the period of high GBI-values. The distribution of oocyte maturity stages differed between diets. The majority of oocytes within S-diet-fed abalone were fully mature stage 8 oocytes compared to a majority of stage 7 oocytes in F-diet-fed abalone. Histology corroborated peaks in GBI-values for abalone fed both diets. There was no significant difference in growth, GBI, visceral index (%) and meat mass index (%) between abalone sorted into monosex and mixed-sex populations. Thus, the presence of the opposite sex did not have an effect on growth and gonad mass in H. midae. The phytoestrogens daidzin, glycitin, genistin, daidzein, glycitein and genistein were present in soybean meal and only traceable amounts were found in the F-diet. This study provided evidence that soybean meal present in formulated feed affected growth and gonad development in H.midae. The difference in the distribution of the maturity stages of oocytes was affected by diet. Sex-sorting abalone into monosex and mixed-sex populations had no influence on weight and length gain and gonad development.

lin-28 was first characterized as a developmental timing regulator in Caenorhabditis elegans. Loss of lin-28 function (lin-28(lf)) mutants skip the hypodermal cell fates specific to the 2nd larval stage. Here, we studied two aspects of lin-28 which had not yet been investigated. First, we show that lin-28(lf) mutants exhibit reduced fertility associated with abnormal somatic gonadal morphology. In particular, the abnormal spermatheca-uterine valve morphology of lin-28(lf) hermaphrodites traps embryos in the spermatheca, which disrupts ovulation and causes embryonic lethality. The same genes downstream of lin-28 in the regulation of hypodermal developmental timing also act downstream of lin-28 in somatic gonadal morphogenesis and fertility. Importantly, we find that hypodermal expression, but not somatic gonadal expression, of lin-28 is sufficient for restoring normal somatic gonadal morphology in lin-28(lf) mutants. We propose that the abnormal somatic gonadal morphogenesis of lin-28(lf) hermaphrodites results from temporal discoordination between the accelerated hypodermal development and normally timed somatic gonadal development. Thus, our findings exemplify how a cell-intrinsic developmental timing program can also control proper development of other interacting tissues, cell non-autonomously. We also investigated the expression patterns and functions of two lin-28 isoforms in C. elegans. Our analysis of spatial expression patterns suggests that lin-28a and lin-28b are co-expressed in diverse tissues. Consistently, neither of isoform specific knock-out mutant, lin-28a(lf) or lin-28b(lf), exhibits defects in hypodermal development, somatic gonad, or fertility, indicating functional redundancy of two isoforms. Our study will contribute to further investigation of lin-28 isoforms by providing the mutants of each isoform as well as the primary analysis of their phenotypes.

The inclusion of soya as a dietary protein source in the formulated feed, Abfeed® S34 (Marifeed Pty (Ltd), Hermanus) for farmed abalone, Haliotis midae has resulted in larger gonads during reproductive seasons compared to the gonads of abalone fed kelp or diets that included fishmeal as the only main protein source. The aim of this study was to determine if the isoflavones present in the soya were responsible for this increase in gonad size and the subsequent effects on farmed abalone growth. Animals weighing between 40-50 g were fed one of seven isonitrogenous and isoenergetic diets containing either 0, 25, 50 or 100 percent of the soya component of the commercial feed (Abfeed® S34, Marifeed Pty (Ltd), Hermanus) from September 2013 to March 2014. An additional three diets were formulated to include crystalline isoflavone (ISO). These diets were identical to the 0 percent soya diet (i.e. the fishmeal only diet - FM), only ISO was included at the same rate that ISO occurred in the three soya diets. Data were analysed using a multiple forward stepwise regression analysis (MSR) to test the effects of ISO concentration, soya concentration, time, sex, time by concentration interaction and sex by concentration interaction on growth and gonad development and to identify those variables that most contributed to the model. The inclusion of crystalline ISO failed to promote larger gonads and had no effect on abalone growth, while growth and gonad development was dose dependent on soya inclusion rates with sex and time contributing to the models. Mean monthly weight gain in males correlated with increasing soya concentrations (c) (MSR, y = 3.24 + 0.002c, r2 = 0.23, p = 0.03), ranging from 3.11 ± 0.55 g abalone-1 month-1 to 4.43 ± 0.46 g abalone-1 month-1, while both male and female monthly length gain was not influenced by soya concentration with an overall mean of 1.62 ± 0.05 mm abalone-1 month-1 (MSR, p = 0.05 and p = 0.81, respectively). By December, the whole body mass, meat mass and visceral mass in both males and females decreased with increasing soya levels. However, by February, female whole body mass, meat mass and visceral mass positively correlated with soya levels. At the end of the study, male abalone fed FM with soya equivalent to the commercial feed had the highest whole body mass (69.00 ± 2.48 g abalone-1), meat mass (41.80 ± 1.12 g abalone-1), visceral mass (9.00 ± 2.47 g abalone-1) and gonad bulk index (42.70 ± 9.82 g abalone-1), while females were not influenced by soya concentrations with an overall whole body mass of 63.46 ± 0.79 g abalone-1. Weight loss was observed in all treatments between February and March, probably due to a spawning event. The moisture content in the meat was not influenced by treatment, however, visceral water loss was effected by both ISO and soya concentration with time and sex contributing to the model. The visceral water loss of females fed graded levels of soya decreased as a function of soya from December to March, and from December to February for males, whereas females fed ISO-enriched diets decreased as a function of ISO concentration (c) at the end of the study from 74.98 ± 0.88 to 73.10 ± 0.75 percent (MSR, y = 74.97 – 0.0025c, r2 = 0.20, p = 0.048). The inclusion of crystalline ISO had no significant effect on oogenesis in female farmed Haliotis midae, while the distribution of the predominant oocyte stage, stage 7 (second last stage prior to spawning) was dose-dependent in abalone fed increasing soya concentration (c) (MSR, y = 33.38 + 0.03c, r2 = 0.32, F(1, 18) = 8.52, p = 0.01). The increase in stage 7 oocytes in abalone fed FM with soya did not reduce the number of oocytes (44.96 ± 3.01 oocytes mm-2) present within the lumen, while the number of oocytes (o) in abalone fed the FM-only based diets decreased with increasing abundance of stage 7 oocytes (MSR, y = 58.28 – 0.48c, r2 = 0.38, F(1, 18) = 12.51, p = 0.002), possibly due to the increase in size of the oocytes with thicker jelly coats. This study provided evidence that crystalline isoflavone had no influence on abalone gonad development over five months, while soya had a dose-dependent effect on growth, gonad mass and oogenesis in farmed Haliotis midae. Formulated abalone feed could be manipulated at certain times of the year to obtain maximum growth. These implications and further studies were discussed.

Abstract The embryonic urogenital system generates the metanephric kidneys, the gonads and the adrenal glands, and its development is based on sequential and reciprocal cell and tissue interactions. The mechanisms which regulate urogenital ontogeny are still poorly understood. In this thesis, the roles of Wnt-4 and ErbB4 functions in gonad and kidney development were analysed by using in vivo functional genomic technologies. Wnt-4 is crucial in female development since its absence leads to a partial female to male sex reversal. We found that Wnt-4 mediated the interactions between the somatic and the germ cells and played a role in meiosis which is regulated in part by the secreted signal retinoic acid (RA). Expression of certain meiosis-controlling genes (Stra8, Spo11) was inhibited in the Wnt-4 deficient germ cells, while certain pluripotency genes (Oct4, Fgf9, Sox2 and Dnmt3l) were activated similarly as in the wild-type male gonad. In addition to this, we noted that a gene encoding for a Cyp26b1 enzyme, which degrades RA in the embryonic testis, was ectopically expressed in the Wnt-4 deficient ovary. Microarray analysis was used to identify candidate Wnt-4 target genes by using the Wnt-4 knock-out mouse. Of these genes, Runx-1 may represent a novel signalling target to mediate Wnt-4 activity in the control female development The role of receptor-tyrosine kinase ErbB4 in kidney development was studied by using both in vivo gain and loss of function approaches. In the gain-of-function situation, we found that certain markers for the epithelial tubules and collecting ducts lost their polarized expression pattern. At the same time, the orientation of the cells in the kidney tubules was deregulated and an increase in cell proliferation was noticed. We suggest that the observed defects gave rise to an increase in the tubule diameter and to cyst formation in the kidney cortex. In the loss-of-function mouse, the lack of ErbB4 expression led to a similar phenotype as with the gain of function, and the renal functions of the mutant adult kidneys were compromised. In conclusion, the results point to specific roles for Wnt-4 and ErbB4 in the control of urogenital development. Wnt-4 appears to be crucial in sustaining proper female somatic cell and germ cell differentiation, and maintenance of gonad development during and after the sex determination event, while ErbB4 activity is critical for the regulation of tubular growth in embryonic kidney development
Tiivistelmä Sekä nisäkkään jälkimunuainen, lisämunuainen että sukurauhanen kehittyvät alkion urogenitaalialueen järjestelmästä ja solu- ja kudosvuorovaikutukset ohjaavat elinkehitysprosessia. Tapahtuman molekyylitason mekanismit ovat kuitenkin huonosti tunnettuja. Tässä väitöskirjatyössä tutkittiin Wnt-4 signaalin tehtäviä sukurauhasen ja ErbB4- proteiinin munuaisen kehityksessä. Wnt-4 signaali on keskeinen naisen sukupuolisuuden kehityksessä, koska signaalin puutos aiheuttaa alkion sukupuolen osittaisen kääntymisen naaraasta koiraaksi. Tarkastelimme aluksi sitä, välittääkö Wnt-4 itusolujen ja sukurauhasen somaattisten solujen vuorovaikutuksia ohjaten itusolujen meioosia, jota mm. A-vitamiini säätelee. Havaitsimme, että Wnt-4 geeni puuttuessa tietyt meioosia säätelevät geenit kuten Stra8 ja Spo11 olivat heikentyneet, kun taas solujen monikykyisyyteen liittyvät geenit kuten Oct4, Fgf9, Sox2 ja Dnmt3l aktivoituivat vastaavalla tavalla kuin havaitaan normaalisti koirasalkion kivesaiheessa. Tämän lisäksi havaitsimme, että Cyp26b1-geeni, joka johtaa A-vitamiinin hajoamiseen alkiossa ja estää normaalisti meioosin koirasalkion kivesaiheessa oli aktivoitunut munuaisrauhasaiheessa, jolta puuttuu Wnt-4 aktiivisuus. Tuloksemme osoittavat, että Wnt-4 säätelee osaltaan naarasalkion itusolujen meioosia. Tarkastelimme myös mikrosirututkimusten avulla niitä geenejä, joita Wnt-4 säätelee sukuelinaiheessa. Identifioimme useissa Wnt ja β-catenin signaalireittiin liittyvissä geeneissa muutoksia. Muuntuneet geenit voivat olla Wnt-4 signaalireitin kohdegeenejä. Näistä Runx-1 saattaa olla keskeinen Wnt signaalitien kohdegeeni, joka säätelee merkittävällä tavalla naaraan munarauhasen kehitystä. Väitöskirjan toisessa osassa tarkastelimme ErbB4-reseptorityrosiinikinaasin tehtäviä munuaisen kehityksen säätelyssä. ErbB4-geenin tehtäviä tutkittiin käyttäen hyväksi siirtogeenisiä malliorganismeja, joissa ErbB4-geenin määrä oli joko koholla tai ajastetusti inaktivoitu. ErbB4- geenin kokeellinen yliaktiivisuus muutti spesifisti tekijöitä, jotka säätelevät osaltaan jälkimunuaisen epiteeliputkien solujen orientaatiota ja solun jakautumista. Solujen orientaatiomuutoksen yhteydessä myös solujen jakautuminen häiriintyi. Oletuksemme on, että nämä epiteelikudoksessa tapahtuneet muutokset ovat syy, miksi kohotettu ErbB4-aktiviteetti muuttaa epiteeliputkien paksuutta ja pituutta erityisesti munuaisen pintakerroksissa. Havaitsimme myös, että ErbB4-geenin ajastettu poistaminen munuaisen epiteelikudoksessa johti hyvin samankaltaisiin, mutta vastakkaisiin muutoksiin kuin ErbB4-aktiviteetin kohottaminen. Muutokset johtivat myös muutoksiin munuaisen toiminnassa. Yhteenvetona toteamme, että näillä Wnt-4 ja ErbB4 solusignallointiin liittyvillä molekyyleillä on keskeinen tehtävä alkion munarauhasen ja munuaisen aiheen kehityksen säätelyssä. Wnt-4 ohjaa sekä itusolujen että somaattisten solujen erilaistumista ja samalla sukupuolen määräytymistä ja jatkokehitystä, kun taas ErbB4-signallointireseptorin tehtävä on avainasemassa munuaisen epiteeliputken kasvun säätelyssä

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

Brominated flame retardants (BFRs) are a group of industrially important chemicals that are incorporated into a wide variety of consumer products in order to increase their flame ignition resistance. These BFRs are readily released into the immediate environment through a multitude of routes, whereupon an appreciable level can be detected in household dust. The ingestion of contaminated dust is the major source of exposure for humans. Rising body burdens of BFRs over the last decades have led to major concern because of the persistence of BFR in the environment, their bioaccumlative and lipophilic properties, and their implication as endocrine disruptors. Elevated levels in humans have been positively correlated with dysregulation of the endocrine system and disruptions of male reproductive function and development. Animal studies have identified similar effects; however, these studies have characterized the consequences of exposure to single BFR congeners or to technical mixtures but not to an environmentally relevant mixture. Therefore, the aim of this thesis was to determine if chronic exposure to a representative mixture of the BFRs found in North American household dust during adulthood or gestation disrupts male adult or fetal gonadal structure or function and development. The BFR mixture was delivered to adult male and female Sprague-Dawley rats through their diet for 70 days in the males and for the duration of a premating and gestation period in the females. Male testicular histology was examined to determine effects on cellular associations and tissue integrity; the expression of key genes was assessed to reflect any effect on testicular function and steroidogenic capacity. Neither testicular histology nor the expression of marker genes was affected by BFR exposure in the adult males. However, alterations in testicular cell morphology and count were observed in testes from fetuses on gestation day 20 after in utero exposure to BFRs. Specifically, BFR exposure affected Sertoli cell and gonocyte morphology, as well as Sertoli cell counts and the ratio of Sertoli cells to gonocytes. Therefore, an environmentally relevant BFR mixture that did not affect the structure or function of the adult male gonad did adversely affect gonad development, suggesting that these chemicals target critical events during differentiation of the male gonad.
Voués à limiter les risques d'incendies, les retardateurs de flammes bromés (BFRs) sont des composés dotés de propriétés physicochimiques qui réduisent l'inflammabilité d'une large gamme de produits de consommation. Relargués dans l'environnement, les BFRs s'accrochent aux particules de poussière d'où résulte une contamination par les BFRs, s'effectuant principalement par ingestion. Un accroissement important de la charge corporelle en BFRs suscitent une inquiétude majeure en raison de leur persistance dans l'environnement et de leurs propriétés lipophiliques et bioaccumulables.En effet, des taux élevés en BFRs détectés chez l'humain ont été associé à une perturbation du système endocrinien, du développement des organes reproducteurs mâles et de la fertilité masculine. Des études menées chez les rongeurs ont mis en évidence des résultats similaires. Cependant, ces études caractérisent les effets d'un congénère ou d'une mixture de BFRs et ne reflètent pas ce qui est mesuré dans l'environnement. Ainsi, l'objectif de cette thèse était de déterminer si une exposition chronique à une mixture de BFRs détectée dans la poussière domestique Nord-américaine, à l'âge adulte ou durant la gestation affectait la structure des organes reproducteurs et leur fonction à l'âge adulte ou, le développement fœtal des gonades, chez le mâle. Des mâles et femelles rats Sprague-Dawley adultes ont été exposé à la mixture de BFRs via leur nourriture pendant 70 jours pour les mâles et pendant la période de pré accouplement et la gestation chez les femelles. Afin d'investiguer les associations cellulaires et l'intégrité des tissus, des coupes histologiques de testicules ont été analysé. L'expression de gènes clés a été quantifié afin d'évaluer la fonction testiculaire et l'activité stéroidogenique. Chez le mâle adulte, aucuns effets induits par l'exposition aux BFRs ont été observés sur l'histologie des testicules ou l'expression de gènes marqueurs. Cependant, la morphologie et le nombre de cellules dans les testicules fœtal à 20 jours de gestation étaient affectés par une exposition in utero aux BFRs. Non seulement la morphologie des cellules de Sertoli et des gonocytes était anormale, mais également le nombre de cellules de Sertoli et, le ratio du nombre de cellules de Sertoli et de gonocytes. Par conséquent, une exposition aux BFRs, à des doses reflétant celles mesurées dans l'environnement, n'affecte pas la structure et la fonction du système reproducteur mâle adulte mais est associée à des conséquences néfastes sur le développement des gonades mâles, suggérant une sensibilité accrue aux BFRs lors de la période de différenciation des gonades mâles.

Chez les mammifères, le développement testiculaire des gonades XY est initié par les facteurs de transcription SRY/SOX9 qui promeuvent la différenciation des cellules de Sertoli. Chez l’embryon XX, la signalisation RSPO1/WNT/beta-catenin contrôle la différenciation des cellules de la granulosa et le développement ovarien. De fait, les souris XY n’exprimant pas Sox9 (KO) développent des ovaires et les souris XX n’exprimant pas Rspo1(KO) développent des ovotestis, constitués d’une partie testiculaire et une ovarienne. Leur formation est due à la différenciation précoce de cellules de granulosa et la reprogrammation d’une partie d’entre elles, en cellules de Sertoli. Chez les souris XX Rspo1 KO, SRY n’est pas nécessaire au développement testiculaire.De plus, les gonades des souris XX et XY présentant une double inactivation des gènes Rspo1 et Sox9 (Double Knockout/DKO) montrent une différenciation testiculaire partielle et complète respectivement, avec un développement d’ovotestis chez les individus XX DKO et un développement de testicules hypoplasiques chez les souris XY DKO. SOX9 et/ou SRY ne sont donc pas nécessaires à la différenciation testiculaire dans ce contexte, suggérant l’implication d’autres facteurs.L’objectif de ma thèse est de tester l’hypothèse selon laquelle SOX8, un facteur de transcription de la même famille que SOX9, pourrait induire le développement testiculaire chez les souris XX et XY DKO Rspo1 Sox9. Afin d’établir l’existence d’une compensation entre ces gènes SOX, nous avons analysé leur expression et le développement des gonades chez la souris DKO pour les gènes Rspo1 et Sox8 ou Sox9. Nous avons ensuite étudié les souris mutantes simultanément pour les gènes Rspo1, Sox8 et Sox9 (triple knockout/TKO). Notre hypothèse est qu’une perte d’expression des gènes Sox8 et Sox9 chez les souris TKO empêche la reprogrammation des cellules de la granulosa en Sertoli et par conséquent le développement testiculaire. Nous avons donc analysé la morphologie des gonades, les caractères sexuels secondaires, ainsi que l’organisation des gonades avec les différentes populations cellulaires qui les constituent par histologie et immuno-marquages à différents stades : à 17.5 jours de développement embryonnaire (E17.5) où la reprogrammation de cellules de granulosa en Sertoli commence dans la souris XX Rspo1 KO; chez les souris juvéniles au jour 10 (P10) où le développement somatique est achevé; et chez les souris adultes 40 jours après la naissance (P40).Nos résultats montrent que SOX8 et SOX9 sont exprimés de manière indépendante dans les gonades des souris XY and XX DKO Rspo1 Sox9 et DKO Rspo1 Sox8 à E17.5 et à P10. De plus, les souris XY et XX DKO Rspo1 Sox8 développent des testicules et des ovotestis indiquant que la perte d’un seul facteur SOX n’altère pas la formation des testicules, comme dans les souris XY et XX DKO Rspo1 Sox9. Cependant, chez les souris XX et XY TKO, la reprogrammation des granulosa en Sertoli à E17.5 et le développement testiculaire postnatal ne sont plus observés, démontrant que SOX8 peut compenser la perte de SOX9. De plus, les gonades des souris XY et XX TKO sont des ovaires atrophiques, indiquant que la différenciation ovarienne peut s’opérer.En résumé, nous avons analysé l’étiologie du développement physiopathologique des gonades chez les souris ayant une perte de fonction de RSPO1. Bien que SOX8 ne soit pas nécessaire à la différenciation testiculaire chez la souris, il peut promouvoir le développement testiculaire en l’absence de SRY et SOX9 en raison de sa redondance fonctionnelle avec SOX9. Chez l’Homme, dans les cas cliniques d’ambiguïtés sexuelles avec différenciation testiculaire, qui ne sont pas expliqués par le défaut d’expression de SRY ou SOX9, SOX8 pourrait ainsi être un facteur causatif
In humans and mice, testicular development in XY gonads involves SRY/SOX9 signaling to promote Sertoli cell differentiation and their formation as testis chords. For ovarian development in XX gonads, RSPO1/WNT/beta-catenin signaling is the main pathway for granulosa cell differentiation and their subsequent assembly into follicles. Indeed, XY Sox9 mutant mice develop ovaries, and XX Rspo1 mutant mice develop ovo-testes, a gonad containing a testicular and an ovarian part. In XX Rspo1 mutant mice, ovo-testicular development involves precocious differentiation of some granulosa cells and their and reprogramming as Sertoli cells. Thus, these single mutant studies demonstrated that SOX9 and RSPO1 are required for testicular and ovarian development respectively, and that SRY is dispensable for testicular development in XX Rspo1 mice. Interestingly, gonad development in XY and XX Rspo1 Sox9 double knockout (DKO) mice has challenged the requirement of SOX9 for testicular development. In XX Rspo1 single mutants, it was assumed that Sertoli cell differentiation was SOX9-dependent, but co-inactivation of Sox9 in DKO mice does not impair the ovo-testicular phenotype. For XY Sox9 single mutant mice developing ovaries, co-inactivation of Rspo1 in XY DKO mice rescues the sex reversal, though the testes are hypo-plastic. Thus, in XY and XX Rspo1 Sox9 DKO mice, SOX9 and/or SRY are dispensable for testicular differentiation, indicating that an alternate testis factor exists. For my research project, we hypothesized that a SOX9-related transcription factor, SOX8, acts redundantly for testicular development in XY and XX Rspo1 Sox9 DKO mice. Thus, to first establish redundancy among the SOX factors, we first analyzed their expression in Rspo1 mutant mice lacking Sox8 or Sox9, and then generated and analyzed gonad development in XY and XX Rspo1 Sox8 DKO mice. Then to test our hypothesis, we studied Rspo1 Sox8 Sox9 triple knockout (TKO) mice. We predicted that a loss of both Sox genes in TKO mice would prevent granulosa cell reprogramming as Sertoli cells and subsequent testicular development. To characterize gonad development and their effects in DKO and TKO mice, we performed analyses in embryonic day 17.5 (E17.5) mice, when granulosa-to-Sertoli cell reprogramming begins in XX Rspo1 single mutants; in juvenile post-natal day 10 (P10) mice, when gonad fate is set; and in young adult P40 mice. We examined a variety of parameters including gonad morphology and secondary sex characteristics, as well as gonad organization and cell population by histological and immunostaining analyses. We report that SOX8 and SOX9 are expressed independently in XY and XX Rspo1 Sox9 DKO and Rspo1 Sox8 DKO gonads in embryonic and juvenile mice. Next, XY and XX Rspo1 Sox8 DKO mice developed testes and ovo-testes, indicating that loss of one SOX factor does not impair testicular differentiation, as in XY and XX Rspo1 Sox9 DKO mice. In XY and XX Rspo1 Sox8 Sox9 TKO mice, granulosa-to-Sertoli cell reprogramming was impaired at E17.5 and post-natal gonads lacked testicular development. Thus, SOX8 can compensate for the loss of SOX9 in Rspo1 Sox9 DKO mice. In addition, gonads in XY and XX TKO mice developed as atrophied ovaries, indicating that ovarian fate is partially maintained.In total, we investigated the etiology of pathophysiological testicular development in RSPO1 loss-of-function mice. Remarkably, though SOX8 is dispensable for male sex determination in mice, it can promote testicular differentiation in the absence of SRY and SOX9 because of functional redundancy with SOX9. Thus, in human cases of sex reversal where testicular development cannot be explained by misexpression of SRY or SOX9, SOX8 could be a causative factor

Le rôle des microenvironnements dans la différenciation embryonnaire à été étudié sous deux aspects, d'une part les intéractions entre deux lignées de cellules d'origines différentes, d'autre part les reconnaissances entre sites de l'embryon et rudiments gréffés en position ectopique. Ces aspects ont été étudiés sur deux ébauches, celle des gonades et celle de l'aorte, dont la paroi est le progéniteur des cellules souches hématopoîques embryonnaires

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.
Includes bibliographical references.
Germ cells are the only cell type to undergo meiosis, a specialized cell division process necessary for the formation of haploid gametes. Timing of this process is sex-specific. Ovarian germ cells initiate meiosis during embryonic development, while testicular germ cells initiate meiosis after birth. In a series of gonad explant culture experiments, I show that retinoic acid (RA) is required for meiotic initiation in embryonic ovaries, because it is necessary for Stra8 (Stimulated by retinoic acid gene 8) expression. Stra8 is required for pre-meiotic DNA replication in embryonic ovaries and it is only expressed in testes after birth. I also show that a cytochrome p450 enzyme CYP26B 1, specifically expressed in embryonic testes but not ovaries, prevents Stra8 expression in testes during embryonic development. To confirm our results in vivo, and to examine if RA is sufficient to induce meiosis in embryonic testes, I generated Cyp26bl-/- and Cyp26bl-/-Stra8-/- mice. I show that germ cells in Cyp26bl-/- embryonic testes initiate a meiotic program but fail to complete meiotic prophase. Instead, germ cells proliferate until birth. RA also causes somatic cell defects. It inhibits Leydig cell differentiation and disturbs testis cord maintenance. Thus, RA has distinct effects in embryonic ovaries and embryonic testes. In ovaries, it is required for meiotic entry with no known effects on somatic cell development. In embryonic testes, RA is not sufficient for functional meiotic prophase and it induces proliferation in germ cells. RA also disrupts embryonic testicular somatic cell development.
by Jana C. Koubova.
Ph.D.

Abstract Although the genetic sex of an embryo is determined at conception by the presence or absence of the Y chromosome, both females and males have bipotential, undifferentiated gonads early in their development. Genes and testicular hormones direct differentiation into either testes or ovaries. The first relevant gene to be identified was the Y-linked master regulatory gene, SRY, since when several other genes have been found to be of importance for sex determination. The primary aim here was to identify the role of Wnt-4 in the development of the gonad and adrenal gland. Wnt-4 was found to be expressed in the developing gonad, the Müllerian duct and the adrenal gland, in addition to the kidney, pituitary gland and mammary gland as observed earlier. Expression in the gonad was found to be regulated in a sex-specific manner. After sex determination Wnt-4 was downregulated in the testis, but the expression persisted until birth in the ovary. Wnt-4-deficient female mice demonstrated a partial female-to-male sex reversal and a reduction in the number of oocytes, while the Müllerian duct was absent from both sexes. Lack of Wnt-4 in the adrenal gland led to reduced aldosterone production, indicating abnormal development of the zona glomerulosa. Flutamide administration to pregnant Wnt-4 heterozygote females was shown to partially restore the sex reversal. The results suggest that female development is not a default pathway but needs active signalling, in which Wnt-4 plays an essential role.

Les analgésiques tel que le paracétamol, ou acétaminophène (ACE), et les anti-inflammatoires non stéroïdiens (AINS) comme l’aspirine et l’ibuprofène, sont largement utilisés dans le monde pour soulager douleur, fièvre ou inflammation. Ces médicaments, vendus sans ordonnance et souvent pris en automédication, sont de plus en plus utilisés par les femmes enceintes depuis les années 1990, notamment aux États-Unis et en Europe. De nombreuses études de cohorte ont mis en avant l’association entre la prise de ces molécules par les femmes durant le premier et deuxième trimestre de grossesse, et l’apparition de malformations congénitales des organes reproducteurs des garçons nouveau-nés, comme la cryptorchidie ou l’hypospadias. Ces analgésiques inhibent l’activité enzymatique des cyclooxygénases (Cox), qui catalysent l’étape clef de la voie de biosynthèse des prostaglandines (PGs), dont la prostaglandine D2 (PGD2), impliquée dans la détermination sexuelle des lignées somatique et germinale de la gonade mâle chez la souris. Afin d’étudier si la gonade embryonnaire peut constituer une cible de ces molécules, l’objectif principal de cette thèse a été d’analyser l’impact de l’exposition in utero à l’ACE, l’aspirine et l’ibuprofène seuls ou en combinaison, pendant la période de détermination sexuelle (de 10,5 à 13,5 jours post coitum (jpc) chez la souris). Des doses « thérapeutiques » similaires à celles utilisées chez l’Homme ont été administrées par voie orale. Mon travail a permis de démontrer que l’exposition in utero à la combinaison des deux drogues ACE et ibuprofène induisait, dans le testicule embryonnaire, une accélération de la différenciation de la lignée germinale du testicule embryonnaire, précurseurs des gamètes à l’âge adulte, par le biais d’une reprogrammation épigénétique avancée, ainsi qu’une augmentation du stockage du glycogène dans les cordons testiculaires, via l’activation de l’expression des gènes de la matrice extracellulaire. De plus, ce projet a permis d’identifier pour la première fois le profil d’expression des prostaglandines dans le testicule embryonnaire de souris. Dans un deuxième temps, l’impact de l’exposition in utero à ces drogues sur la stéroïdogenèse a été étudié, en parallèle avec l’étude sur le rôle de la PGD2 dans les cellules stéroïdogènes (cellules de Leydig) dans le testicule fœtal de souris à 17,5 jpc. Ces résultats pourraient conduire à modifier l’utilisation de ces médicaments chez la femme, au cours des deux premiers trimestres de grossesse
Mild analgesics such as paracetamol, or acetaminophen (ACE), and nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen, are widely used worldwide for relieving pain, fever or inflammation. These over-the-counter drugs are often taken as self-medication, and are increasingly used by pregnant women since the 1990s, particularly in the United States and Europe. Many cohort studies have highlighted the association between intake of these molecules by women during the first and second trimester of pregnancy, and the occurrence of birth defects of the reproductive organs of newborn boys, such as cryptorchidism or hypospadias. These analgesics inhibit the enzymatic activity of cyclooxygenases (Cox), which catalyze key step in the biosynthetic pathway of prostaglandins (PGs), whose prostaglandin D2 (PGD2), involved in the male sex determination of both somatic and germinal lineages in mice. To investigate whether the embryonic gonad can be a target of these molecules, the main objective of this thesis was to analyze the impact of in utero exposure to ACE, aspirin and ibuprofen alone or in combination, during the period of sex determination (10.5 to 13.5 days post coitum (dpc) in mice). "Therapeutic" doses similar to those used in humans were orally administered. My work has shown that in utero exposure to the combination of both ACE and ibuprofen drugs induced acceleration of the differentiation of the embryonic testis germline, the precursors of gametes in adulthood, through an advanced epigenetic reprogramming, and an increase of glycogen storage in the testicular cords, via activation of extracellular matrix gene expression. In addition, this project has identified for the first time the profile of prostaglandins expression in mouse embryonic testis. Secondly, the impact of in utero exposure to these drugs on steroidogenesis was evaluated, in parallel with the study on the role of PGD2 in steroidogenic cells (Leydig cells) in mouse fetal 17.5 dpc testis. These results could lead to change the use of these drugs in women, during the first two trimesters of pregnancy

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Diabrotica speciosa (Germar, 1824), causa danos diretos por alimentação e indiretos como vetor de vírus para diversos grupos de plantas. A principal forma de controle são os agroquímicos, portanto visando o manejo populacional sem impacto ambiental, o presente trabalho teve como objetivo determinar a dose de radiação gama que proporciona a esterilidade de machos, o seu consumo foliar e as alterações histológicas em suas gônadas. Os adultos foram submetidos à radiação gama (60Co) no terceiro dia após a emergência nas doses de 0, 25, 50, 75 e 100 Gy a uma taxa de 0,808 KGy/hora, totalizando 20 repetições/ dose. A dose esterilizante baseou-se na fertilidade de fêmeas sexualmente maduras acasaladas por machos irradiados. Os casais foram individualizados em \"arenas\" e alimentados com folíolos de feijão (Phaseolus vulgaris L.) com gaze preta umedecida para oviposição. Os ovos foram tratados e dispostos em recipientes plásticos forrados com papel de filtro. Após a eclosão, as larvas foram transferidas para um recipiente maior com tampa telada contendo vermiculita fina e plântulas de milho (Zea mays L.) que foram substituídas a cada 10 dias, até a emergência do adulto. Após o 4º dia de irradiação disponibilizou-se um disco foliar de 3,2 cm de diâmetro por 24 horas, para cada casal. Os discos foram digitalizados e analisados no software ImageJ. Para avaliação das gônadas foram utilizados 3 machos por dose com 8 dias de idade dissecados em PBS e através da técnica de Hematoxilina Eosina as laminas foram avaliadas em microscópio óptico. Verificou-se que a esterilidade dos machos ocorreu a partir de 75 Gy e sua longevidade média foi de 12,5 dias. O consumo da área foliar dos casais constituídos por um macho estéril foi de 42,9% e a analise histológica testicular demonstrou desorganização nos tecidos e lacunas entre as células germinativas nas maiores doses de 75 Gy e 100 Gy.
Dissertação (Mestrado em Tecnologia Nuclear)
IPEN/D
Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

碩士
國立陽明大學
生命科學系暨基因體科學研究所
102
In zebrafish, the molecular mechanism of sex determination and early gonadal development is unclear. In our lab, we found that female gonad started to be bigger than male gonad at 12 dpf (days post fertilization). This indicates that the growth rate of germ cells may be different between male and female gonads prior to 12 dpf. Recent evidence showes that the PI3K/AKT pathway participates in germ cell migration at 24 hpf. However, it remains unclear whether the PI3K/AKT pathway is involved in gonadal growth and development. Here I showed that pAKT was activated in the female gonad from 8 dpf. After I blocked PI3K activity by inhibitor LY294002 from 7 dpf to 12 dpf, I found that some region of gonad lacked germ cells marker vasa, and mitotic cells were decreased in gonads. The gonadal morphology was affected, and the number of germ cells and gonadal mitotic cells were decreased, suggesting that the PI3K/AKT pathway may regulate germ cell proliferation. Furthermore, the dimorphic expression of gsdf and dmrt1a, the female gonadal somatic cell markers were also reduced in LY-treated gonads at 12 dpf and 10 dpf, indicating that the PI3K/AKT pathway is involved in gonadal differentiation. Moreover, the pAkt was activated and gsdf expression was enhanced in Akt activator-(SC-79) treated gonad. In conclusion, my results showed that the PI3K/Akt pathway was important for gonadal cell growth and somatic cell differentiation.

碩士
國立陽明大學
生命科學暨基因體科學研究所
99
In zebrafish, the sex chromosome has not been found and little is known about the mechanisms about sex determination and the sexually dimorphic gonad differentiation. Most of studies about gonad differentiation focus on the stages after three weeks when oocytes are already formed. However, recent study in our lab shows that the earliest sign of sexual dimorphism starts at 12 dpf when male and female gonads start to have different sizes. This indicates that this earlier period is the critical stage for gonad development, and that at this time the growth rate of germ cells and the expression of certain genes should start to be different. I examined the expression of two genes in the gonad by in situ hybridization. One is dmrt1a, which is an evolutionarily conserved gene involved in sex determination and sex differentiation in many species. I found that it started to be expressed in zebrafish gonad at 10 dpf, while dimorphic expression level was observed after 10 dpf. Furthermore, the expression of dmrt1a was always weaker in 17α-methyltestosterone-treated gonads. The other one is gsdf, which is a novel member of TGF-β superfamily. It started to be expressed at 8 dpf, while dimorphic expression level was observed after 12 dpf. The expression levels of gsdf were always weaker in 17α-methyltestosterone-treated gonads. Moreover, in dnd morphants, in which germ cells were depleted, the expression levels of gsdf were also weaker. This indicates that germ cells may affect the expression of gsdf during early gonadal development. To understand the relationship between germ cell number and gonad development, I examined the number of germ cell by counting vasa-positive germ cells, and found germ cell number started to increase at 8 dpf. In order to trace germ cells in individual larva, mCherry-nanos 3’UTR transgenic fish with red fluorescent germ cells were generated. Germ cell proliferation was also observed after 8 dpf by counting the number of red fluorescent germ cells. Furthermore, larvae could be segregated into two groups according to the number of red fluorescent germ cells after 8 dpf. One group has higher germ cell number, while the other group has lower germ cell number, likely representing the female and male groups. These results indicate that during this critical period, the growth rates of germ cells are dimorphic and certain genes are dimorphically expressed between male and females. These differences may further lead to the development toward sexual dimorphic gonad differentiation pathway in each individual larva

碩士
國立中興大學
生命科學系所
99
Sex determination is a special system that decides dimorphic sex characteristic development in male and female. The difference between the two sexes involves gene expression, karyotype, gonad morphology, hormone expression, psychology and social behavior. The sex determination of birds is chromosomally based, as in mammals, but the sex chromosomes are different and the mechanism of avian sex determination remains poorly understood. In the chicken and all other birds, the male carries two Z sex chromosomes, and the female carries one Z and one W sex chromosome. In the mammal’s model, the mechanism of testis formation is now comparably well understood, but understanding of the molecular pathways specifying gonadal differentiation in female is still incomplete. The differentiation of the bipotential genital ridge into a testis requires the Y-encoded gene Sry. Sox9 and Sf1 have been shown to play a role during testis differentiation as candidates. Wnt4 and DAX1 were involved in female fate determination and Wnt4 were proposed as a candidate of female sex determining genes in 1999. The later studies indicate the importance that Wnt4 be in sex determination. Because of the avian transegene technique is not mature, the function of some sex determinate genes in gonad are not complete known. We want to study the influence of Wnt4 in chicken gonad, so we try to use different methods to provide ectopic Wnt4 in chicken gonad, and to observe gonad development. Our result indicate Wnt4 can’t change male gonad morphological structure and there‘s no aromatase expression in the male gonad. We use Sox9 as a male somatic cell marker and found SOX9 still could be detected in the Sertoli and Leydig cells of the male gonad. This analysis means that Wnt4 can’t suppress SOX9 in chicken male gonad. These are suggested that the ectopic Wnt4 expression in the chicken male gonad is not sufficient to induce sex reversal.

The process of sex-determination can be better understood through examinations of developing organs and cells, which are involved in the formation of undifferentiated gonad. This mechanisms show in fish a broad variety, ranging from hermaphroditism to gonochorism and environmental to genetic sex determination. Hormones and abiotic factors such as temperature and pH can influence teleost development and reproductive traits. These factors are vulnerable to pollutants and climate changes. Therefore, it is important to examine gonad development and sex-determination/differentiation in teleost fish. Teleost fish are the largest known group of vertebrates with approximately 25,000 species and are used for such kind of examinations as model organisms. Recently, in Oryzias latipes (medaka), dmrt1bY (or dmy), a member of the Dmrt gene family, has been described as testis-determining gene. However, this gene is not the universal master sex-determining gene in teleost fish. Although dmrt1bY is present in the most closely related species of the genus, namely Oryzias curvinotous, it is absent from other Oryzias species, like Oryzias celebensis, and other fish. During my thesis, I studied gonad development in medaka and in the closely related species Oryzias celebensis. Germ cell specification in medaka seems to be dependent on maternally provided cytoplasmatic determinants, so called germ plasm. Nanos and vasa are such germ cell specific genes. In zebrafish they are asymmetrically localized in the early embryo. I have shown that nanos mRNA is evenly distributed in the early embryo of medaka. A similar pattern has been already described for the medaka vasa homolog, olvas. This suggests differences in PGC specification in zebrafish and medaka. Further, the vasa homolog was isolated and the expression pattern examined in O. celebensis. The results show that it can be used as a germ cell specific marker. Additionally, the primordial germ cell migration in O. celebensis was followed, which is similar to medaka PGC migration. Primordial germ cell migration in vertebrates is dependent on the chemokine stromal cell-derived factor 1 (Sdf-1). Medaka has two different sdf-1 genes, sdf-1a and sdf-1b. Both genes are expressed in the lateral plate mesoderm (LPM). During late embryonic development, I could show that sdf-1a is expressed in newly formed somites and not longer in the LPM. Sdf-1b expression persisted in the posterior part of the lateral plate mesoderm in the developing gonad. In terms of early and late functions, this suggests subfunctionalization of sdf-1a and sdf-1b. In “higher” vertebrates, genes that are involved in the process of gonad development have been studied in detail, e.g. Wt1, Sox9, and Amh. I have analyzed the expression pattern of wt1 and sox9 co-orthologs and amh. In both, the medaka and O. celebensis, wt1a transcripts were localized in the LPM and its expression was similar to sdf-1a gene expression in medaka. Wt1b expression was restricted to the developing pronephric region. During later embryonic development, wt1a is specifically expressed in the somatic cells of the gonad primordium in both sexes. This is the first time that in fish wt1 gene expression in developing gonads has been described. Therefore, this result suggests that wt1a is involved in the formation of the bipotential gonad. Furthermore, I have analyzed the gonad specific function of the wt1 co-orthologs in medaka. I could show that a conditional co-regulation mechanism between Wt1a and Wt1b ensures PGC maintenance and/or survival. The expression of sox9 genes in medaka and sox9b in O. celebensis were detected in the somatic cells of the gonad primordium of both sexes. Additionally, I have shown that amh and amhrII in medaka are expressed in somatic cells of the gonad primordium of both sexes. This suggests that sox9b, amh and amhrII are involved in gonad development and have specific functions in the adult gonad. In O. celebensis I could detect an expression of dmrt1 already six days after fertilization in half of the embryos, which is similar to the dmrt1bY expression in medaka. Whether the expression of dmrt1 is male specific in O. celebensis is currently under investigation. Altogether, the obtained results provide new insights into gene expression patterns during the processes of gonad development. Furthermore, no differences in the expression pattern of wt1a and sox9b during gonad development between the medaka and O. celebensis could be detected. This might indicate that the genetic mechanisms during gonad development are similar in both species
Die Untersuchung der Keimzellwanderung in O. celebensis zeigte hohe Ähnlichkeiten zu der bereits Beschriebenen im Medaka. Die Keimzellwanderung in Wirbeltieren ist abhängig von stromal cell-derived factor 1 (Sdf-1), einem chemotaktisch wirkendem Zytokinin. Im Medaka existieren zwei sdf-1 Gene, sdf-1a und sdf-1b, die während der embryonalen Entwicklung im Seitenplattenmesoderm (LPM) exprimiert werden. Die Expression der beiden Gene unterscheiden sich jedoch zeitlich und auch örtlich im LPM. Dies lässt vermuten, dass sich im Verlauf der Evolution eine frühe und eine späte keimzellspezifische Funktion zwischen sdf-1a und sdf-1b aufgeteilt hat. In „höheren“ Wirbeltieren wurden schon verschiedene Gene, z.B. Wt1, Sox9 und Amh, in dem Prozess der Gonadenentwicklung beschrieben. Die Expressionsmuster von wt1 und sox9 Co-Orthologen und amh habe ich während meiner Arbeit untersucht. Im Medaka und in O. celebensis wird wt1a im LPM transkribiert und ähnelt der von sdf-1a im Medaka. Die Expression von wt1b erfolgt hingegen nur in der Region der Vorläufer-Niere. Im weiteren Verlauf der Embryogenese ließen sich wt1a Transkripte erstmalig in somatischen Zellen des Gonaden-Vorläufers nachweisen. Wt1a spielt vermutlich eine Rolle in der Entwicklung der bipotentialen Gonade. Die funktionelle Analyse von wt1 Genen im Medaka zeigte, dass durch eine konditionale Co-Regulation zwischen wt1a und wt1b die Keimzellen überleben bzw. erhalten bleiben. Die Expression von sox9b im Medaka und in O. celebensis ließ sich in somatischen Zellen des Gonaden-Vorläufers nachweisen. Zusätzlich werden amh und amhrII ebenfalls in somatischen Zellen beider Geschlechter exprimiert, daher kann man eine wichtige Rolle dieser Gene während der Gonadenentwicklung und in der adulten Gonade annehmen. Die Expression von dmrt1 in O. celebensis konnte ich, in etwa der Hälfte der beobachteten Embryonen, bereits schon früh in der embryonalen Entwicklung (6 Tage nach der Befruchtung) nachweisen. Das Transkriptionsmuster von dmrt1 in O. celebensis ist ähnlich der Expression von dmrt1bY im Medaka. Inwieweit diese Expression in O. celebensis spezifisch für Männchen ist wird zurzeit noch untersucht. Die erhaltenen Ergebnisse zeigen neue Einblicke in die Genexpressionsmuster der Gonadenentwicklung von Medaka und O. celebensis und weisen neue Möglichkeiten für weitere Forschungen auf. Des Weiteren konnte ich im Verlauf der Gonadenentwicklung keine Unterschiede in der Genexpression von wt1a und sox9b zwischen Medaka und O. celebensis nachweisen. Dies deutet an, dass die genetischen Mechanismen der Gonadenentwicklung zwischen den beiden nahverwandten Arten sehr ähnlich sind

碩士
國立臺灣海洋大學
水產養殖學系
97
The objectives of this study were to investigate the heat shock protein 70（Hsp70）and Gonadotropin-releasing hormone （GnRH）in scleractinian coral, Euphyllia ancora. The possible roles of Hsp70 and GnRH in the gonad development and maturation of coral were also studied. By applying “cDNA library construct” were cloned two heat shock protein 70-like genes fragment（hsp70-like） in Euphyllia ancora, and temporarily to named hsp70a and hsp70b. Heat shock protein 70（Hsp70）was stress and gametogenesis-related gene in vetebrate and invertebrate, but the possible roles of heat shock protein 70 in coral were not clear. The objectives of were to investigate the expression and relationship of Hsp70 in the gonad development and maturation of coral. In the study, hsp70a in female and male had no significantly change from non-spawning season to spawning season, but female presented higher expression of hsp70a than male. In male corals the expression of hsp70b decreased from non-spawning season to spawning season, but in female corals the expression of hsp70b increased from non-spawning season to spawning season. It’s relationship with oogenesis. Another the study by the cultured of gonad cell to track to metabolic products of 「3H」Pregnenolone during gonad maturation, and the objectives of were to investigate the possbible roles of GnRH in the gonad development and maturation of coral. In the study, we found the sex steroid hormone metabolic products of 11KT、20βS、S、E2、AD、DOC＆T、17OHP、DHP、P5 , and in male gonad tissue the concentration of 11-ketotestosterone increased by LHRH-A treated. In female ovary the concentration of Estradiol 、 Estrone和Androstenedione increased by LHRH-A treated. GnRH had the directly regulative function to the coral gonad, and extrapolated the coral should have the completely mechanism of sex steroidogenesis.

碩士
國立東華大學
海洋生物多樣性及演化研究所
97
Polybrominated diphenyl ethers (PBDEs) are a group of emerging persistent organic pollutants (POPs) widely used as brominated flame retardants. Among the 209 possible congeners, PBDE-47 is the most common PBDE congener found in biological samples. Recent studies have shown that PBDEs may cause toxic effects to interfere with endocrine systems and reproduction, or to neurobehavioral damages in animals. However, related research on fish is scarce. In this study we chose zebrafish as the model species. Zebrafish were fed food dosed with PBDE-47 (control, solvent control, 10, 100 and 1000 ng/g) from 21 days post hatch (dph). Fish length, weigh and morphology were recorded at 38 and 90 dph, and their swimming behavior was assessed at 54 dph. All samples were fixed for whole fish histological analysis after the end of PBDE-47 exposure at 90 dph. There was no significant difference within five treatments both on length and weight each at 38 and 90 dph. In the behavioral analysis, there was no significant difference on maximum swimming speed. However, both total swimming distance and percent time active were significantly decreased in 1000 ng/g group than that in control. Histological analysis showed that PBDE-47 exposure caused no significant effect on gonad development in both male and female fish. In summary, this study showed that chronic dietary PBDE-47 exposure did not affect growth or gonad development but caused negative effect on swimming performance of zebrafish.

碩士
國立臺灣海洋大學
生物科技研究所
95
The main function of sperm is transporting genetic material into egg. The activity of sperm influences the ability of eugenesis. Sperm moves on by the wiggle of the tail, and there are three parts of sperm-tail. The central one is axoneme which is close to outer dense fiber, and the outside part is fibrous sheath. The promoter of sperm-tail has high specificity in sperm. We made GFP fusion with it, and use the GFP-Sperm-T fusion constract for transgenesis zebrafish. After selection, breed, and propagation, we can get the transgenic line. Because the characteristic between transgenic and endogenic is almost similar, it could stably transmit to next generation. The aim of this study is the generation of Sperm-T-GFP-rtTA-2S-M2 zebrafish transgenic fish, and use fluorescent microscope to observe the formation of testis momentary. The final goal of this experiment is to get the sterility of zabrafish, and to apply GMO policy. The method is cutting the tail fin to get genomic DNA, and make sure it is a transgenic fish（F0）. Then F0 fish were inter-crossed to genered the F1 transgenic fish. We also use the same way to confirm F1 fish. Besides we use 14-days-fish total RNA, and select the high expressive levels of transgenic fish by RT-PCR. Further, use whole-mount in situ hybridization to confirm the expression of sperm-Tail. In the further, we will carry on mating of the sperm-T and TRE-Tight-RFP-mono-Bax-V5-pA transgenic fish. Bax gene is one of the reasons for causing apoptosis and atrophy of testis. Under normal state, Bax gene is inhibited until induced.

Sex determination is a critical biological process for all sexually reproducing animals. Despite its significance, evolution has provided a vast array of mechanisms by which sexual phenotype is determined and elaborated even within amniote vertebrates. The most prevalent systems of sex determination in this clade are genetic and temperature dependent sex determination. These two systems are sometimes consistent within large groups of species, such as the mammals who nearly ubiquitously utilize XY genetic sex determination, or they can be much more mixed as in reptiles that use genetic or temperature dependent systems and even both simultaneously. The turtles are a particularly diverse group in the way they determine sex with multiple different genetic and temperature based systems having been described. We investigated the nature of the temperature based sex determination system in Trachemys scripta elegans to ascertain whether it behaved as a purely temperature based system or if some other global source of sex determining information might be apparent within thermal regions insufficient to fully induce male or female development. These experiments found that sex determination in this species is much more complex and early acting than previously thought and that each gonad within an individual has the same sexual fate established enough that it can persist even without further communication between. We established a best practice for the assembly and annotation of de novo whole transcriptomes from T. scripta RNA-seq and utilized the technique to quantify the gene regulatory events that occur across the thermal sensitive period.

Evidence is entirely lacking on the resolution of TSD when eggs are incubated at the pivitol temperature in which equal numbers or males and females are produces. We have produced a timecourse data set that allowed for the elucidation of the gene expression events that occur at both the MPT and FPT over the course of the thermal sensitive period. Our data suggests that early establishment of a male or female fate is possible when temperature is sufficiently strong enough as at MPT and FPT. We see a strong pattern of mutually antagonistic gene expression patterns emerging early and expanding over time through the end of the period of gonad plasticity. In addition, we have identified a strong pattern of differential expression in the early embryo at stages prior to the formation of the gonad. Even without the known systemic signaling attributed to sex hormones emanating from the gonad, the early embryo has a clear male and female gene expression pattern. We discuss how this early potential masculinization or feminization of the embryo may indicate that the influence of temperature may extend beyond the determination of gonadal sex or even metabolic adjustments and how this challenges the well-defined paradigm in which gonadal sex determines peripheral sexual characteristics.

Dissertation

碩士
國立臺灣海洋大學
水產養殖學系
98
Sox family has an important HMG (higher mobility group) domain which can bind with the DNA structure and regulate target genes. They were conserved in many species, and with many functions such as sex determination, testis formation, neuronal development, lymphocyte differentiation, sex differentiation and chondrogenesis. Black porgy, Acanthopagrus schlegeli bleeker, a marine protandrous hermaphrodite fish, have a complicated mechanism in sex differentiation and development. Juvenile fish processed male sex-differentiation about 4 month after hatch and developed to a functional male in 1st spawing season. After spawning, testis regressed and ovary grow up, testis developed again until the 2nd pre-spawning season. Nevertheless, about 40% fish change sex(male to female) in 3rd spawning season. In 0+ yr-old fish, sox3 expression had no obvious variation during sex differentiation, sox5 expression decreased during sex differentiation, sox8 expression increased after sex differetiation and sox9 expression had increased during testis development. E2＆AI treatment induecd ovary development in 0+-yr-old fish, sox3 expression had an obvious variation during ovary development, sox5 expression seemed to be related to aromatase expression, sox8 expression had the same results with control experiment and sox9 expression had no obvious variation. In the gonadal regression and development experiment, sox3 expression increased with the ovary development. Sox5 expression increased with the gonadal regression. Sox8 expression had no obvious variation. Sox9 maintain high expression in testis and rapidly decreased in the mature-sperm stage. Sox3 expression appeared to be related to ovary development, especially at primary oocyte stage. Sox3 expressed in primary oocyte by in situ hybridization and immunohistochemical stainning, suggested that sox3 is related to primary oocyte development. Sox5 increased at the regressive stage of ganad and decreased at the developing stage of gonad, suggested that sox5 is related to the regression in testis and ovary both. Sox9 remained at high expression during spermatogenesis and decreased at the mature-sperm stage, suggested that sox9 is related to early spermatogenesis. Sox8 had no obvious variation during gonadal development and regression. However sox8 decreased dramatically after testis excision in 2+-yr-old fish, suggested sox8 might be regulated by androgen.

碩士
國立成功大學
生命科學系
102
Studies about early germ cells development and gonad formation of grouper species were limited. Therefore, this study characterized the ontology of primordial germ cells (PGCs) and gonadogenesis by cloning the potential germ cell marker Vasa from the giant grouper, Epinephelus lanceolatus. The full length of Vasa contained 1,929 bp of open reading frame which generated 643 amino acid protein with the eight conserved domains belong to DEAD box family. Phylogenetic tree analysis and comparisons of the deduced amino acid sequence with other vertebrates revealed the high homology (73-85%) and belonged to marine teleost. Moreover, giant grouper Vasa gene was highly expressed in gonad, and increased with gonadal development. IHC stain showed ggVasa distributed in the cytoplasm of germ cells. Whole-mount in situ hybridization and immunostaining data indicated PGCs migrated to germinal ridge at 3 dpf. Besides, the roles of HSC70 and SPARC were investigated as well. The results of qRT-PCR and IHC stain showed HSC70 was significantly higher during germ cell proliferation. Besides, SPARC expression was higher and strongly detected in the reproductive tract of 3 month-old giant grouper. Consequently, HSC70 may be involved in gametogenesis, and SPARC may play specific roles in reproductive tract formation.

碩士
國立臺灣海洋大學
水產養殖學系
96
Anti-Mullerian hormone (AMH) plays an important role in the process of mammalian male development, which major function is inhibition of Mullerian duct activity of female reproduction tissue. Aecently, amh and its type II receptor, amhrII have been cloned from teleosts, which do not contain the tissue of Mullerian duct. The functions of Amh and AmhrII in teleost are not clean. The present study aimed to clone amhrII and investigate the amh and amhrII expression levels in testis and ovary during the male development, artificial-induced female development, and sex change in black porgy. Two gene related to the development status of testis and ovary, one was pcna, which is related to cell proliferation, and the other was caspase-3, which is related to cell apoptosis. The data showed that the expression level is of amh and amhrII in testis were hisher than those in ovary during male development. However, the expression levels of amh and amhrII in ovary were hisher than those in testis during nature sex reverse or artificial-induced female development. This result suggested that high expression levels of amh and amhrII in testis are related to the growth of male germ cell in early one to two years. Thus, amhrII had low expression level in ovary in early one or two years. When black porgy grew to the three year, the sex reversed-black porgy had high expression level of amhrII in ovary than that in testis. These data suggest that amhrII has the function to regulate amh and has the effect to promote growing of male and female germ cells in black porgy.

Understanding transcriptional regulation in development and disease is one of the central questions in modern biology. The current working model is that Transcription Factors (TFs) combinatorially bind to specific regions of the genome and drive the expression of groups of genes in a cell-type specific fashion. In organisms with large genomes, particularly mammals, TFs bind to enhancer regions that are often several kilobases away from the genes they regulate, which makes identifying the regulators of gene expression difficult. In order to overcome these obstacles and uncover transcriptional regulatory networks, we used an approach combining expression profiling and genome-wide identification of enhancers followed by motif analysis. Further, we applied these approaches to uncover the TFs important in mammalian sex determination.

Using expression data from a panel of 19 human cell lines we identified genes showing patterns of cell-type specific up-regulation, down-regulation and constitutive expression. We then utilized matched DNase-seq data to assign DNase Hypersensitivity Sites (DHSs) to each gene based on proximity. These DHSs were scanned for matches to motifs and compiled to generate scores reflecting the presence of TF binding sites (TFBSs) in each gene's putative regulatory regions. We used a sparse logistic regression classifier to classify differentially regulated groups of genes. Comparing our approach to proximal promoter regions, we discovered that using sequence features in regions of open chromatin provided significant performance improvement. Crucially, we discovered both known and novel regulators of gene expression in different cell types. For some of these TFs, we found cell-type specific footprints indicating direct binding to their cognate motifs.

The mammalian gonad is an excellent system to study cell fate determination processes and the dynamic regulation orchestrated by TFs in development. At embryonic day (E) 10.5, the bipotential gonad initiates either testis development in XY embryos, or ovarian development in XX embryos. Genetic studies over the last 3 decades have revealed about 30 genes important in this process, but there are still significant gaps in our understanding. Specifically, we do not know the network of TFs and their specific combinations that cause the rapid changes in gene expression observed during gonadal fate commitment. Further, more than half the cases of human sex reversal are as yet unexplained.

To apply the methods we developed to identify regulators of gene expression to the gonad, we took two approaches. First, we carried out a careful dissection of the transcriptional dynamics during gonad differentiation in the critical window between E11.0 and E12.0. We profiled the transcriptome at 6 equally spaced time points and developed a Hidden Markov Model to reveal the cascades of transcription that drive the differentiation of the gonad. Further, we discovered that while the ovary maintains its transcriptional state at this early stage, concurrent up- and down-regulation of hundreds of genes are orchestrated by the testis pathway. Further, we compared two different strains of mice with differential susceptibility to XY male-to-female sex reversal. This analysis revealed that in the C57BL/6J strain, the male pathway is delayed by ~5 hours, likely explaining the increased susceptibility to sex reversal in this strain. Finally, we validated the function of Lmo4, a transcriptional co-factor up-regulated in XY gonads at E11.6 in both strains. RNAi mediated knockdown of Lmo4 in primary gonadal cells led to the down-regulation of male pathway genes including key regulators such as Sox9 and Fgf9.

To find the enhancers in the XY gonad, we conducted DNase-seq in E13.5 XY supporting cells. In addition, we conducted ChIP-seq for H3K27ac, a mark correlated with active enhancer activity. Further, we conducted motif analysis to reveal novel regulators of sex determination. Our work is an important step towards combining expression and chromatin profiling data to assemble transcriptional networks and is applicable to several systems.

Dissertation

碩士
國立臺灣海洋大學
水產養殖學系
96
This experiment was designed to investigate the effects of temperature, photoperiod and dietary β-carotene level on somatic growth and gonad development of sea urchin? Tripneustes gratilla. A 2x3x3 factorial feeding experiment was conducted with the two levels for temperatures at 25℃ and 29℃? three levels for photoperiods：10L/14D? 12L/12D, 14L/10D and the three dietary β-carotene levels：0mg/kg, 60mg/kg, 120mg/kg with duplicated treatment. Prepared diets were fed to the urchins once daily at a ration of 0.5% body wet weight for 120 days. Temperature effect was observed on the sea urchins all with higher values held at 25℃：PWTG(38.16%)? survival rate(95%)? FCR(2.25)? PER(1.98)? SGR(0.27) than those held at 29℃(PWTG, 25.41%., survival rate, 86.67%., FCR, 3.26., PER, 1.40., SGR, 0.19). However, no temperature effect on the gonad color? GSI(Gonad index) and GDS(Gonad development stage) between urchins held in the two temperatures. Photoperiod with light exposure shorter than 12hrs, 10L/14D(GSI, 18.78) exhibited significant lower GSI values than those groups exposed to longer day light conditions, 12L/12D(GSI, 24.03) and 14L/10D(GSI, 23.50). The 12L/12D urchin group had the highest GDS(2.45) than the two photoperiod groups, 10L/14D(GDS, 1.82) and 14L/10D(GDS, 2.00). However? there were no significant differences on PWTG, survival rate, FCR, PER, SGR, and gonad color among the urchins confined in the three levels of photoperiods. Concentrations of dietary β-carotene also significantly affected the urchin’s PWTG, FCR, SGR and gonad color. Dietary β-carotene level at 60ppm resulted in values for the highest PWTG(37.44)? FCR(2.12)?and SGR(0.26)? than those urchins fed at 0ppm(PWTG, 25.64%., FCR, 3.47., SGR, 0.19) and 120ppm(PWTG, 32.28., FCR, 2.68., SGR, 0.23). Dietary β-carotene level had no effect on the survival rate, PER, GSI and GDS of urchins. Urchins received β-carotene level exceeded 60ppm exhibited significantly higher gonad color values, 120ppm(Gonad color, 3.49), 60ppm(Gonad color, 3.39) than those fed 0ppm(Gonad color, 2.48). The interaction between temperature and photoperiod was well reflected on the WTG of urchins. Higher WTG value was observed on the urchins held at 25℃ then 29℃, and shorter day light exposure less then 12L exhibited better growth rate. However, the enhanced effect on the growth when urchins held at 25℃ and 10L/14D was observed. The other confliction of the two factors was 14L/10D exhibited better growth performance than the 12L/12D photoperiod group. Interaction of the three factors occurred on the GDS of urchins which resulted in a best combination of 25℃, 12L/12D and 60ppm β-carotene treatment.

The gonad is a unique primordial organ that retains the ability to adopt one of two morphological fates through much of mammalian embryonic development. Previous work in our lab found that dimorphic vascular remodeling was one of the earliest steps during sex-specific morphogenesis. In particular, vessels in XY gonads display highly ordered behavior that coincides with testis cord formation. It was unknown how the vasculature may influence testis cord morphogenesis and, if so, how this was mechanistically related to sex determination. The work in this thesis addresses a single over-arching hypothesis: Male-specific vascular remodeling is required for testis morphogenesis and orchestrates differentiation of the XY gonad.

To address this question we have modified and developed techniques that allow us to isolate aspects of vascular behavior, gene expression, and endothelial influence on surrounding cells. In particular, the application of live imaging was instrumental to understanding the behavior of various gonadal cell-types in relation to remodeling vessels. It is difficult to grasp the complexity of an organ without understanding the dynamics of its constituents. A critical aim of my work was to identify specific inhibitors of the vasculature that do not affect the early stages of sex determination. Combining inhibitors, live imaging, cell sorting, qRT-PCR, mouse models, and whole organ culture has led to a far richer understanding of how the vasculature behaves and the cell-types that mediate its influence on organ morphogenesis. The beauty of our system is that we do not have to settle for a snapshot of the fate of cells in vivo, but can document their journeys and their acquaintances along the way.

Vascular migration is required for testis cord morphogenesis. Specific inhibitors revealed that in the absence of vessels, testis cords do not form. The work below shows that vessels establish a feedback loop with mesenchymal cells that results in both endothelial migration and subsequent mesenchymal proliferation. Interstitial control of testis morphogenesis is a new model within the field. The mechanisms regulating this process include Vegf mediated vascular remodeling, Pdgf induced proliferation, and Wnt repression of coordinated endothelial-mesenchymal dynamics. Our work also suggests that vascular patterning underlies testis patterning and, again, is mediated by signals within the interstitial space not within testis cords themselves.

A final aspect of my work has been focused on how vessels continue to influence morphology of the testis and the fate of surrounding cells. Jennifer Brennan, a graduate student in our lab, previously showed that loss of Pdgfrα antagonizes cord formation and development of male-specific lineages. The mechanisms and cell-types related to this defect were not clear. I began to reanalyze Pdgfrα mutants after finding remarkable similarity to gonads after vascular inhibition. This work is providing data suggesting that vessels are not simply responsible for testis morphology but also for the fate of specialized cells within the testis. On the whole, this thesis describes specific roles for endothelial cells during gonad development and mechanisms by which they are regulated.

Dissertation

The Caenorhabditis elegans somatic gonad is patterned by the activity of regulatory cell types, which govern its morphology, serve as the germline niche, and pattern its connection to the outside. All regulatory cell types are specified by activity of the basic helix-loop-helix gene hlh-2/E/Daughterless, and differences in how functions are assigned between the regulatory cells in males and hermaphrodites lead directly to their sexually-dimorphic gonads. Here, I present evidence that a code of bHLH genes function together with hlh-2 to promote the specification and function of each regulatory cell type except for the hermaphrodite anchor cell, which is specified by HLH-2 activity alone. Each regulatory cell type expresses an overlapping but distinct set of bHLH genes, which we find are required for its specification and associated functions. Notably, ectopic expression of regulatory cell bHLH complements are sufficient to transform cells with anchor cell potential into the expected regulatory cell, albeit transiently, suggesting that they are master regulators of regulatory cell fate. As all nematode species pattern their gonads through cognate regulatory cells and bHLH genes are highly conserved, we hypothesized that a similar bHLH code might function in specifying the regulatory cells of other species. In some nematode species the anchor cell, which remains stationary in C. elegans, is able to migrate. In C. elegans, the bHLH gene hlh-12 is necessary for proper migration of hermaphrodite distal tip cells and male linker cell, the two migrating regulatory cell types; addition of hlh-12 to the C. elegans anchor cell causes it to become displaced in a manner dependent on the endogenous hermaphrodite distal tip cell and male linker cell machinery, suggesting that the anchor cell gains the ability to migrate with the addition of hlh-12. We thus hypothesized that ectopic expression of an hlh-12 ortholog in these species might have led them to evolve migrating anchor cells. However, phylogenetic analysis of the bHLH genes of several other species, including the ones with migratory anchor cells, suggests that hlh-12 may be novel to the Caenorhabditis genus and does not have orthologs in the species with migrating anchor cells, raising the possibility that either these species use another bHLH gene for migration or that their regulatory cells are specified in a bHLH-independent manner.

Organisms are comprised of many cells with multiple distinct cell types, each of which must be decided precisely to ensure proper formation of a functional organism. In C. elegans, the basic helix-loop helix transcription factor HLH-2 is required for the specification of the anchor cell, or AC. The AC arises from a group of four somatic gonad cells, all of which initially express HLH-2. Two of the four cells, which we call β cells, lose AC competence early and instead become ventral uterine precursor cells, or VUs. We call the remaining two cells α cells. One α cell becomes the AC, while the other becomes a VU. Which α cell becomes the AC is random—50% of the time one α cell becomes the AC, while the other 50% of the time the other α cell becomes the AC. The choice of which cell becomes the AC and which becomes the VU is called the AC/VU decision, and occurs through reciprocal signaling by LIN-12/Notch and its ligand LAG-2/DSL. At first, both α cells express similar levels of lin-12 and lag-2. As the AC/VU decision progresses, the AC expresses higher levels of lag-2, and the VU expresses higher levels of lin-12. By this time, HLH-2 is only present in the specified AC, while it is post-translationally degraded in VUs. The mechanism by which HLH-2 is degraded and the consequences of disrupting its degradation on AC specification are unknown. In this work, we studied the function and regulation of HLH-2 during two stages of somatic gonad development. First, we used long-term fluorescence microscopy to visualize HLH-2 over the course of somatic gonad development. We found that HLH-2 expression begins in the parents of the α and β cells a consistent amount of time after their birth, and that the parent cell that first expresses HLH-2 almost always gives rise to the α cell that becomes the VU, while the second cell to express HLH-2 gives rise to the AC. This led us to study the effect of a loss of hlh-2 activity in the α and β cells. We generated an α and β cell-specific hlh-2(0) allele using genome editing tools and found that LIN-12 protein is not present in the absence of hlh-2 activity. Based on this discovery, we conceived a model where HLH-2 expression biases the first-expressing cell towards the VU fate by endowing it with an edge in lin-12 activity. Next, we focused on restriction of HLH-2 to the AC. Typically, HLH-2 protein is degraded in VUs, which we hypothesized was a crucial step in restriction of the AC fate to a single cell. We found that in a lin-12(0) background, HLH-2 is stabilized in VUs even when the resulting cell does not become an AC, indicating that lin-12 directly promotes HLH-2 degradation. This led us to search for a lin-12-regulated factor that targets HLH-2 for degradation in VUs. We identified seven ubiquitin-related genes whose depletion resulted in stabilized HLH-2 in VUs, but surprisingly did not cause an AC/VU defect. We suspect that HLH-2 degradation in VUs is one of multiple negative regulatory mechanisms that ensure the robustness of the AC/VU decision. The following research contributes new insights into how stochastic cell fate decisions amplify noise to ensure a consistent and reproducible outcome.