<p>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.</p><p>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.</p> / Dissertation

Macrodon ancylodon é um cienídeo demersal distribuído desde a Venezuela até a costa Nordeste do Brasil, sendo um dos principais recursos pesqueiros da costa Norte brasileira. Este trabalho estimou os parâmetros de crescimento, através de métodos diretos e indiretos, assim como descreveu os aspectos reprodutivos utilizando análises macro- e microscópicas dos ovários. As amostras foram obtidas no período de 1998 a 2001, provenientes de atividades pesqueiras industriais e artesanais na costa Norte. Para o estudo do crescimento, pelo método direto, foram realizados cortes transversais dos otólitos sagittae, e, para o método indireto, distribuições modais da freqüência de comprimento. Os parâmetros de crescimento para duas coortes encontradas foram: coorte 1) L¥= 47,4 cm, k= 0,42 e to= -0,3442; coorte 2) L¥= 46,8 cm, k= 0,44 e to= - 0,3302, nascidas em junho e dezembro, respectivamente. As avaliações qualitativas e quantitativas das gônadas indicaram que o período de desova é prolongado, com picos em julho-agosto e dezembro-fevereiro, coincidindo com o final do período de transição do regime de pluviosidade. As fêmeas atingem o comprimento médio de início de primeira maturação gonadal (L50) aos 25,08 cm, com 1,5 anos de idade, e todas estão participando do processo reprodutivo ao atingir 34,00 cm (L100). O estudo histológico indicou existência de estádios maturacionais intermediários aos da classificação macroscópica bem como erros na identificação macroscópica do estádio \"B\", da ordem de 40%. / Macrodon ancylodon is a demersal marine Sciaenidae largely distributed along the Brazilian coast. It is one of the major fisheries resources, mainly in the Northern and Southern regions of the Brazil. This work aims to estimate the growth parameters trough direct and indirect methods as well as estimate the reproductive aspects trough macroscopic and microscopic analyses of the gonads. The samples were obtained in the 1998-2001 period, derived from industrial and artisanal fishing activities that work along the Northern Brazilian coast. In order to conduct the growth study by the direct method, transversal sections were employed over the otolith sagitta. By the indirect method the length frequency modal distribution were observed during the period. The histological study was conducted on macroscopically classified females \"semi-mature\". It was verified the variation of the gonadosomatic relationship, the variation of the condition factor and the frequencies of the maturation stages. The growth parameters couldn\'t be estimated due to the absence of modal distribution in the length frequency and because the otolith sections were unreadable. The macroscopic valuation indicate a prolonged spawning season and has peaks in June-July and November-December, which coincide with the transition of the pluviometric period. The histological study indicate that the female enter this stages \"semi-mature\" more than once per cycle. The female reach the first maturation mean length (L50) of 25,08 cm and all of them participate in the reproductable process when reach 34,00 cm (L100).

A partir da hipófise de Astyanax altiparanae foram clonados e caracterizados os cDNAs completos para fshb, lhb e gpha. Das gônadas, foram clonados os cDNAs para fshr e lhr. As análises filogenéticas destes genes demonstram uma proximidade com as sequências homólogas das ordens Siluriformes e Cypriniformes. Por meio de qPCR, detectamos que a expressão dos mRNAs para as gonadotropinas foi restrita à hipófise, ao passo que dos receptores foi restrita às gônadas. Foi detectado que os mRNAs/proteínas para subunidades beta são expressas por diferentes populações de células gonadotrópicas. Nas gônadas, ambos receptores foram localizados nas células de Sertoli e Leydig, bem como nas células foliculares e da teca de oócitos a partir de crescimento secundário. Então, os níveis de expressão gênica das subunidades das gonadotropinas e seus receptores, juntamente como os níveis plasmáticos de esteroides sexuais foram avaliados e relacionados com a cinética da gametogênese durante o ciclo reprodutivo anual de machos e fêmeas em cativeiro. / From Astyanax altiparanae pituitaries, we cloned the full-leght cDNAs coding for fshb, lhb e gpha. From gonads, we cloned the full-length cDNAs for fshr and lhr. Phylogenetic analysis revealed that all five sequences showed the highest identity degree with homologous sequences from Siluriformes and Cypriniformes, respectively. Using qPCR, we observed that gonadotropin subunits expression were restricted to the pituitary gland, just like their receptors were restricted to the gonads. We also observed that both gonadotropin beta subunits mRNAs/proteins are expressed by distinct populations of gonadotropes at pituitary. In the gonads, both receptors were detected on Sertoli and Leydig cell, as well as on ovaries were located on follicular and theca cells, in oocytes starting from secondary growth. Then, gene expression levels of gonadotropins subunits mRNAs and their receptors were assessed, in parallel to plasma level of sex steroids, and evaluated according to gemetogenesis during the annual reproductive cycle of males and females kept in captivity.

Haematopoietic stem cells (HSC) are invaluable, due to their potential to treat malignant and non-malignant diseases. Modern medicine requires a reliable source of human HSCs (hHSCs) for efficient transplantations, which in many cases cannot be obtained from a single donor. Therefore, the ability to amplify donor hHSCs ex vivo would be an ideal alternative. Past attempts to expand hHSCs in vitro, demonstrated that the protocols developed so far have limited success. My research studied the factors which can affect the optimal culture of transplantable HSCs using a 3D culture system that had previously been used to culture HSCs derived from the aorta-gonad-mesonephros (AGM) region of the mouse embryo. This system involved cell culturing at the gas-liquid interface which is particularly sensitive to mechanical disturbances. To overcome this problem, floating Polypropylene support (rings) were designed and tested and I demonstrated that this was able to prolong aggregate culturing for up to 21 days. Further optimisation tests included altering factors such as oxygen levels, and the presence of antioxidants and apoptosis inhibitors in mouse HSCs culture. I have shown that moderate hypoxia (6% O2) did not affect HSCs in culture, while 2% of O2 led to a significant decrease of HSCs activity. Normoxia resulted in higher reactive oxygen species generation, which would likely be detrimental to cells. However, unexpectedly no improvement in repopulation efficiency of cultured HSCs was achieved by the addition of antioxidant. I also found that when the AGM region was dissociated and co-aggregated in the presence of Rho kinase inhibitor a higher level of repopulation was achieved. In addition, troloxpifitrin-a and p38 inhibitor blocked HSC development without affecting progenitor frequency or the total number of live cells. Subclones of mouse stromal cell line (OP9) were used to create a defined haematopoietic niche for hHSC. Functional screening of these lines in co-aggregate culture re- vealed that 3 of the 34 subclones tested were able to maintain hHSC in culture and repopulate immunodeficient mice at a comparable level to uncultured CD34+ cells. The repopulation in engrafted recipients persisted for over 6 months and showed both myeloid and lymphoid potential. These 3 subclones therefore appeared to create a functional niche for hHSCs and were subsequently used to study the impact of a number of factors including SCF, rock inhibitor, TGFb inhibitor, StemRegenin1 (SR), and prolonged culture technique on hHSC expansion. A significant level of fluctuation between experiments was observed and no definitive conclusions could be drawn. I also attempted to establish stromal cell lines from the human AGM region, more specifically from the ventral (AoV) and dorsal (AoD) regions of the dorsal aorta. Despite attempts to immortalise primary stromal cells, all lines went through a growth crisis. Nevertheless, 30 lines were screened for their ability to support haematopoietic cells in co-aggregate culture with results suggesting that lines derived from AoV expanded haematopoietic precursors more efficiently than AoD lines and OP9 control. Many of the tested lines were able to maintain long-term repopulating human HSCs but the level of repopulation was not as high as that achieved from uncultured CD34+ cells. Unfortunately, these human stromal cell lines have an unstable karyotype which may have an impact on their functional characteristics and they may not represent the nature of the primary cells.

Macrodon ancylodon é um cienídeo demersal distribuído desde a Venezuela até a costa Nordeste do Brasil, sendo um dos principais recursos pesqueiros da costa Norte brasileira. Este trabalho estimou os parâmetros de crescimento, através de métodos diretos e indiretos, assim como descreveu os aspectos reprodutivos utilizando análises macro- e microscópicas dos ovários. As amostras foram obtidas no período de 1998 a 2001, provenientes de atividades pesqueiras industriais e artesanais na costa Norte. Para o estudo do crescimento, pelo método direto, foram realizados cortes transversais dos otólitos sagittae, e, para o método indireto, distribuições modais da freqüência de comprimento. Os parâmetros de crescimento para duas coortes encontradas foram: coorte 1) L¥= 47,4 cm, k= 0,42 e to= -0,3442; coorte 2) L¥= 46,8 cm, k= 0,44 e to= - 0,3302, nascidas em junho e dezembro, respectivamente. As avaliações qualitativas e quantitativas das gônadas indicaram que o período de desova é prolongado, com picos em julho-agosto e dezembro-fevereiro, coincidindo com o final do período de transição do regime de pluviosidade. As fêmeas atingem o comprimento médio de início de primeira maturação gonadal (L50) aos 25,08 cm, com 1,5 anos de idade, e todas estão participando do processo reprodutivo ao atingir 34,00 cm (L100). O estudo histológico indicou existência de estádios maturacionais intermediários aos da classificação macroscópica bem como erros na identificação macroscópica do estádio \"B\", da ordem de 40%. / Macrodon ancylodon is a demersal marine Sciaenidae largely distributed along the Brazilian coast. It is one of the major fisheries resources, mainly in the Northern and Southern regions of the Brazil. This work aims to estimate the growth parameters trough direct and indirect methods as well as estimate the reproductive aspects trough macroscopic and microscopic analyses of the gonads. The samples were obtained in the 1998-2001 period, derived from industrial and artisanal fishing activities that work along the Northern Brazilian coast. In order to conduct the growth study by the direct method, transversal sections were employed over the otolith sagitta. By the indirect method the length frequency modal distribution were observed during the period. The histological study was conducted on macroscopically classified females \"semi-mature\". It was verified the variation of the gonadosomatic relationship, the variation of the condition factor and the frequencies of the maturation stages. The growth parameters couldn\'t be estimated due to the absence of modal distribution in the length frequency and because the otolith sections were unreadable. The macroscopic valuation indicate a prolonged spawning season and has peaks in June-July and November-December, which coincide with the transition of the pluviometric period. The histological study indicate that the female enter this stages \"semi-mature\" more than once per cycle. The female reach the first maturation mean length (L50) of 25,08 cm and all of them participate in the reproductable process when reach 34,00 cm (L100).

A partir da hipófise de Astyanax altiparanae foram clonados e caracterizados os cDNAs completos para fshb, lhb e gpha. Das gônadas, foram clonados os cDNAs para fshr e lhr. As análises filogenéticas destes genes demonstram uma proximidade com as sequências homólogas das ordens Siluriformes e Cypriniformes. Por meio de qPCR, detectamos que a expressão dos mRNAs para as gonadotropinas foi restrita à hipófise, ao passo que dos receptores foi restrita às gônadas. Foi detectado que os mRNAs/proteínas para subunidades beta são expressas por diferentes populações de células gonadotrópicas. Nas gônadas, ambos receptores foram localizados nas células de Sertoli e Leydig, bem como nas células foliculares e da teca de oócitos a partir de crescimento secundário. Então, os níveis de expressão gênica das subunidades das gonadotropinas e seus receptores, juntamente como os níveis plasmáticos de esteroides sexuais foram avaliados e relacionados com a cinética da gametogênese durante o ciclo reprodutivo anual de machos e fêmeas em cativeiro. / From Astyanax altiparanae pituitaries, we cloned the full-leght cDNAs coding for fshb, lhb e gpha. From gonads, we cloned the full-length cDNAs for fshr and lhr. Phylogenetic analysis revealed that all five sequences showed the highest identity degree with homologous sequences from Siluriformes and Cypriniformes, respectively. Using qPCR, we observed that gonadotropin subunits expression were restricted to the pituitary gland, just like their receptors were restricted to the gonads. We also observed that both gonadotropin beta subunits mRNAs/proteins are expressed by distinct populations of gonadotropes at pituitary. In the gonads, both receptors were detected on Sertoli and Leydig cell, as well as on ovaries were located on follicular and theca cells, in oocytes starting from secondary growth. Then, gene expression levels of gonadotropins subunits mRNAs and their receptors were assessed, in parallel to plasma level of sex steroids, and evaluated according to gemetogenesis during the annual reproductive cycle of males and females kept in captivity.

Social status influences reproductive physiology in many species, and sex change in marine teleost fishes provides an excellent model to understand how an organism can modulate its reproductive system in response to social stimuli. The series of experiments presented in this dissertation has focused on the proximate mechanisms underlying sex change and, in particular, the neuroendocrine factors that might translate social information into physiological changes. The bluebanded goby (Lythrypnus dalli) is a sexually plastic fish, and the dominant female typically changes sex when the male is removed from the social group. The direct physical interactions between the male and the females were found to be the main sensory cues that inhibit sex change. Sex steroids can both modulate and be modulated by behavior, and as a result they have been the most obvious candidates for a key role in the regulation of sex change. Males and females showed similar diurnal patterns for steroid hormones, but females had significantly higher water-borne estrogen levels. Concentrations of estradiol, testosterone and 11-ketotestosterone presented sex and tissue differences in brain, gonad and muscle, and they varied in complex ways in different tissues during sex change. The neurotransmitter serotonin (5-HT) has been suggested to be involved in the inhibition of socially regulated sex change because of its role in the modulation of both reproductive and aggressive behavior. None of the pharmacological manipulations performed in L. dalli to alter serotonergic activity was able to overcome the input from the social environment and affect sex change. Neither monoamine levels nor the area or number of 5-HT immunoreactive neurons were different between males, females and sex changers or between dominant and subordinate females. The results do not support the hypothesis of a serotonergic inhibition on sex change in L. dalli, but show that rapid changes in brain androgen levels might be implicated in inducing behavioral or morphological changes associated with sex reversal. Also, steroids respond to changes in the social environment in different ways in different tissues so local steroid synthesis should receive greater attention, and caution is required when using circulating levels to understand behavioral regulation.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.