Incorporation of extracellular polysaccharide produced by Xanthomonas campestris into milk powders : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University

The purpose of the research was to investigate the functional properties of milk powders following exopolysaccharide (EPS) addition to milk solutions and their subsequent spray-drying. The aim was to replace some of the milk proteins with polysaccharide in dairy products while maintaining or improving the functional characteristics. Both commercial xanthan EPS and ferment xanthan EPS were incorporated into whole milk powder (WMP), skim milk powder (SMP), and milk protein concentrate (MPC). Ferment EPS was produced from a by-product of the dairy industry, milk permeate, through the hydrolysis of the lactose and fermentation with a strain of Xanthomonas campestris. Ferment EPS had a characteristic and unpleasant odour. The main compound responsible for this odour was p-cresol which, in milk, is largely bound in the conjugate form. Xanthomonas campestris hydrolyses these conjugates releasing the odour compounds. Ultrafiltration (UF) of the ferment or passing the ferment through a bed of activated carbon was effective in reducing the odour. UF was proven to reduce the levels of p-cresol in the ferment from 138ppb to less than 5ppb after 98 concentration factors. Milk powders made with UF ferment were more acceptable to the consumer sensory panel than those made with untreated ferment. The incorporation of EPS into milk powders has beneficial effects on the product with small additions increasing the viscosity of reconstituted SMP and WMP considerably. The EPS addition could result in a thickened milk product or alternatively, substitute for some of the milk solids. Sensory testing showed that 13.3% WMP solution, containing 0.02% commercial EPS, was not detectably different from a 15% WMP solution. The addition of both commercial and ferment EPS into milk powders leads to the formation of separate flocculated casein and polysaccharide phases with reconstituted milk. Confocal microscopy showed that casein flocculation occurred at all EPS concentrations tested, but this only resulted in sedimentation at intermediate EPS concentrations. At high EPS concentrations of approximately 0.2% the high viscosity limited flocculation and prevented sedimentation. At low EPS concentrations of approximately 0.05% flocculation was insufficient to overcome Brownian motion. Fresh cheese (Panela) made from MPC containing either ferment or commercial EPS showed greatly decreased whey loss. This was attributed to (i) the increased viscosity of the continuous phase limiting the flow of liquid through the pores of the cheese, and (ii) diminished casein interaction in the presence of EPS leading to a looser curd and lower contraction forces. For example the incorporation of 0.161% ferment EPS decreased the whey lost by approximately 75%. Negative effects were also apparent. The addition of EPS led to a granular appearance, which became more apparent with increasing EPS concentration. Cheese firmness was also decreased by approximately 40% by the addition of the ferment EPS at 0.161%. This could also be attributed to the localised aggregation of protein during renneting and the increased heterogeneity of the network. Sensory testing of cheeses made with 15.6% MPC + 0.045% commercial EPS compared with cheese made with 17.37% MPC alone showed that the consumers had no significant preference for one cheese over the other, but did notice a difference in texture. For reasons of safety and health, the sensory testing of milk and cheese in this research was confined to commercial xanthan. Future sensory testing of milk and cheese should be conducted with ferment EPS after odour removal rather than commercial EPS, and use consumers familiar with these cheese and milk products. For commercial production of dairy powders containing UF ferment EPS it is vital that either the xanthan or casein micelle structure be altered to prevent casein flocculation. If this is not feasible then an alternative use of the product may need to be found. A potential option involves the addition of the powder containing UF ferment EPS into food products as a minor food constituent. This may limit the occurrence of phase separation while improving the functionality of the product. Commercialisation is also limited by the increasing costs caused by ferment EPS purification and the lower solids concentrations required for spray-drying. As such the viability of the powder production must be determined

Perinatal germ cell development and differentiation in the male marmoset (Callithrix jacchus):similarities with the human and differences from the rat

STUDY QUESTION: Is perinatal germ cell (GC) differentiation in the marmoset similar to that in the human? SUMMARY ANSWER: In a process comparable with the human, marmoset GC differentiate rapidly after birth, losing OCT4 expression after 5–7 weeks of age during mini-puberty. WHAT IS KNOWN ALREADY: Most of our understanding about perinatal GC development derives from rodents, in which all gonocytes (undifferentiated GC) co-ordinately lose expression of the pluripotency factor OCT4 and stop proliferating in late gestation. Then after birth these differentiated GC migrate to the basal lamina and resume proliferation prior to the onset of spermatogenesis. In humans, fetal GC differentiation occurs gradually and asynchronously and OCT4(+) GC persist into perinatal life. Failure to switch off OCT4 in GC perinatally can lead to development of carcinoma in situ (CIS), the precursor of testicular germ cell cancer (TGCC), for which there is no animal model. Marmosets show similarities to the human, but systematic evaluation of perinatal GC development in this species is lacking. Similarity, especially for loss of OCT4 expression, would support use of the marmoset as a model for the human and for studying CIS origins. STUDY DESIGN, SIZE AND DURATION: Testis tissues were obtained from marmosets (n = 4–10 per age) at 12–17 weeks' gestation and post-natal weeks 0.5, 2.5, 5–7, 14 and 22 weeks, humans at 15–18 weeks' gestation (n = 5) and 4–5 weeks of age (n = 4) and rats at embryonic day 21.5 (e21.5) (n = 3) and post-natal days 4, 6 and 8 (n = 4 each). PARTICIPANTS/MATERIALS, SETTING AND METHODS: Testis sections from fetal and post-natal marmosets, humans and rats were collected and immunostained for OCT4 and VASA to identify undifferentiated and differentiated GC, respectively, and for Ki67, to identify proliferating GC. Stereological quantification of GC numbers, differentiation (% OCT4(+) GC) and proliferation were performed in perinatal marmosets and humans. Quantification of GC position within seminiferous cords was performed in marmosets, humans and rats. MAIN RESULTS AND ROLE OF CHANCE: The total GC number increased 17-fold from birth to 22 post-natal weeks in marmosets; OCT4(+) and VASA(+) GC proliferated equally in late gestation and early post-natal life. The percentage of OCT4(+) GC fell from 54% in late fetal life to <0.5% at 2.5 weeks of age and none were detected after 5–7 weeks in marmosets. In humans, the percentage of OCT4(+) GC also declined markedly during the equivalent period. In marmosets, GC had begun migrating to the base of seminiferous cords at ∼22 weeks of age, after the loss of GC OCT4 expression. LIMITATIONS, REASONS FOR CAUTION: There is considerable individual variation between marmosets. Although GC development in marmosets and humans was similar, there are differences with respect to proliferation during fetal life. The number of human samples was limited. WIDER IMPLICATIONS OF THE FINDINGS: The similarities in testicular GC differentiation between marmosets and humans during the perinatal period, and their differences from rodents, suggest that the marmoset may be a useful model for studying the origins of CIS, with relevance for the study of TGCC. STUDY FUNDING/COMPETING INTERESTS: This work was supported by Grant G33253 from the Medical Research Council, UK. No external funding was sought and there are no competing interests

Diethylstilboestrol exposure does not reduce testosterone production in human fetal testis xenografts

In rodents, in utero exposure to exogenous estrogens including diethylstilboestrol (DES) results in major suppression of steroidogenesis in fetal testes. Whether similar effects occur in the human fetal testis is equivocal. Based on the results of the rodent studies, we hypothesised that exposure of human fetal testes to DES would result in a reduction in testosterone production. We show, using a xenograft approach, that testosterone production is not reduced in human fetal testis following DES exposure. Human fetal testes (15-19 weeks' gestation, n = 6) were xenografted into castrate male nude mice which were then treated for 35 days with vehicle or 100 µg/kg DES three times a week. For comparison, similar treatment was applied to pregnant rats from e13.5-e20.5 and effects on fetal testes evaluated at e21.5. Xenograft testosterone production was assessed by measuring host seminal vesicle (SV) weights as an indirect measure over the entire grafting period, and single measurement of serum testosterone at termination. Human fetal testis xenografts showed similar survival in DES and vehicle-exposed hosts. SV weight (44.3 v 26.6 mg, p = 0.01) was significantly increased in DES compared to vehicle-exposed hosts, respectively, indicating an overall increase in xenograft testosterone production over the grafting period, whilst serum testosterone at termination was unchanged. In contrast intra-testicular testosterone levels were reduced by 89%, in fetal rats exposed to DES. In rats, DES effects are mediated via Estrogen Receptor α (ESR1). We determined ESR1 protein and mRNA expression in human and rat fetal testis. ESR1 was expressed in rat, but not in human, fetal Leydig cells. We conclude that human fetal testis exposure to DES does not impair testosterone production as it does in rats, probably because ESR1 is not expressed in human fetal Leydig cells. This indicates that DES exposure is likely to pose minimal risk to masculinization of the human fetus

A Normative Model of Serum Inhibin B in Young Males

RTM is supported by a Wellcome Trust Intermediate Clinical Fellowship (Grant No: 098522).Inhibin B has been identified as a potential marker of Sertoli cell function in males. The aim of this study is to produce a normative model of serum inhibin B in males from birth to seventeen years. We used a well-defined search strategy to identify studies containing data that can contribute to a larger approximation of the healthy population. We combined data from four published studies (n = 709) and derived an internally validated model with high goodness-of-fit and normally distributed residuals. Our results show that inhibin B increases following birth to a post-natal peak of 270 pg/mL (IQR 210–335 pg/mL) and then decreases during childhood followed by a rise at around 8 years, peaking at a mean 305 pg/mL (IQR 240–445 pg/mL) at around age 17. Following this peak there is a slow decline to the standard mature adult normal range of 170 pg/mL (IQR 125–215 pg/mL). This normative model suggests that 35% of the variation in Inhibin B levels in young males is due to age alone, provides an age-specific reference range for inhibin B in the young healthy male population, and will be a powerful tool in evaluating the potential of inhibin B as a marker of Sertoli cell function in pre-pubertal boys.Publisher PDFPeer reviewe

A novel formulation of inhaled sodium cromoglicate (PA101) in idiopathic pulmonary fibrosis and chronic cough: a randomised, double-blind, proof-of-concept, phase 2 trial

Background Cough can be a debilitating symptom of idiopathic pulmonary fibrosis (IPF) and is difficult to treat. PA101 is a novel formulation of sodium cromoglicate delivered via a high-efficiency eFlow nebuliser that achieves significantly higher drug deposition in the lung compared with the existing formulations. We aimed to test the efficacy and safety of inhaled PA101 in patients with IPF and chronic cough and, to explore the antitussive mechanism of PA101, patients with chronic idiopathic cough (CIC) were also studied. Methods This pilot, proof-of-concept study consisted of a randomised, double-blind, placebo-controlled trial in patients with IPF and chronic cough and a parallel study of similar design in patients with CIC. Participants with IPF and chronic cough recruited from seven centres in the UK and the Netherlands were randomly assigned (1:1, using a computer-generated randomisation schedule) by site staff to receive PA101 (40 mg) or matching placebo three times a day via oral inhalation for 2 weeks, followed by a 2 week washout, and then crossed over to the other arm. Study participants, investigators, study staff, and the sponsor were masked to group assignment until all participants had completed the study. The primary efficacy endpoint was change from baseline in objective daytime cough frequency (from 24 h acoustic recording, Leicester Cough Monitor). The primary efficacy analysis included all participants who received at least one dose of study drug and had at least one post-baseline efficacy measurement. Safety analysis included all those who took at least one dose of study drug. In the second cohort, participants with CIC were randomly assigned in a study across four centres with similar design and endpoints. The study was registered with ClinicalTrials.gov (NCT02412020) and the EU Clinical Trials Register (EudraCT Number 2014-004025-40) and both cohorts are closed to new participants. Findings Between Feb 13, 2015, and Feb 2, 2016, 24 participants with IPF were randomly assigned to treatment groups. 28 participants with CIC were enrolled during the same period and 27 received study treatment. In patients with IPF, PA101 reduced daytime cough frequency by 31·1% at day 14 compared with placebo; daytime cough frequency decreased from a mean 55 (SD 55) coughs per h at baseline to 39 (29) coughs per h at day 14 following treatment with PA101, versus 51 (37) coughs per h at baseline to 52 (40) cough per h following placebo treatment (ratio of least-squares [LS] means 0·67, 95% CI 0·48–0·94, p=0·0241). By contrast, no treatment benefit for PA101 was observed in the CIC cohort; mean reduction of daytime cough frequency at day 14 for PA101 adjusted for placebo was 6·2% (ratio of LS means 1·27, 0·78–2·06, p=0·31). PA101 was well tolerated in both cohorts. The incidence of adverse events was similar between PA101 and placebo treatments, most adverse events were mild in severity, and no severe adverse events or serious adverse events were reported. Interpretation This study suggests that the mechanism of cough in IPF might be disease specific. Inhaled PA101 could be a treatment option for chronic cough in patients with IPF and warrants further investigation

Intratubular germ cell neoplasia of the human testis:heterogeneous protein expression and relation to invasive potential

Testicular germ cell cancer develops from premalignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4(+)/MAGEA4(-)) into pre-spermatogonia (OCT4(-)/MAGEA4(+)). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesized that cells expressing an immature (OCT4(+)/MAGEA4(-)) germ cell profile would exhibit an increased proliferation rate compared with those with a mature profile (OCT4(+)/MAGEA4(+)). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2γ, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with preinvasive disease, seminoma, and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4(+)/MAGEA4(-) cells showed a significantly increased rate of proliferation compared with the OCT4(+)/MAGEA4(+) population (12.8 versus 3.4%, P<0.0001) irrespective of histological tumor type, reflected in the predominance of OCT4(+)/MAGEA4(-) cells in the invasive tumor component. Surprisingly, OCT4(+)/MAGEA4(-) cells in patients with preinvasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 versus 10.2 versus 7.2%, P<0.05, respectively). In conclusion, this study has demonstrated that OCT4(+)/MAGEA4(-) cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas

A Validated Age-Related Normative Model for Male Total Testosterone Shows Increasing Variance but No Decline after Age 40 Years

The diagnosis of hypogonadism in human males includes identification of low serum testosterone levels, and hence there is an underlying assumption that normal ranges of testosterone for the healthy population are known for all ages. However, to our knowledge, no such reference model exists in the literature, and hence the availability of an applicable biochemical reference range would be helpful for the clinical assessment of hypogonadal men. In this study, using model selection and validation analysis of data identified and extracted from thirteen studies, we derive and validate a normative model of total testosterone across the lifespan in healthy men. We show that total testosterone peaks [mean (2.5-97.5 percentile)] at 15.4 (7.2-31.1) nmol/L at an average age of 19 years, and falls in the average case [mean (2.5-97.5 percentile)] to 13.0 (6.6-25.3) nmol/L by age 40 years, but we find no evidence for a further fall in mean total testosterone with increasing age through to old age. However we do show that there is an increased variation in total testosterone levels with advancing age after age 40 years. This model provides the age related reference ranges needed to support research and clinical decision making in males who have symptoms that may be due to hypogonadism.Publisher PDFPeer reviewe

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Physical, cognitive, and mental health impacts of COVID-19 after hospitalisation (PHOSP-COVID): a UK multicentre, prospective cohort study

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research

Germ cell development in the human and marmoset fetal testis and the origins of testicular germ cell tumours

Normal germ cell development in the human testis is crucial for subsequent fertility and reproductive health. Disruption of testis development in fetal life can result in deleterious health consequences such as testicular dysgenesis syndrome (TDS), which includes disorders, such as cryptorchidism, hypospadias, infertility and testicular germ cell tumours (TGCT). A rat model of TDS in which rats are exposed to phthalates in utero has been validated, but does result in the development of TGCT. In humans, TGCTs result from transformation of pre-neoplastic carcinoma in-situ (CIS) cells and these CIS cells are believed to arise from human fetal germ cells during their transition from gonocyte to spermatogonia, based on their morphology and protein expression profile. It has been proposed asynchronous differentiation of germ cells in the human fetal testis may predispose fetal germ cells to become CIS cells. Studying the development of these tumours in humans is difficult because of their fetal origins and prolonged duration from initiation of impaired development to invasive disease. For this reason the use of relevant animal models that can mimic normal and abnormal germ cell development may provide new insight into how TGCT develop. The Common Marmoset monkey, a New World primate exhibits many similarities to the human in terms of reproductive biology and could represent such a model. This thesis aimed to further characterise the origins of CIS cells in the human testis by investigating the protein expression profile of CIS cells in patients with TGCT and comparing them to established markers of human fetal germ cell types using immunohistochemistry and immunofluorescence. Quantification of the various subpopulations of CIS and proliferation within these populations was performed. The thesis also investigated the Common Marmoset monkey as a potential model of normal testis and germ cell development by comparing the differentiation and proliferation profile of germ cells with those of the human during fetal and early postnatal life. During the present studies methods were successfully developed that enabled us to use testicular xenografts to recapitulate normal development of immature testes from marmoset and human. This involved grafting pieces of testis tissue subcutaneously under the dorsal skin of immunodeficient mice and retrieving them several weeks later to investigate their development during the grafting period. Xenografts using tissue from fetal, neonatal and juvenile marmosets were performed in addition to testes from first and second trimester human fetuses. Finally the present studies aimed to use the marmoset and the xenografting approach as systems in which to examine the effects of gonadotrophin suppression and phthalate treatment on germ cell differentiation and proliferation, with particular attention to the potential for development of CIS and TGCT. Heterogeneous phenotypes of CIS cells were identified, mostly consistent with those seen in the normal human fetal testis, however some of these CIS cells did not exhibit the same phenotype as germ cells identified in normal fetal testes. In addition it was shown that some of the proteins considered to be ‘classical’ markers of CIS cells, such as the pluripotent transcription factor OCT4, were not expressed in a proportion of the CIS cells. The proliferation index of CIS cells is also significantly higher in those subpopulations with the most ‘undifferentiated’ phenotype (i.e. OCT4+/VASA-). The present studies have generated novel data showing that the marmoset is a good model of fetal and neonatal germ cell development, with similarities to the human in terms of an asynchronous and prolonged period of differentiation and proliferation of germ cells from gonocyte to spermatogonia. This feature is also common to the human, but not a characteristic of the rodent. Fetal, neonatal and pre-pubertal germ cell development can be re-capitulated by xenografting tissue from marmoset and human testes into nude mouse hosts. Human fetal testis grafts produced testosterone and were responsive to hCG stimulation. First trimester human testis xenografts that have not developed fully formed seminiferous cords prior to grafting can complete the process of cord formation whilst grafted in host mice. In addition, germ cells in fetal human and marmoset xenografts can differentiate and proliferate in a similar manner to that seen in the intact non-grafted testis. In the intact neonatal marmoset, suppression of gonadotrophins resulted in a 30% decrease in proliferation, however differentiation of gonocytes is not affected. In-utero treatment of neonatal marmosets with mono-n-butyl phthalate was associated with unusual ‘gonocyte’ clusters, however, di-n-butyl phthalate treatment of mice carrying fetal marmoset xenografts resulted in no visible effects on germ cell differentiation or proliferation and did not result in the development of CIS or TGCT. In conclusion, this thesis has shown that there are many subpopulations of CIS cells of which many have not been previously described. These subpopulations have different characteristics, such as variable proliferation rates and this may indicate the potential for progression or invasiveness. These subpopulations have similar protein expression phenotypes to normal human fetal germ cells although the present studies have identified some CIS cells with phenotypes that are not found in the normal human testis. This thesis has demonstrated that the marmoset is a comparable model to the human in terms of asynchronous fetal germ cell development, which may predispose this species to the development of CIS/TGCT. In addition to the use of intact marmosets, these studies have also demonstrated for the first time that testis xenografting provides a comparable system for testis cord formation, germ cell differentiation and proliferation in fetal/postnatal marmosets and fetal human testis. In addition the marmoset and xenografting models have indicated that phthalates may have minor effects on testis development in the human and marmoset but do not result in CIS or TGCT. These model systems are suitable for further investigation of normal and disrupted testis development.EThOS - Electronic Theses Online ServiceGBUnited Kingdo