Relationship of transcriptional markers to Leydig cell number in the mouse testis

ObjectivesThe current study aims to identify markers that would reflect the number of Leydig cells present in the testis, to help determine whether labour-intensive methods such as stereology are necessary. We used our well-characterised Sertoli cell ablation model in which we have empirically established the size of the Leydig cell population, to try to identify transcriptional biomarkers indicative of population size.ResultsFollowing characterisation of the Leydig cell population after Sertoli cell ablation in neonatal life or adulthood, we identified Hsd3b1 transcript levels as a potential indicator of Leydig cell number with utility for informing decision-making on whether to engage in time-consuming stereological cell counting analysis

Anogenital distance (AGD) plasticity in adulthood:Implications for its use as a biomarker of fetal androgen action

Androgen action during the fetal masculinization programming window (MPW) determines the maximum potential for growth of androgen-dependent organs (eg, seminal vesicles, prostate, penis, and perineum) and is reflected in anogenital distance (AGD). As such, determining AGD in postnatal life has potential as a lifelong easily accessible biomarker of overall androgen action during the MPW. However, whether the perineum remains androgen responsive in adulthood and thus responds plastically to perturbed androgen drive remains unexplored. To determine this, we treated adult male rats with either the antiandrogen flutamide or the estrogen diethylstilbestrol (DES) for 5 weeks, followed by a 4-week washout period of no treatment. We determined AGD and its correlate anogenital index (AGI) (AGD relative to body weight) at weekly intervals across this period and compared these with normal adult rats (male and female), castrated male rats, and appropriate vehicle controls. These data showed that, in addition to reducing circulating testosterone and seminal vesicle weight, castration significantly reduced AGD (by ∼17%), demonstrating that there is a degree of plasticity in AGD in adulthood. Flutamide treatment increased circulating testosterone yet also reduced seminal vesicle weight due to local antagonism of androgen receptor. Despite this suppression, surprisingly, flutamide treatment had no effect on AGD at any time point. In contrast, although DES treatment suppressed circulating testosterone and reduced seminal vesicle weight, it also induced a significant reduction in AGD (by ∼11%), which returned to normal 1 week after cessation of DES treatment. We conclude that AGD in adult rats exhibits a degree of plasticity, which may be mediated by modulation of local androgen/estrogen action. The implications of these findings regarding the use of AGD as a lifelong clinical biomarker of fetal androgen action are discussed

Sertoli cells modulate testicular vascular network development, structure and function to influence circulating testosterone concentrations in adult male mice

The testicular vasculature forms a complex network, providing oxygenation, micronutrients, and waste clearance from the testis. The vasculature is also instrumental to testis function because it is both the route by which gonadotropins are delivered to the testis and by which T is transported away to target organs. Whether Sertoli cells play a role in regulating the testicular vasculature in postnatal life has never been unequivocally demonstrated. In this study we used models of acute Sertoli cell ablation and acute germ cell ablation to address whether Sertoli cells actively influence vascular structure and function in the adult testis. Our findings suggest that Sertoli cells play a key role in supporting the structure of the testicular vasculature. Ablating Sertoli cells (and germ cells) or germ cells alone results in a similar reduction in testis size, yet only the specific loss of Sertoli cells leads to a reduction in total intratesticular vascular volume, the number of vascular branches, and the numbers of small microvessels; loss of germ cells alone has no effect on the testicular vasculature. These perturbations to the testicular vasculature leads to a reduction in fluid exchange between the vasculature and testicular interstitium, which reduces gonadotropin-stimulated circulating T concentrations, indicative of reduced Leydig cell stimulation and/or reduced secretion of T into the vasculature. These findings describe a new paradigm by which the transport of hormones and other factors into and out of the testis may be influenced by Sertoli cells and highlights these cells as potential targets for enhancing this endocrine relationship. The testicular vasculature forms a complex capillary bed, interdigitating between the seminiferous tubules to provide oxygenation, delivery of micronutrients, and clearance of waste from the testis. Impairment of the testicular vasculature, for example, the reduction in venous drainage observed in cases of varicocele, causes intratesticular hypoxia and germ cell apoptosis (1). The vasculature is also instrumental to the endocrine function of the testis because it is the route by which pituitary gonadotropins are delivered to the testis to support T production and spermatogenesis (2). Conversely, alongside the lymphatic system, the vascular system is important for transport of T to other body systems; a reduced testis and vascular volume is associated with a reduction in circulating T concentrations (3). Our understanding of the mechanisms by which the testis controls local vascular function in adulthood is extremely limited. There is some evidence that testicular mast cells can influence vascular blood flow through release of 5-hydroxytryptamine (4), but perhaps the most well-studied factor influencing testicular vascular function is T. T is a well-established regulator of testicular vasomotion (rhythmical contraction and relaxation of blood vessels, independent of heartbeat) (5, 6) via direct T-mediated activation of the androgen receptor in smooth muscle cells of the testicular vasculature (7). Speculation that Sertoli cells may influence the testicular vasculature is supported by some indirect evidence (5) and in vitro studies (8), but confirmation of a direct role for Sertoli cells in the regulation of the testicular vasculature in vivo has never been demonstrated unequivocally. Recently we developed a unique model system that uses diphtheria toxin to specifically and acutely ablate Sertoli cells from the testis (9, 10). This model has revealed several important, yet previously unknown, roles that Sertoli cells play in neonatal and adult life (reviewed in reference 11). In this study we used models of acute Sertoli cell ablation and acute germ cell ablation, to address whether Sertoli cells actively influence vascular function in the adult testis. Our findings suggest that Sertoli cells play a key role in supporting the structure of the testicular vasculature and describe a new paradigm by which the transport of hormones and other factors into and out of the testis can be influenced by Sertoli cells and highlights these cells as potential targets for enhancing this endocrine relationship

Assessment of abdominal aortic aneurysm biology using magnetic resonance imaging and positron emission tomography-computed tomography.

Background Although abdominal aortic aneurysm (AAA) growth is non-linear, serial measurements of aneurysm diameter are the mainstay of aneurysm surveillance and contribute to decisions on timing of intervention. Aneurysm biology plays a key part in disease evolution but is not currently routinely assessed in clinical practice. Magnetic Resonance Imaging (MRI) and Positron Emission Tomography-Computed Tomography (PET-CT) provide insight into disease processes on a cellular or molecular level, and represent exciting new imaging biomarkers of disease activity. Macrophage-mediated inflammation may be assessed using ultrasmall superparamagnetic particles of iron oxide (USPIO) MRI and the PET radiotracer 18FSodium Fluoride (18F-NaF) identifies microcalcification which is a response to underlying necrotic inflammation. The central aim of this thesis was to investigate these imaging modalities in patients with AAA. Methods and Results USPIO MRI: MULTI-CENTRE STUDY In a prospective multi-centre observational cohort study, 342 patients (85.4% male, mean age 73.1±7.2 years, mean AAA diameter 49.6±7.7mm) with asymptomatic AAA ≥4 cm anteroposterior diameter underwent MRI before and 24-36 hours after intravenous administration of USPIO. Colour maps (depicting the change in T2* caused by USPIO) were used to classify aneurysms on the basis of the presence of USPIO uptake in the aneurysm wall, representing mural inflammation. Intra- and inter-observer agreement were found to be very good, with proportional agreement of 0.91 (kappa 0.82) and 0.83 (kappa 0.66), respectively. At 1 year, there was 29.3% discordant classification of aneurysms on repeated USPIO MRI and at 2 years, discordance was 65%, suggesting that inflammation evolves over time. In the observational study, after a mean of 1005±280 days of follow up, there were 126 (36.8%) aneurysm repairs and 17 (5.0%) ruptures. Participants with USPIO enhancement (42.7%) had increased aneurysm expansion rates (3·1±2·5 versus 2·5±2·4 mm/year; difference 0·6 [95% confidence intervals (CI), 0·02 to 1·2] mm/year, p=0·0424) and had higher rates of aneurysm rupture or repair (69/146=47·3% versus 68/191=35·6%; difference 11·7%, 95% CI 1·1 to 22·2%, p=0·0308). USPIO MRI was therefore shown to predict AAA expansion and the composite of rupture or repair, however this was not independent of aneurysm diameter (c-statistic, 0·7924 to 0·7926; unconditional net reclassification -13·5%, 95% confidence intervals -36·4% to 9·3%). 18F-NaF PET-CT: SINGLE-CENTRE STUDY A sub-group of 76 patients also underwent 18F-NaF PET-CT, which was evaluated using the maximum tissue-to-background ratio (TBRmax) in the most diseased segment (MDS), a technique that showed very good intra- (ICC 0.70-0.89) and inter-observer (ICC 0.637-0.856) agreement. Aneurysm tracer uptake was compared firstly in a case-control study, with 20 patients matched to 20 control patients for age, sex and smoking status. 18F-NaF uptake was higher in aneurysm when compared to control aorta (log2TBRmax 1.712±0.560 vs. 1.314±0.489; difference 0.398 (95% CI 0.057, 0.739), p=0.023), or to non-aneurysmal aorta in patients with AAA (log2TBRmax 1.647±0.537 vs. 1.332±0.497; difference 0.314 (95% CI 0.0685, 0.560), p=0.004). An ex vivo study was performed on aneurysm and control tissue, which demonstrated that 18F-NaF uptake on microPET-CT was higher in the aneurysm hotspots and higher in aneurysm tissue compared to control tissue. Histological analysis suggested that 18F-NaF was highest in areas of focal calcification and necrosis. In an observational cohort study, aneurysms were stratified by tertiles of TBRmax in the MDS and followed up for 510±196 days, with 6 monthly serial ultrasound measurements of diameter. Those in the highest tertile of tracer uptake expanded more than 2.5 times more rapidly than those in the lowest tertile (3.10 [3.58] mm/year vs. 1.24 [2.41] mm/year, p=0.008) and were also more likely to experience repair or rupture (15.3% vs. 5.6%, log-rank p=0.043). In multivariable analyses, 18F-NaF uptake on PET-CT emerged as an independent predictor of AAA expansion (p=0.042) and rupture or repair (HR 2.49, 95% CI1.07, 5.78; p=0.034), even when adjusted for age, sex, body mass index, systolic blood pressure, current smoking and, crucially, aneurysm diameter. Conclusion These are the largest USPIO MRI and PET-CT studies in AAA disease to date and the first to investigate 18F-NaF. Both USPIO MRI and 18F-NaF PET-CT are able to predict AAA expansion and the composite of rupture and repair, with 18F-NaF PETCT emerging as the first imaging biomarker that independently predicts expansion and AAA events, even after adjustment for aneurysm diameter. This represents an exciting new predictor of disease progression that adds incremental value to standard clinical assessments. Feasibility and randomised clinical trials are now required to assess the potential of this technique to change the management and outcome of patients with AAA

Pituitary androgen receptor signalling regulates prolactin but not gonadotrophins in the male mouse

Production of the androgen testosterone is controlled by a negative feedback loop within the hypothalamic-pituitary-gonadal (HPG) axis. Stimulation of testicular Leydig cells by pituitary luteinising hormone (LH) is under the control of hypothalamic gonadotrophin releasing hormone (GnRH), while suppression of LH secretion by the pituitary is controlled by circulating testosterone. Exactly how androgens exert their feedback control of gonadotrophin secretion (and whether this is at the level of the pituitary), as well as the role of AR in other pituitary cell types remains unclear. To investigate these questions, we exploited a transgenic mouse line (Foxg1 Cre/+; AR fl/y) which lacks androgen receptor in the pituitary gland. Both circulating testosterone and gonadotrophins are unchanged in adulthood, demonstrating that AR signalling is dispensable in the male mouse pituitary for testosterone-dependent regulation of LH secretion. In contrast, Foxg1 Cre/+; AR fl/y males have a significant increase in circulating prolactin, suggesting that, rather than controlling gonadotrophins, AR-signalling in the pituitary acts to suppress aberrant prolactin production in males

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Sertoli cells maintain leydig cell number and peritubular myoid cell activity in the adult mouse testis

The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health

No changes are seen in some pituitary hormone mRNAs or circulating products in Foxg1^Cre/+; AR^fl/y mice.

<p>(A) There is no significant difference in transcript of glucocorticoid receptor (<i>Gr</i>) between Foxg1^Cre/+; AR^fl/y and control pituitaries. (B) There is no significant difference in transcript of pro-opiomelanocortin (<i>Pomc</i>) between Foxg1^Cre/+; AR^fl/y and control pituitaries. (C) No significant difference was seen between Foxg1^Cre/+; AR^fl/y and control plasma levels of ACTH (Control 18.42 ±5.52, Foxg1^Cre/+; AR^fl/y 16.30 ±2.97 pg/ml). (D) There is no significant difference in transcript of growth hormone (<i>Gh</i>), between Foxg1^Cre/+; AR^fl/y and control pituitaries. (E) No significant difference was seen between Foxg1^Cre/+; AR^fl/y and control plasma levels of GH (Control 1.48 ±0.51, Foxg1^Cre/+; AR^fl/y 0.50 ±0.16 ng/ml). (F) There is no significant difference in transcript of thyroid-stimulating hormone-β (<i>Tsh</i>) between Foxg1^Cre/+; AR^fl/y and control pituitaries. (G) No significant difference was seen between Foxg1^Cre/+; AR^fl/y and control plasma levels of TSH (Control 1.06 ±0.11, Foxg1^Cre/+; AR^fl/y 1.20 ±0.14 ng/ml).</p