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

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

Randomized, controlled trial of a best-practice individualized behavioral program for treatment of childhood overweight : Scottish Childhood Overweight Treatment Trial (SCOTT)

The objective of this study was to determine whether a generalizable best-practice individualized behavioral intervention reduced BMI z score relative to standard dietetic care among overweight children. The design consisted of an assessor-blinded, randomized, controlled trial involving 134 overweight children (59 boys, 75 girls; BMI ≥ 98th centile relative to United Kingdom 1990 reference data for children aged 5–11 years) who were randomly assigned to a best-practice behavioral program (intervention) or standard care (control). The intervention used family-centered counseling and behavioral strategies to modify diet, physical activity, and sedentary behavior. BMI z score, weight, objectively measured physical activity and sedentary behavior, fat distribution, quality of life, and height z score were recorded at baseline and at 6 and 12 months. The intervention had no significant effect relative to standard care on BMI z score from baseline to 6 months and 12 months. BMI z score decreased significantly in both groups from baseline to 6 and 12 months. For those who complied with treatment, there was a significantly smaller weight increase in those in the intervention group compared with control subjects from baseline to 6 months. There were significant between-group differences in favor of the intervention for changes in total physical activity, percentage of time spent in sedentary behavior, and light-intensity physical activity. A generalizable, best-practice individualized behavioral intervention had modest benefits on objectively measured physical activity and sedentary behavior but no significant effect on BMI z score compared with standard care among overweight children. The modest magnitude of the benefits observed perhaps argues for a longer-term and more intense intervention, although such treatments may not be realistic for many health care systems

ESR1 expression in human fetal testis and endometrium.

<p>A) Relative <i>ESR1</i> mRNA expression in adult human endometrium compared with human fetal testis (n = 3). w = weeks. Note: data presented on a log-scale. ESR1 protein expression in B) a 20 week gestation ungrafted human fetal testis and human endometrium (positive control; inset.) and C) e21.5 rat fetal testis. SC = seminiferous cords, arrows indicate Leydig cells. Original magnification 20×. Scale bar = 50 µm.</p

Effect of exposure to DES on testosterone production by human fetal testis xenografts.

<p>Seminal vesicle weight A) and serum testosterone B) in ungrafted (n = 6) and xenografted (n = 15) mice. Mean ± SEM. Bars with letters in common are not significantly different. Data analysed by unpaired t-test. Base-line variation in seminal vesicle weight C) and serum testosterone D) for host mice xenografted with individual fetuses (n = 6). Mean ± SEM. Data analysed by two-way ANOVA.</p

Graft survival and total graft weight of human fetal testis xenografts recovered from host mice exposed to either vehicle or DES at six weeks after xenografting.

<p>A) Graft survival (%). B) Total graft weight (mg). Data analysed by unpaired t-test; Mean ± SEM for n = 15, p>0.05.</p

Effect of exposure to DES from e13.5–e20.5 on intratesticular testosterone (ITT) at e21.5 in fetal rat testes.

<p>Data is presented as litter means (unpaired t-test, p = 0.004).</p