Gender-Specific Adverse Effects of Mono-Ethylhexyl Phthalate on Steroidogenesis in Immature Granulosa Cells and Rat Leydig cell Progenitors in vitro

Di-(2-ethylhexyl) phthalate, one of the phthalates most widely distributed in the environment, causes reproductive toxicity that is attributable to the action of its primary metabolite, mono-(2-ethylhexyl) phthalate (MEHP). Here, we have investigated the effects of MEHP on steroidogenesis by primary cultures of rat Leydig cell progenitors and immature granulosa cells. This phthalate stimulated basal steroidogenesis and steroidogenic acute regulatory protein (StAR) expression in both types of steroidogenic cells. However, when MEHP was incubated with (Bu)2cAMP, steroid production was increased in granulosa cells and suppressed in Leydig cell progenitors, a process associated with up-regulation of StAR expression. Our data suggest that MEHP exerts gender-specific adverse effects on the hormonal function of the developing gonads. This may be involved in the development of pathological conditions including disorders of prenatal sex development that may attenuate future reproductive health

Placental Apoptosis in Small for Gestational Age Babies: A Comparison between Swedish and Pakistani Populations

Background: Foeto-placental growth is regulated by a complex balance of growth promoting and growth inhibiting factors and hormones, namely the insulin-like growth factors (IGF) and the intracellular caspase proteins. Changes in the IGF-axis appear to affect this balance, with deficiencies possibly triggering apoptosis.Aim: To ascertain levels of apoptosis in the placenta of infants born small for gestational age (SGA) and appropriate for gestational age (AGA), comparing samples from two population groups, Pakistani and Swedish, in an attempt to better understand the mechanism behind foetal-placental growth restriction.Methods: Placental samples were taken immediately following delivery in both Karachi and Stockholm. In total 36 samples were included for further analysis (Pakistani: SGA n = 12, AGA n = 12; Swedish: SGA n = 7, AGA n = 5). Protein extraction was conducted for cell-death ELISA, and the remaining tissue samples were then paraffin embedded for further immunohistochemical and immunoflourescent analysis, looking at the apoptotic proteins, p53, caspase 8, and caspase 3. Furthermore, we compared maternal and newborn anthropometry between populations.Results: A higher apoptotic index, for caspase 8 and caspase 3, was seen in the Pakistani samples, as compared to the Swedish samples (p<0.01). TUNEL assays showed higher levels of apoptosis in the Pakistani population as compared to the Swedish population (p<0.01). Cell death ELISA analysis showed greater apoptotic activity in placenta from the Pakistani population as compared to the Swedish groups (p<0.05) as well as increased apoptotic activity in the SGA groups as compared to the AGA groups within each population (ELISA, p<0.05). No differences were seen in p53 levels as assessed by immunohistochemistry. Pakistani mothers were, on average, shorter than their Swedish counterparts (p<0.01).Conclusion: Increased apoptotic activity in placenta of the Pakistani population, as compared to their Swedish counterparts, may be associated with decreased foetal-placental growth seen in this population, particularly in babies born SGA. These findings, along with previously published results of the IGF-axis, and birth weight outcomes, suggests that lower IGF levels may be involved in the extracellular triggering of apoptosis, through caspase 8. This may further suggest a possible mechanism of foetal-placental growth restriction

Testicular organoid generation by a novel in vitro three-layer gradient system.

A system that models the testicular microenvironment and spermatogonial stem-cell (SSC) niche in vitro has not been produced yet. Here, we developed and characterized a novel three-dimensional multilayer model, the Three-Layer Gradient System (3-LGS), which permits the generation of rat testicular organoids with a functional blood-testis barrier (BTB) and germ cell establishment and proliferation. The model is unique as regards the formation of cellular organizations that more closely represent the in vivo germ-to-somatic cell associations in vitro. Moreover, we also verified the roles of retinoic acid (RA), IL-1α, TNFα and RA inhibitors in germ cell maintenance and BTB organization in vitro. Treatment with RA was beneficial for germ cell maintenance, while IL-1α and TNFα were observed to impair the formation of testicular organoids and germ cell maintenance. Taking in account our characterization and validation results, we propose the 3-LGS as a new platform to investigate the SSC niche in vitro and to search for novel unknown factors involved in germ cell proliferation and differentiation. Moreover, we suggest that this model can be used in other scientific fields to study organogenesis and development by the generation of organoids

Self-organising human gonads generated by a Matrigel-based gradient system.

BACKGROUND: Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro. Here we extend the model to human first-trimester embryonic gonadal tissue. RESULTS: Testicular cell suspensions reorganised into testis-like organoids with distinct seminiferous-like cords situated within an interstitial environment after 7 days. In contrast, tissue reorganisation failed to occur when mesonephros, which promotes testicular development in vivo, was included in the tissue digest. Organoids generated from dissociated female gonad cell suspensions formed loosely organised cords after 7 days. In addition to displaying testis-specific architecture, testis-like organoids demonstrated evidence of somatic cell differentiation. Within the 3-LGS, we observed the onset of AMH expression in the cytoplasm of SOX9-positive Sertoli cells within reorganised testicular cords. Leydig cell differentiation and onset of steroidogenic capacity was also revealed in the 3-LGS through the expression of key steroidogenic enzymes StAR and CYP17A1 within the interstitial compartment. While the 3-LGS generates a somatic cell environment capable of supporting germ cell survival in ovarian organoids germ cell loss was observed in testicular organoids. CONCLUSION: The 3-LGS can be used to generate organised whole gonadal organoids within 7 days. The 3-LGS brings a new opportunity to explore gonadal organogenesis and contributes to the development of more complex in vitro models in the field of developmental and regenerative medicine

Primary Human Testicular Cells Self-Organize into Organoids with Testicular Properties.

So far, successful de novo formation of testicular tissue followed by complete spermatogenesis in vitro has been achieved only in rodents. Our findings reveal that primary human testicular cells are able to self-organize into human testicular organoids (TOs), i.e., multi-cellular tissue surrogates, either with or without support of a biological scaffold. Despite lacking testis-specific topography, these mini-tissues harbored spermatogonia and their important niche cells, which retained specific functionalities during long-term culture. These observations indicate the posibility of in vitro re-engineering of a human testicular microenvironment from primary cells. Human TOs might help in the development of a biomimetic testicular model that would exert a tremendous impact on research and development, clinical treatment of infertility, and screening in connection with drug discovery and toxicology

Increases in bioactive IGF do not parallel increases in total IGF-I during growth hormone treatment of children born SGA.

BACKGROUND: Some children born small for gestational age (SGA) experience supra-physiological insulin-like growth factor-I (IGF-I) concentrations during GH treatment. However, measurements of total IGF-I concentrations may not reflect the bioactive fraction of IGF-I which reaches the IGF-I receptor at target organs. We examined endogenous IGF-bioactivity using an IGF-I kinase receptor activation (KIRA) assay that measures the ability of IGF-I to activate the IGF-IR in vitro. AIM: To compare responses of bioactive IGF and total IGF-I concentrations in short GH treated SGA children in the North European Small for Gestational Age Study (NESGAS). RESULTS: Bioactive IGF increased with age in healthy pre-pubertal children (n=94). SGA children had low-normal bioactive IGF levels at baseline (-0.12 (1.8 SD), increasing significantly after one year of high-dose GH treatment to 1.1 (1.4) SD, p2SD (mean IGF-I 2.8 SDS), whereas only 15% (n=15) had levels of bioactive IGF slightly above normal reference values. At baseline, bioactive IGF (SDS) was significantly correlated to height (SDS) (r=0.29, p=0.005), in contrast to IGF-I (SDS) (r=0.17, p=0.10). IGF-I (SDS) was inversely correlated to delta height (SDS) after one year of high-dose GH treatment (r=-0.22, p=0.02). CONCLUSION: In contrast to total IGF-I concentrations, bioactive IGF stayed within the normal reference ranges for most SGA children during the first year of GH treatment

Genetic markers of insulin sensitivity and insulin secretion are associated with spontaneous postnatal growth and response to growth hormone treatment in short SGA children: the North European SGA Study (NESGAS).

PURPOSE: The wide heterogeneity in the early growth and metabolism of children born small for gestational age (SGA), both before and during GH therapy, may reflect common genetic variations related to insulin secretion or sensitivity. METHOD: Combined multiallele single nucleotide polymorphism scores with known associations with insulin sensitivity or insulin secretion were analyzed for their relationships with spontaneous postnatal growth and first-year responses to GH therapy in 96 short SGA children. RESULTS: The insulin sensitivity allele score (GS-InSens) was positively associated with spontaneous postnatal weight gain (regression coefficient [B]: 0.12 SD scores per allele; 95% confidence interval [CI], 0.01-0.23; P = .03) and also in response to GH therapy with first-year height velocity (B: 0.18 cm/y per allele; 95% CI, 0.02-0.35; P = .03) and change in IGF-1 (B: 0.17 SD scores per allele; 95% CI, 0.00-0.32; P = .03). The association with first-year height velocity was independent of reported predictors of response to GH therapy (adjusted P = .04). The insulin secretion allele score (GS-InSec) was positively associated with spontaneous postnatal height gain (B: 0.15; 95% CI, 0.01-0.30; P = .03) and disposition index both before (B: 0.02; 95% CI, 0.00-0.04; P = .04) and after 1 year of GH therapy (B: 0.03; 95% CI, 0.01-0.05; P = .002), but not with growth and IGF-1 responses to GH therapy. Neither of the allele scores was associated with size at birth. CONCLUSION: Genetic allele scores indicative of insulin sensitivity and insulin secretion were associated with spontaneous postnatal growth and responses to GH therapy in short SGA children. Further pharmacogenetic studies may support the rationale for adjuvant therapies by informing the mechanisms of treatment response.This study was funded by a research grant from The Danish Council for Independent Research/ Medical Sciences and a research grant from Novo Nordisk A/S.This is the accepted manuscript. The final version is available at http://dx.doi.org/10.1210/jc.2014-3469

Alternative (backdoor) androgen production and masculinization in the human fetus

Funding: The study was supported by the following grants: Chief Scientist Office (Scottish Executive, CZG/4/742) (PAF and PJOS) (http://www.cso.scot.nhs.uk/funding-2/); NHS Grampian Endowments 08/02 (PAF and PJOS) and 15/1/010 (PAF, PF, US, and PJOS) (https://www.nhsgcharities.com/); the Glasgow Children’s Hospital Research Charity Research Fund, YRSS/PHD/2016/05 (NW, MB, PJOS, and PAF) (http://www.glasgowchildrenshospitalcharity.org/research/glasgow-childrens-hospital-charity-research-fund); the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement number 212885 (PAF) (https://ec.europa.eu/research/fp7/index_en.cfm); Medical Research Council Grants MR/L010011/1 (PAF and PJOS) and MR/K501335/1 (MB, PAF, and PJOS) (https://mrc.ukri.org/); and the Kronprinsessan Lovisas Foundation, “Stiftelsen Gunvor och Josef Anérs,” the “Stiftelsen Jane och Dan Olssons,” and the “Stiftelsen Tornspiran” (KS and OS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Adiposity in Children Born Small for Gestational Age Is Associated With β-Cell Function, Genetic Variants for Insulin Resistance, and Response to Growth Hormone Treatment.

BACKGROUND: Genetic susceptibility to insulin resistance is associated with lower adiposity in adults. Insulin resistance, and therefore adiposity, may alter sensitivity to GH. We aimed to determine the relationship between adiposity, genetic susceptibility to insulin resistance or insulin secretion, and response to GH treatment in short children born small for gestational age (SGA). METHODS: In 89 short prepubertal SGA children (age, 6.2 ± 1.6 y; 55 boys) treated with GH for 1 year in a multicenter study, body fat percentage was estimated at baseline and 1 year using dual-energy x-ray absorptiometry. The main outcome measures were treatment-related changes in height, IGF-1 standard deviation score, insulin sensitivity, insulin secretion, and disposition index. Combined multiallele gene scores based on single nucleotide polymorphisms with known associations with lower insulin sensitivity (gene scores for insulin resistance [GS-InRes]) and insulin secretion (gene scores for insulin secretion [GS-InSec]) were analyzed for their relationships with adiposity. RESULTS: Mean percentage body fat at baseline was low compared to normative data (P = .045) and decreased even further on GH treatment (baseline vs 1-year z-scores, -0.26 ± 1.2 vs -1.23 ± 1.54; P < .0001). Baseline percentage body fat was positively associated with IGF-1 responses (p-trends = .042), first-year height gains (B [95% confidence interval], 0.61 cm/y [0.28,0.95]; P < .0001), insulin secretion at baseline (p-trends = .020) and 1 year (p-trends = .004), and disposition index at 1 year (p-trends = .024). GS-InRes was inversely associated with body mass index (-0.13 SD score per allele [-0.26, -0.01]; P = .040), body fat (-0.49% per allele [-0.97, -0.007]; P = .047), and limb fat (-0.81% per allele [-1.62, 0.00]; P = .049) at baseline. During GH treatment, GS-InRes was related to a lesser decline in trunk fat (0.38% per allele [0.16, 0.59]; P = .001) and a higher trunk-limb fat ratio at 1 year (0.04 per allele [0.01, 0.08]; P = .008). GS-InSec was positively associated with truncal fat (0.36% per allele [0.09, 0.63]; P = .009). CONCLUSIONS: Adiposity in SGA children has favorable effects on GH sensitivity and glucose metabolism. The associations with multiallele scores support a causal role of insulin resistance in linking lesser body fat to reduced sensitivity to exogenous GH.This study was funded by research grants from the Danish Council for Independent Research/Medical Sciences and Novo Nordisk A/S. The research work was also supported by International Center for Research and Research Training in Endocrine Disrupting Effects on Male Reproduction and Child Health (EDMaRC), Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Denmark.This is the author accepted manuscript. The final version is available from Endocrine Society via http://dx.doi.org/10.1210/jc.2015-301