Preconceptional and in utero exposure of sheep to a real-life environmental chemical mixture disrupts key markers of energy metabolism in male offspring.

Over recent decades, an extensive array of anthropogenic chemicals have entered the environment and have been implicated in the increased incidence of an array of diseases, including metabolic syndrome. The ubiquitous presence of these environmental chemicals (ECs) necessitates the use of real-life exposure models to the assess cumulative risk burden to metabolic health. Sheep that graze on biosolids-treated pastures are exposed to a real-life mixture of ECs such as phthalates, per- and polyfluoroalkyl substances, heavy metals, pharmaceuticals, pesticides, and metabolites thereof, and this EC exposure can result in metabolic disorders in their offspring. Using this model, we evaluated the effects of gestational exposure to a complex EC mixture on plasma triglyceride (TG) concentrations and metabolic and epigenetic regulatory genes in tissues key to energy regulation and storage, including the hypothalamus, liver, and adipose depots of 11-month-old male offspring. Our results demonstrated a binary effect of EC exposure on gene expression particularly in the hypothalamus. Principal component analysis revealed two subsets (B-S1 [n = 6] and B-S2 [n = 4]) within the biosolids group (B, n = 10), relative to the controls (C, n = 11). Changes in body weight, TG levels, and in gene expression in the hypothalamus, and visceral and subcutaneous fat were apparent between biosolid and control and the two subgroups of biosolids animals. These findings demonstrate that gestational exposure to an EC mixture results in differential regulation of metabolic processes in adult male offspring. Binary effects on hypothalamic gene expression and altered expression of lipid metabolism genes in visceral and subcutaneous fat, coupled with phenotypic outcomes, point to differences in individual susceptibility to EC exposure that could predispose vulnerable individuals to later metabolic dysfunction.

Sexually dimorphic impact of preconceptional and gestational exposure to a real-life environmental chemical mixture (biosolids) on offspring growth dynamics and puberty in sheep.

Humans are ubiquitously exposed to complex mixtures of environmental chemicals (ECs). This study characterised changes in post-natal and peripubertal growth, and the activation of the reproductive axis, in male and female offspring of sheep exposed to a translationally relevant EC mixture (in biosolids), during pregnancy. Birthweight in both sexes was unaffected by gestational biosolids exposure. In contrast to females (unaffected), bodyweight in biosolids males was significantly lower than controls across the peripubertal period, however, they exhibited catch-up growth eventually surpassing controls. Despite weighing less, testosterone concentrations were elevated earlier, indicative of early puberty in the biosolids males. This contrasted with females in which the mean date of puberty (first progesterone cycle) was delayed. These results demonstrate that developmental EC-mixture exposure has sexually dimorphic effects on growth, puberty and the relationship between body size and puberty. Such programmed metabolic/reproductive effects could have significant impacts on human health and wellbeing.

Scoping review to assess online information available to new dog owners.

BACKGROUND: Many dog owners turn to the internet for pet care advice. As internet resources can positively or negatively influence owners' decision making and thus impact dogs' welfare, the identification of trustworthy information online is crucial. METHODS: A scoping review was conducted in 2014, and repeated in 2021, to assess the availability and quality indicators of information resources generated from Google searches using empirically derived dog owner internet search terms. RESULTS: A total of 121 unique resources were identified from 300 search hits in 2014, compared to 102 in 2021. On both occasions, the resources identified covered most aspects of dog care and related to all ages and breeds of dog. Flesch Kincaid readability scores indicated the majority to be easily understandable. However, many resources did not offer supporting evidence or a reference list, and the minority of resources stated an author. Of the resources identified in 2014, only 10% (n = 12/121) were dated, compared to 45% in 2021 (n = 46/102). CONCLUSION: This study found that while a great deal of information on caring for a new dog or puppy is available online, reliable indicators of quality were lacking, posing a barrier to owners' identification of trustworthy information.

Ovine fetal testis stage-specific sensitivity to environmental chemical mixtures.

Exposure of the fetal testis to numerous individual environmental chemicals (ECs) is frequently associated with dysregulated development, leading to impaired adult reproductive competence. However, 'real-life' exposure involves complex mixtures of ECs. Here we test the consequences, for the male fetus, of exposing pregnant ewes to EC mixtures derived from pastures treated with biosolids fertiliser (processed human sewage). Fetal testes from continuously exposed ewes were either unaffected at day 80 or exhibited a reduced area of testis immunostained for CYP17A1 protein at day 140. Fetal testes from day 140 pregnant ewes that were exposed transiently for 80-day periods during early (0-80 days), mid (30-110 days), or late (60-140 days) pregnancy had fewer Sertoli cells and reduced testicular area stained for CYP17A1. Male fetuses from ewes exposed during late pregnancy also exhibited reduced fetal body, adrenal and testis mass, anogenital distance, and lowered testosterone; collectively indicative of an anti-androgenic effect. Exposure limited to early gestation induced more testis transcriptome changes than observed for continuously exposed day 140 fetuses. These data suggest that a short period of EC exposure does not allow sufficient time for the testis to adapt. Consequently, testicular transcriptomic changes induced during the first 80 days of gestation may equate with phenotypic effects observed at day 140. In contrast, relatively fewer changes in the testis transcriptome in fetuses exposed continuously to ECs throughout gestation are associated with less severe consequences. Unless corrected by or during puberty, these differential effects would predictably have adverse outcomes for adult testicular function and fertility.

The ovarian follicle of ruminants: the path from conceptus to adult.

This review resulted from an international workshop and presents a consensus view of critical advances over the past decade in our understanding of follicle function in ruminants. The major concepts covered include: (1) the value of major genes; (2) the dynamics of fetal ovarian development and its sensitivity to nutritional and environmental influences; (3) the concept of an ovarian follicle reserve, aligned with the rise of anti-Müllerian hormone as a controller of ovarian processes; (4) renewed recognition of the diverse and important roles of theca cells; (5) the importance of follicular fluid as a microenvironment that determines oocyte quality; (6) the 'adipokinome' as a key concept linking metabolic inputs with follicle development; and (7) the contribution of follicle development to the success of conception. These concepts are important because, in sheep and cattle, ovulation rate is tightly regulated and, as the primary determinant of litter size, it is a major component of reproductive efficiency and therefore productivity. Nowadays, reproductive efficiency is also a target for improving the 'methane efficiency' of livestock enterprises, increasing the need to understand the processes of ovarian development and folliculogenesis, while avoiding detrimental trade-offs as greater performance is sought.

Environmental chemicals in dog testes reflect their geographical source and may be associated with altered pathology.

In humans and dogs, a temporal decline in semen quality and increased incidence of testicular cancer is hypothesised to be associated with exposure to anthropogenic chemicals, particularly during fetal development. Human studies suggest that differential exposures to environmental chemicals may be associated with geographical differences in male reproductive health. Here we investigate testicular chemical profiles and pathologies in dogs residing in the UK [West Midlands (WM), East Midlands (EM), South East (SE)], Denmark (Copenhagen) and Finland (Vantaa). Testes, surplus from routine castrations, contained region specific differences in relative concentrations of diethylhexyl phthalate (DEHP), polybrominated diphenyl ethers (PBDE) and polychlorinated biphenyls (PCB). Relative to UK regions, testes from dogs living in Finland and Denmark had higher concentrations of PBDE and lower concentrations of DEHP and PCBs. Regional differences in the UK in PCB concentrations were also observed. Dog testes from Finland had fewer pathologies, reduced testicular area stained for Sertoli and germ cells and evidence of reduced cellular proliferation. Since the geographical differences in testis pathologies in dogs parallel reports of regional differences in human testicular cancer, we postulate that this may reflect chemical effects within the testis and that this may be related to environmental influences on male reproductive function.

Independent and combined effects of diethylhexyl phthalate and polychlorinated biphenyl 153 on sperm quality in the human and dog.

A temporal decline in human and dog sperm quality is thought to reflect a common environmental aetiology. This may reflect direct effects of seminal chemicals on sperm function and quality. Here we report the effects of diethylhexyl phthalate (DEHP) and polychlorinated biphenyl 153 (PCB153) on DNA fragmentation and motility in human and dog sperm. Human and dog semen was collected from registered donors (n = 9) and from stud dogs (n = 11) and incubated with PCB153 and DEHP, independently and combined, at 0x, 2x, 10x and 100x dog testis concentrations. A total of 16 treatments reflected a 4 × 4 factorial experimental design. Although exposure to DEHP and/or PCB153 alone increased DNA fragmentation and decreased motility, the scale of dose-related effects varied with the presence and relative concentrations of each chemical (DEHP.PCB interaction for: DNA fragmentation; human p < 0.001, dog p < 0.001; Motility; human p < 0.001, dog p < 0.05). In both human and dog sperm, progressive motility negatively correlated with DNA fragmentation regardless of chemical presence (Human: P < 0.0001, r = -0.36; dog P < 0.0001, r = -0.29). We conclude that DEHP and PCB153, at known tissue concentrations, induce similar effects on human and dog sperm supporting the contention of the dog as a sentinel species for human exposure.

Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism.

Adverse temporal trends in human semen quality and cryptorchidism in infants have been associated with exposure to environmental chemicals (ECs) during development. Here we report that a population of breeding dogs exhibit a 26 year (1988-2014) decline in sperm quality and a concurrent increased incidence of cryptorchidism in male offspring (1995-2014). A decline in the number of males born relative to the number of females was also observed. ECs, including diethylhexyl phthalate (DEHP) and polychlorinated biphenyl 153 (PCB153), were detected in adult dog testes and commercial dog foods at concentrations reported to perturb reproductive function in other species. Testicular concentrations of DEHP and PCB153 perturbed sperm viability, motility and DNA integrity in vitro but did not affect LH stimulated testosterone secretion from adult testis explants. The direct effects of chemicals on sperm may therefore contribute to the decline in canine semen quality that parallels that reported in the human.

The fetal ovary exhibits temporal sensitivity to a 'real-life' mixture of environmental chemicals.

The development of fetal ovarian follicles is a critical determinant of adult female reproductive competence. Prolonged exposure to environmental chemicals (ECs) can perturb this process with detrimental consequences for offspring. Here we report on the exposure of pregnant ewes to an environmental mixture of ECs derived from pastures fertilized with sewage sludge (biosolids): a common global agricultural practice. Exposure of pregnant ewes to ECs over 80 day periods during early, mid or late gestation reduced the proportion of healthy early stage fetal follicles comprising the ovarian reserve. Mid and late gestation EC exposures had the most marked effects, disturbing maternal and fetal liver chemical profiles, masculinising fetal anogenital distance and greatly increasing the number of altered fetal ovarian genes and proteins. In conclusion, differential temporal sensitivity of the fetus and its ovaries to EC mixtures has implications for adult ovarian function following adverse exposures during pregnancy.

Maternal undernutrition does not alter Sertoli cell numbers or the expression of key developmental markers in the mid-gestation ovine fetal testis.

BACKGROUND: The aim of this study was to determine the effects of maternal undernutrition on ovine fetal testis morphology and expression of relevant histological indicators. Maternal undernutrition, in sheep, has been reported, previously, to alter fetal ovary development, as indicated by delayed folliculogenesis and the altered expression of ovarian apoptosis-regulating gene products, at day 110 of gestation. It is not known whether or not maternal undernutrition alters the same gene products in the day 110 fetal testis. DESIGN AND METHODS: Mature Scottish Blackface ewes were fed either 100% (Control; C) or 50% (low; L) of estimated metabolisable energy requirements of a pregnant ewe, from mating to day 110 of gestation. All pregnant ewes were euthanized at day 110 and a sub-set of male fetuses was randomly selected (6 C and 9 L) for histology studies designed to address the effect of nutritional state on several indices of testis development. Sertoli cell numbers were measured using a stereological method and Ki67 (cell proliferation index), Bax (pro-apoptosis), Mcl-1 (anti-apoptosis), SCF and c-kit ligand (development and apoptosis) gene expression was measured in Bouins-fixed fetal testis using immunohistochemistry. RESULTS: No significant differences were observed in numbers of Sertoli cells or testicular Ki67 positive cells. The latter were localised to the testicular cords and interstitium. Bax and Mcl-1 were localised specifically to the germ cells whereas c-kit was localised to both the cords and interstitium. SCF staining was very sparse. No treatment effects were observed for any of the markers examined. CONCLUSIONS: These data suggest that, unlike in the fetal ovary, maternal undernutrition for the first 110 days of gestation affects neither the morphology of the fetal testis nor the expression of gene products which regulate apoptosis. It is postulated that the effects of fetal undernutrition on testis function may be expressed through hypothalamic-pituitary changes.

Ovine corpus luteum proteins, with functions including oxidative stress and lipid metabolism, show complex alterations during implantation.

Progesterone (P(4)) secreted by the corpus luteum (CL) is critical for in utero embryo survival and development, although CL proteins are key regulatory factors during the luteal phase. We, therefore, characterised protein expression patterns in ovine CL of pregnancy (days 12, 16 and 20) compared with those of controls, CL of oestrous cycle (days 12 and 16), using two-dimensional gel electrophoresis (2DE) gel-based proteomics. Proteins in 24 significantly altered spots were identified by tandem mass spectroscopy. At the time of embryo implantation (day 16), 77 spots were up-regulated and 101 spots were down-regulated in CL of pregnancy compared with regressed CL. Vimentin, lamin A/C (LMNA), [Mn] superoxide dismutase (SOD2), isocitrate dehydrogenase 1, annexin A1 and elongation factor Tu, mitochondrial (TUFM) altered during CL regression, whereas glutathione S-transferase A1, apolipoprotein A-1, myxovirus resistance protein 1, ornithine aminotransferase and enoyl-CoA hydratase, mitochondrial (ECHS1) tended to be altered during CL maintenance. biliverdin reductase B (BLVRB), FDXR, guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (GNB2) and cytochrome b-c1 complex subunit 1, mitochondrial (UQCRC1) showed divergent expression during CL regression and maintenance. The expression of two representative proteins, SOD2 and BLVRB, by western blot increased in CL of non-pregnant ewes on day 16 compared with that on day 12. SOD2 and BLVRB were localised in the large and small luteal cells and endothelial cells of CL over peri-implantation periods. 2DE gel and mass spectrometry have been used, for the first time, to study ovine CL function. We have identified proteins involved in key pathways, including oxidative stress, steroidogenesis, signal transduction and apoptosis, which have not previously been associated with changes occurring in the CL during the peri-implantation period. These proteins are most likely involved with mechanisms allowing the CL to produce P(4) during early pregnancy.

Novel aspects of endometrial function: a biological sensor of embryo quality and driver of pregnancy success.

Successful pregnancy depends on complex biological processes that are regulated temporally and spatially throughout gestation. The molecular basis of these processes have been examined in relation to gamete quality, early blastocyst development and placental function, and data have been generated showing perturbations of these developmental stages by environmental insults or embryo biotechnologies. The developmental period falling between the entry of the blastocyst into the uterine cavity to implantation has also been examined in terms of the biological function of the endometrium. Indeed several mechanisms underlying uterine receptivity, controlled by maternal factors, and the maternal recognition of pregnancy, requiring conceptus-produced signals, have been clarified. Nevertheless, recent data based on experimental perturbations have unveiled unexpected biological properties of the endometrium (sensor/driver) that make this tissue a dynamic and reactive entity. Persistent or transient modifications in organisation and functionality of the endometrium can dramatically affect pre-implantation embryo trajectory through epigenetic alterations with lasting consequences on later stages of pregnancy, including placentation, fetal development, pregnancy outcome and post-natal health. Developing diagnostic and prognostic tools based on endometrial factors may enable the assessment of maternal reproductive capacity and/or the developmental potential of the embryo, particularly when assisted reproductive technologies are applied.

Effects of omega-3 and -6 polyunsaturated fatty acids on ovine follicular cell steroidogenesis, embryo development and molecular markers of fatty acid metabolism.

We previously reported increased follicular fluid progesterone (P(4)) concentrations in ewes fed an n-3 compared to an n-6 polyunsaturated fatty acid (PUFA)-enriched diet, but detected no differential effect of n-3 and n-6 PUFA-enriched high-density lipoproteins (HDL) on granulosa cell (GC) steroidogenesis in vitro. Moreover, net n-6 PUFA-enriched HDL reduced early embryo development, but in the absence of a net uptake of FA. Consequently, we hypothesised that a) effects of n-3 PUFA on ovarian steroidogenesis are mediated by theca rather than GCs and b) during embryo culture lipids are acquired solely from the albumin fraction of serum, so that albumin-delivered n-3 and n-6 PUFA exert a greater differential effect on embryo development than either low-density lipoprotein (LDL)- or HDL-delivered PUFA. Data confirmed that n-3 PUFA increases P(4) production solely in theca cells and that this is associated with an increase in STAR transcript expression. Furthermore, LDL- and HDL-delivered n-3 PUFA are equally efficacious in this regard during the first 96 h of culture, but thereafter only HDL-delivered n-3 PUFA induces this effect in partially luteinised theca cells. We also demonstrate that albumin is the sole serum fraction that leads to a net uptake of FA during embryo culture. PUFA-enriched serum and albumin increased the yield of morphologically poorer quality blastocysts with increased transcript expression for the antioxidant enzyme SOD1. Important differential effects of n-3 and n-6 PUFA on ovarian steroidogenesis acting solely on theca cells are identified, but differential effects of PUFA on embryo development are less apparent.

Equine transcriptome quantification using human GeneChip arrays can be improved using genomic DNA hybridisation and probe selection.

Affymetrix GeneChip arrays are a powerful tool for transcriptome profiling and have been applied to a wide range of species. A genomic DNA (gDNA)-based probe selection method has been developed which broadens the range of species to which GeneChips may be successfully applied. This study demonstrated that gDNA-based probe selection on the Affymetrix U133+2 GeneChip array can be used to study the equine transcriptome which, to date, has received only limited attention. More than 29,000 transcripts can be detected in equine brain and liver and in primary cultures of equine articular chondrocytes. Gene ontology analysis of differentially expressed genes revealed the presence of expected categories within each tissue. The level of gene expression could also be correlated with the phenotypes and specialised functions of each tissue. The results demonstrated that probe selection on a human chip can be successfully used to study the equine transcriptome.

Gene expression analysis of human fetal ovarian primordial follicle formation.

CONTEXT: Primordial follicle formation dictates the maximal potential female reproductive capacity and establishes the ovarian reserve. Currently, little is known about this process in the human. OBJECTIVE: The aim of the study was to identify genes associated with the onset of human fetal primordial follicle formation in morphologically normal human fetuses. DESIGN: We conducted an observational study of the female fetal gonad, comparing gene expression before and during primordial follicle formation. SETTING: The study was conducted at the Universities of Aberdeen, Glasgow, and Nottingham. PATIENTS/PARTICIPANTS: Ovaries were collected from 51 morphologically normal human female fetuses of women undergoing elective termination of normal second trimester pregnancies. MAIN OUTCOME MEASURES: We performed fetal ovarian transcript expression by Affymetrix array and quantitative RT-PCR and gene product expression and localization by Western blot and immunohistochemistry. RESULTS: Five transcripts were down-regulated and 61 were up-regulated in ovaries from older fetuses (18-20 wk) in which primordial follicle formation had started compared with younger (15-16 wk) fetuses in which no primordial follicles were observed. The altered genes contribute to major functions, including gene expression, tissue morphology, and apoptosis, that are essential for ovarian development. NALP5, the most highly regulated transcript, is an oocyte-specific maternal effect gene that is regulated downstream of FIGLA. CONCLUSIONS: NALP5 probably plays a key role in the onset of human primordial follicle formation and thus the establishment of ovarian reserve in women.

In utero exposure to low doses of environmental pollutants disrupts fetal ovarian development in sheep.

Epidemiological studies of the impact of environmental chemicals on reproductive health demonstrate consequences of exposure but establishing causative links requires animal models using 'real life' in utero exposures. We aimed to determine whether prolonged, low-dose, exposure of pregnant sheep to a mixture of environmental chemicals affects fetal ovarian development. Exposure of treated ewes (n = 7) to pollutants was maximized by surface application of processed sewage sludge to pasture. Control ewes (n = 10) were reared on pasture treated with inorganic fertilizer. Ovaries and blood were collected from fetuses (n = 15 control and n = 8 treated) on Day 110 of gestation for investigation of fetal endocrinology, ovarian follicle/oocyte numbers and ovarian proteome. Treated fetuses were 14% lighter than controls but fetal ovary weights were unchanged. Prolactin (48% lower) was the only measured hormone significantly affected by treatment. Treatment reduced numbers of growth differentiation factor (GDF9) and induced myeloid leukaemia cell differentiation protein (MCL1) positive oocytes by 25-26% and increased pro-apoptotic BAX by 65% and 42% of protein spots in the treated ovarian proteome were differently expressed compared with controls. Nineteen spots were identified and included proteins involved in gene expression/transcription, protein synthesis, phosphorylation and receptor activity. Fetal exposure to environmental chemicals, via the mother, significantly perturbs fetal ovarian development. If such effects are replicated in humans, premature menopause could be an outcome.

Maternal smoking during pregnancy specifically reduces human fetal desert hedgehog gene expression during testis development.

CONTEXT: Maternal cigarette smoking during gestation increases cryptorchidism and hypospadias and reduces testis size and fertility in sons by unknown mechanisms. OBJECTIVE: The objective of the study was to determine whether maternal smoking is linked with changes in male human fetal endocrinology, testis gene expression, and liver concentrations of cigarette smoke chemicals. DESIGN: This was an observational study of the male fetus, comparing pregnancies during which the mothers either did or did not smoke. SETTING: The study was conducted at the universities of Aberdeen, Glasgow, and Nottingham and Macaulay Institute (Aberdeen). PATIENTS/PARTICIPANTS: Testes, blood, and livers were collected from 69 morphologically normal human male fetuses of women undergoing elective termination of normal second-trimester pregnancies. MAIN OUTCOME MEASURES: Testosterone, human chorionic gonadotropin, LH, and cotinine; expression of 30 reproductive/developmental genes; liver concentrations of 16 polycyclic aromatic hydrocarbons; and Leydig, Sertoli. and germ cell numbers were determined. RESULTS: There were no significant differences in fetal size, testis weight, cell numbers, seminiferous tubule diameter, or circulating LH and testosterone. Fetuses from smoking mothers had smoking range cotinine levels and liver concentrations of polycyclic aromatic hydrocarbons that were significant predictors of maternal smoking (P < 0.001). Only the Sertoli cell-specific gene, desert hedgehog (DHH), was significantly altered by maternal smoking (reduced 1.8-fold, P = 0.013). CONCLUSIONS: The consequences of reduced DHH signaling in men and mice are consistent with epidemiology for effects of gestational maternal smoking on sons. Given the absence of other observed effects of maternal smoking, we concluded that reduced DHH is part of a mechanism linking maternal gestational smoking with impaired reproductive development in male offspring.

DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status.

A complex combination of adult health-related disorders can originate from developmental events that occur in utero. The periconceptional period may also be programmable. We report on the effects of restricting the supply of specific B vitamins (i.e., B(12) and folate) and methionine, within normal physiological ranges, from the periconceptional diet of mature female sheep. We hypothesized this would lead to epigenetic modifications to DNA methylation in the preovulatory oocyte and/or preimplantation embryo, with long-term health implications for offspring. DNA methylation is a key epigenetic contributor to maintenance of gene silencing that relies on a dietary supply of methyl groups. We observed no effects on pregnancy establishment or birth weight, but this modest early dietary intervention led to adult offspring that were both heavier and fatter, elicited altered immune responses to antigenic challenge, were insulin-resistant, and had elevated blood pressure-effects that were most obvious in males. The altered methylation status of 4% of 1,400 CpG islands examined by restriction landmark genome scanning in the fetal liver revealed compelling evidence of a widespread epigenetic mechanism associated with this nutritionally programmed effect. Intriguingly, more than half of the affected loci were specific to males. The data provide the first evidence that clinically relevant reductions in specific dietary inputs to the methionine/folate cycles during the periconceptional period can lead to widespread epigenetic alterations to DNA methylation in offspring, and modify adult health-related phenotypes.

Human fetal testis Leydig cell disruption by exposure to the pesticide dieldrin at low concentrations.

BACKGROUND: Declining human reproductive health over the last 60 years has been proposed to be due to effects of environmental chemicals, especially endocrine disrupting compounds, on fetal development. We investigated whether a model pesticide, dieldrin, at concentrations within both maternal circulation and environmental ranges (1 pmol/l = 0.0004 p.p.b. = 380.9 pg/l), could disrupt the human fetal testis. METHODS: Human fetal testes were collected during the second trimester, a critical period of male sexual differentiation (development and masculinization). Testis explants were cultured for 24 h in the presence and absence of LH (10-1000 IU LH/l) and dieldrin (1 pmol and 1 nmol/l). Endocrine, immunohistological and proteome characteristics of the tissues were investigated. RESULTS: Exposure to dieldrin reduced LH-induced testosterone secretion (P < 0.05) and tissue protein concentrations of LH receptor and steroid acute regulatory protein (P < 0.05). Dieldrin altered proteins associated with cancer, apoptosis, transcription and development. Wnt-2b was reduced 3-fold and immunolocalized to Leydig and Sertoli cells. Dieldrin also reversed some LH-induced changes in protein expression, supporting the conclusion that Leydig cell function is at risk from environmental chemicals. CONCLUSIONS: Our findings indicate that exposure to very low, biologically relevant, concentrations of environmental chemicals could affect the fetal human Leydig cell, reducing testosterone secretion and potentially leading to subtle dysregulation of reproductive development and adult fecundity.