Genome-wide expression changes induced by bisphenol A, F and S in human stem cell derived hepatocyte-like cells.

The debate about possible adverse effects of bisphenol A (BPA) has been ongoing for decades. Bisphenol F (BPF) and S (BPS) have been suggested as "safer" alternatives. In the present study we used hepatocyte-like cells (HLCs) derived from the human embryonic stem cell lines Man12 and H9 to compare the three bisphenol derivatives. Stem cell-derived progenitors were produced using an established system and were exposed to BPA, BPF and BPS for 8 days during their transition to HLCs. Subsequently, we examined cell viability, inhibition of cytochrome P450 (CYP) activity, and genome-wide RNA profiles. Sub-cytotoxic, inhibitory concentrations (IC50) of CYP3A were 20, 9.5 and 25 µM for BPA, BPF and BPS in Man12 derived HLCs, respectively. The corresponding concentrations for H9-derived HLCs were 19, 29 and 31 µM. These IC50 concentrations were used to study global expression changes in this in vitro study and are higher than unconjugated BPA in serum of the general population. A large overlap of up- as well as downregulated genes induced by the three bisphenol derivatives was seen. This is at least 28-fold higher compared to randomly expected gene expression changes. Moreover, highly significant correlations of expression changes induced by the three bisphenol derivatives were obtained in pairwise comparisons. Dysregulated genes were associated with reduced metabolic function, cellular differentiation, embryonic development, cell survival and apoptosis. In conclusion, no major differences in cytochrome inhibitory activities of BPA, BPF and BPS were observed and gene expression changes showed a high degree of similarity.

Identification of Sertoli cell-specific transcripts in the mouse testis and the role of FSH and androgen in the control of Sertoli cell activity.

BACKGROUND: The Sertoli cells act to induce testis differentiation and subsequent development in fetal and post-natal life which makes them key to an understanding of testis biology. As a major step towards characterisation of factors involved in Sertoli cell function we have identified Sertoli cell-specific transcripts in the mouse testis and have used the data to identify Sertoli cell-specific transcripts altered in mice lacking follicle-stimulating hormone receptors (FSHRKO) and/or androgen receptors (AR) in the Sertoli cells (SCARKO). RESULTS: Adult iDTR mice were injected with busulfan to ablate the germ cells and 50 days later they were treated with diphtheria toxin (DTX) to ablate the Sertoli cells. RNAseq carried out on testes from control, busulfan-treated and busulfan + DTX-treated mice identified 701 Sertoli-specific transcripts and 4302 germ cell-specific transcripts. This data was mapped against results from microarrays using testicular mRNA from 20 day-old FSHRKO, SCARKO and FSHRKO.SCARKO mice. Results show that of the 534 Sertoli cell-specific transcripts present on the gene chips, 85% were altered in the FSHRKO mice and 94% in the SCARKO mice (mostly reduced in both cases). In the FSHRKO.SCARKO mice additive or synergistic effects were seen for most transcripts. Age-dependent studies on a selected number of Sertoli cell-specific transcripts, showed that the marked effects in the FSHRKO at 20 days had largely disappeared by adulthood although synergistic effects of FSHR and AR knockout were seen. CONCLUSIONS: These studies have identified the Sertoli cell-specific transcriptome in the mouse testis and have shown that most genes in the transcriptome are FSH- and androgen-dependent at puberty although the importance of FSH diminishes towards adulthood.

Timing of Maternal Exposure and Foetal Sex Determine the Effects of Low-level Chemical Mixture Exposure on the Foetal Neuroendocrine System in Sheep.

We have shown that continuous maternal exposure to the complex mixture of environmental chemicals (ECs) found in human biosolids (sewage sludge), disrupts mRNA expression of genes crucial for development and long-term regulation of hypothalamic-pituitary gonadal (HPG) function in sheep. The present study investigated whether exposure to ECs only during preconceptional period or only during pregnancy perturbed key regulatory genes within the hypothalamus and pituitary gland and whether these effects were different from chronic (life-long) exposure to biosolid ECs. The findings demonstrate that the timing and duration of maternal EC exposure influences the subsequent effects on the foetal neuroendocrine system in a sex-specific manner. Maternal exposure prior to conception, or during pregnancy only, altered the expression of key foetal neuroendocrine regulatory systems such as gonadotrophin-releasing hormone and kisspeptin to a greater extent than when maternal exposure was 'life-long'. Furthermore, hypothalamic gene expression was affected to a greater extent in males than in females and, following EC exposure, male foetuses expressed more 'female-like' mRNA levels for some key neuroendocrine genes. This is the first study to show that 'real-life' maternal exposure to low levels of a complex cocktail of chemicals prior to conception can subsequently affect the developing foetal neuroendocrine system. These findings demonstrate that the developing neuroendocrine system is sensitive to EC mixtures in a sex-dimorphic manner likely to predispose to reproductive dysfunction in later life.

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy.

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.

Reproduction Symposium: does grazing on biosolids-treated pasture pose a pathophysiological risk associated with increased exposure to endocrine disrupting compounds?

Biosolids (processed human sewage sludge), which contain low individual concentrations of an array of contaminants including heavy metals and organic pollutants such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), and polychlorinated dibenzodioxins/polychlorinated dibenzofurans known to cause physiological disturbances, are increasingly being used as an agricultural fertilizer. This could pose a health threat to both humans and domestic and wild animal species. This review summarizes results of a unique model, used to determine the effects of exposure to mixtures of environmentally relevant concentrations of pollutants, in sheep grazed on biosolids-treated pastures. Pasture treatment results in nonsignificant increases in environmental chemical (EC) concentrations in soil. Whereas EC concentrations were increased in some tissues of both ewes and their fetuses, concentrations were low and variable and deemed to pose little risk to consumer health. Investigation of the effects of gestational EC exposure on fetal development has highlighted a number of issues. The results indicate that gestational EC exposure can adversely affect gonadal development (males and females) and that these effects can impact testicular morphology, ovarian follicle numbers and health, and the transcriptome and proteome in adult animals. In addition, EC exposure can be associated with altered expression of GnRH, GnRH receptors, galanin receptors, and kisspeptin mRNA within the hypothalamus and pituitary gland, gonadotroph populations within the pituitary gland, and regional aberrations in thyroid morphology. In most cases, these anatomical and functional differences do not result in altered peripheral hormone concentrations or reproductive function (e.g., lambing rate), indicating physiological compensation under the conditions tested. Physiological compensation is also suggested from studies that indicate that EC effects may be greater when exposure occurs either before or during gestation compared with EC exposure throughout life. With regard to human and animal health, this body of work questions the concept of safe individual concentration of EC when EC exposure typically occurs as complex mixtures. It suggests that developmental EC exposure may affect many different physiological systems, with some sex-specific differences in EC sensitivity, and that EC effects may be masked under favorable physiological conditions.

Identification of Leydig cell-specific mRNA transcripts in the adult rat testis.

The adult population of Leydig cells acts to secrete testosterone which is essential for reproductive health and fertility in the adult male. However, other physiological functions of these cells are uncertain, and to address this issue a cell ablation model has been used to identify Leydig cell-specific mRNA transcripts. Ethane dimethane sulphonate (EDS) was synthesised by a novel process and was used to ablate Leydig cells in adult male rats previously treated with butane dimethane sulphonate (busulphan) to delete the germ cell population. Levels of mRNA transcripts were measured in the testis using microarrays 1, 3, 5, 8 and 12 days after EDS injection. During this period, there was a significant change in the levels of 2200 different transcripts with a marked decline in the levels of canonical Leydig cell transcripts, such as Cyp11a1, Cyp17a1 and Insl3. A total of 95 transcripts showed a similar decline in expression after EDS treatment, suggesting that they have a Leydig cell-specific origin. Analysis of selected transcripts confirmed that they were expressed specifically in Leydig cells and showed that most had a late onset of expression during adult Leydig cell development. Apart from transcripts encoding components of the steroidogenic apparatus, the most common predicted function of translated proteins was endogenous and xenotoxicant metabolism. In addition, a number of transcripts encode acute-phase proteins involved in reduction of oxidative stress. Results show that, in addition to androgen secretion, Leydig cells may have a critical role to play in protecting the testis from damage caused by toxicants or stress.

Proteomic analysis of the sheep caruncular and intercaruncular endometrium reveals changes in functional proteins crucial for the establishment of pregnancy.

The expression and regulation of endometrial proteins are crucial for conceptus implantation and development. However, little is known about site-specific proteome profiles of the mammalian endometrium during the peri-implantation period. We utilised a two-dimensional gel electrophoresis/mass spectrometry-based proteomics approach to compare and identify differentially expressed proteins in sheep endometrium. Caruncular and intercaruncular endometrium were collected on days 12 (C12) and 16 (C16) of the oestrous cycle and at three stages of pregnancy corresponding to conceptus pre-attachment (P12), implantation (P16) and post-implantation (P20). Abundance and localisation changes in differentially expressed proteins were determined by western blot and immunohistochemistry. In caruncular endometrium, 45 protein spots (5% of total spots) altered between day 12 of pregnancy (P12) and P16 while 85 protein spots (10% of total spots) were differentially expressed between P16 and C16. In intercaruncular endometrium, 31 protein spots (2% of total spots) were different between P12 and P16 while 44 protein spots (4% of total spots) showed differential expression between C12 and C16. The pattern of protein changes between caruncle and intercaruncle sites was markedly different. Among the protein spots with implantation-related changes in volume, 11 proteins in the caruncular endometrium and six proteins in the intercaruncular endometrium, with different functions such as protein synthesis and degradation, antioxidant defence, cell structural integrity, adhesion and signal transduction, were identified. Our findings highlight the different but important roles of the caruncular and intercaruncular proteins during early pregnancy.

Steroidogenic enzyme expression in the human fetal liver and potential role in the endocrinology of pregnancy.

The human feto-maternal unit produces large amounts of steroid hormones, particularly estrogens, during the second and third trimesters. The fetal adrenal gland and the placenta are considered the principal tissues driving steroid production but the fetal liver is likely to play an essential role in this process. This study was designed to measure transcript expression of proteins involved in steroid synthesis, metabolism, conjugation and signalling in the human fetal liver and to examine sex differences and effects of maternal smoking. Liver samples were taken from 55 normal fetuses from women undergoing second trimester elective termination. Levels of 23 mRNA transcripts encoding steroid synthesis/metabolic/conjugation enzymes and steroid receptors were measured by real-time PCR. The expression of representative proteins was confirmed by western blotting and immunohistochemistry. The human fetal livers expressed high levels of CYP19A1, SULT2A1, SULT1E1, HSD17B2, SRD5A3 and CYP3A7. Lower levels of SULT1A1, STS, UGT2B17, GPER, AKR1C3, UGT2B15, AR, CYP11A1, CYP21A2, HSD17B3, HSD17B1 and SRD5A1 were also detectable. The expression of ESR, ESR2, CYP17A1 and HSD3B transcripts was undetectable in most fetal livers, although HSD3B was shown to be present by western blotting. Sex differences were limited to SRD5A3 (lower in females) and UGT2B17 (higher in females). Maternal smoking increased the expression of CYP19A1, SULT2A1, UGT2B17, HSD17B2 and AKR1C3 and reduced the expression of SRD5A3 in the male fetal liver. This study shows that the human fetal liver is likely to have an extensive effect on circulating steroid levels in the human fetus and mother. The most important of these effects will be alterations to the species, conjugation and availability of estrogens in the fetus. Maternal smoking is likely to reduce circulating androgen bioactivity in male fetuses.

Effects of exposure to environmental chemicals during pregnancy on the development of the male and female reproductive axes.

There is a large body of literature describing effects of environmental chemicals (ECs), many of them anthropogenic with endocrine-disrupting properties, on development in rodent laboratory species, some of which lead to impaired reproduction and adverse health. This literature joins extensive human epidemiological data and opportunistic wildlife findings on health effects of ECs. In contrast, the effect of endocrine disruption on foetal development and reproductive performance in domestic species is less extensively documented. This applies both to domestic farm and to companion species even though the former is critical to food production and the latter share our homes and many aspects of the modern developed human lifestyle. In domestic species, the nature of chemicals exposure in utero and their consequences for animal health and production are poorly understood. A complication in our understanding is that the pace of development, ontogeny and efficiency of foetal and maternal hepatic and placental activity differs between domestic species. In many ways, this reflects the difficulties in understanding human exposure and consequences of that exposure for the foetus and subsequent adult from epidemiological and largely rodent-based data. It is important that domestic species are included in research into endocrine disruption because of their (i) wide variety of exposure to such chemicals, (ii) greater similarity of many developmental processes to the human, (iii) economic importance and (iv) close similarities to developed world human lifestyle in companion species.

Foetal and post-natal exposure of sheep to sewage sludge chemicals disrupts sperm production in adulthood in a subset of animals.

Exposure to ubiquitous, environmental chemicals (ECs) has been hypothesized as a cause for declining male reproductive health. Understanding the long-term effects of EC exposure on reproductive health in humans requires animal models and exposure to 'real life', environmentally relevant, mixtures during development, a life stage of particular sensitivity to ECs. The aim of this study was to evaluate the effects of in utero and post-natal exposure to environmentally relevant levels of ECs, via sewage sludge application to pasture, on the adult male sheep testis. Hormones, liver concentrations of candidate ECs and Sertoli and germ cell numbers in testes of adult rams that were exposed to ECs in sewage sludge in utero, and until weaning via maternal exposure, and post-weaning via grazing pastures fertilized with sewage sludge, were quantified. Evaluated as a single group, exposure to sludge ECs was without significant effect on most parameters. However, a more detailed study revealed that 5 of 12 sludge-exposed rams exhibited major spermatogenic abnormalities. These consisted of major reductions in germ cell numbers per testis or per Sertoli cell and more Sertoli cell-only tubules, when compared with controls, which did not show any such changes. The sludge-related spermatogenic changes in the five affected animals were significantly different from controls (p < 0.001); Sertoli cell number was unaffected. Hormone profiles and liver candidate EC concentrations were not measurably affected by exposure. We conclude that developmental exposure of male sheep to real-world mixtures of ECs can result in major reduction in germ cell numbers, indicative of impaired sperm production, in a proportion of exposed males. The individual-specific effects are presumed to reflect EC effects on a heterogeneous population in which some individuals may be more susceptible to adverse EC effects. Such effects of EC exposure in humans could have adverse consequences for sperm counts and fertility in some exposed males.

Maternal and fetal tissue accumulation of selected endocrine disrupting compounds (EDCs) following exposure to sewage sludge-treated pastures before or after conception.

Liver concentrations of selected pollutant classes were determined in groups of sheep fetuses and their dams, at 55 (Experiment 1) and 110 (Experiment 2) days of gestation (term = 145 d) following exposure, throughout their breeding lives and after mating, to pasture treated with either inorganic fertiliser (control, CC) or with sewage sludge (treated, TT). In a unique study designed to separate the respective contributions of environmental sources and mobilised tissue to the available EDC burden, in additional groups of animals, pollutant burdens at 110 days gestation were assessed following exposure to the respective treatments, either throughout their breeding lives until mating, but not thereafter (TC), or only between mating and slaughter (CT) (Experiment 3). With very few exceptions, maternal and fetal liver concentrations of diethylhexyl phthalate (DEHP) and selected polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDE) and polycyclic aromatic hydrocarbons (PAHs) were not significantly affected by sludge exposure in any group. In some cases, maternal and fetal tissue EDC concentrations were different but the differences were not consistent, and maternal and fetal concentrations of none of the classes of chemical were significantly correlated. It was not possible to identify a single chemical, or class of chemical, that may be responsible for previously observed physiological effects of exposure to sludge-treated pastures. It is concluded that exposure of sheep to pastures fertilised with sewage sludge was not associated with increased liver concentrations of EDCs, irrespective of the stage of development at which they were measured and of maternal tissue mobilisation and EDC release during gestation. Thus, retrospective measurements of EDC tissue burdens could not be used to accurately assess earlier fetal EDC insults.

Effects of environmental pollutants on the reproduction and welfare of ruminants.

Anthropogenic pollutants comprise a wide range of synthetic organic compounds and heavy metals, which are dispersed throughout the environment, usually at low concentrations. Exposure of ruminants, as for all other animals, is unavoidable and while the levels of exposure to most chemicals are usually too low to induce any physiological effects, combinations of pollutants can act additively or synergistically to perturb multiple physiological systems at all ages but particularly in the developing foetus. In sheep, organs affected by pollutant exposure include the ovary, testis, hypothalamus and pituitary gland and bone. Reported effects of exposure include changes in organ weight and gross structure, histology and gene and protein expression but these changes are not reflected in changes in reproductive performance under the conditions tested. These results illustrate the complexity of the effects of endocrine disrupting compounds on the reproductive axis, which make it difficult to extrapolate between, or even within, species. Effects of pollutant exposure on the thyroid gland, immune, cardiovascular and obesogenic systems have not been shown explicitly, in ruminants, but work on other species suggests that these systems can also be perturbed. It is concluded that exposure to a mixture of anthropogenic pollutants has significant effects on a wide variety of physiological systems, including the reproductive system. Although this physiological insult has not yet been shown to lead to a reduction in ruminant gross performance, there are already reports indicating that anthropogenic pollutant exposure can compromise several physiological systems and may pose a significant threat to both reproductive performance and welfare in the longer term. At present, many potential mechanisms of action for individual chemicals have been identified but knowledge of factors affecting the rate of tissue exposure and of the effects of combinations of chemicals on physiological systems is poor. Nevertheless, both are vital for the identification of risks to animal productivity and welfare.

Foetal hypothalamic and pituitary expression of gonadotrophin-releasing hormone and galanin systems is disturbed by exposure to sewage sludge chemicals via maternal ingestion.

Animals and humans are chronically exposed to endocrine disrupting chemicals (EDCs) that are ubiquitous in the environment. There are strong circumstantial links between environmental EDC exposure and both declining human/wildlife reproductive health and the increasing incidence of reproductive system abnormalities. The verification of such links, however, is difficult and requires animal models exposed to 'real life', environmentally relevant concentrations/mixtures of environmental contaminants (ECs), particularly in utero, when sensitivity to EC exposure is high. The present study aimed to determine whether the foetal sheep reproductive neuroendocrine axis, particularly gondotrophin-releasing hormone (GnRH) and galaninergic systems, were affected by maternal exposure to a complex mixture of chemicals, applied to pasture, in the form of sewage sludge. Sewage sludge contains high concentrations of a spectrum of EDCs and other pollutants, relative to environmental concentrations, but is frequently recycled to land as a fertiliser. We found that foetuses exposed to the EDC mixture in utero through their mothers had lower GnRH mRNA expression in the hypothalamus and lower GnRH receptor (GnRHR) and galanin receptor (GALR) mRNA expression in the hypothalamus and pituitary gland. Strikingly, this, treatment had no significant effect on maternal GnRH or GnRHR mRNA expression, although GALR mRNA expression within the maternal hypothalamus and pituitary gland was reduced. The present study clearly demonstrates that the developing foetal neuroendocrine axis is sensitive to real-world mixtures of environmental chemicals. Given the important role of GnRH and GnRHR in the regulation of reproductive function, its known role programming role in utero, and the role of galanin in the regulation of many physiological/neuroendocrine systems, in utero changes in the activity of these systems are likely to have long-term consequences in adulthood and represent a novel pathway through which EC mixtures could perturb normal reproductive function.

Primordial follicular assembly in humans--revisited.

Recent interest in the initial phases of ovarian follicular formation and development has lead to a number of publications in this area, most of which address the autocrine and paracrine factors involved in primordial follicle activation to primary follicle. Primordial follicle assembly (first step in follicle formation) determines the lifetime supply of primordial follicles and remains a poorly understood phenomenon. Despite a number of recent articles that are concentrating on immuno-histochemistry, basic steps in the process are not clear. Hence, we feel it is time to take a step back and see what is available in the literature and identify the gaps in which future research about primordial follicle assembly in humans needs to be directed.

Developmental changes in human fetal testicular cell numbers and messenger ribonucleic acid levels during the second trimester.

CONTEXT: Normal fetal testis development is essential for masculinization and subsequent adult fertility. The second trimester is a critical period of human testicular development and masculinization, but there is a paucity of reliable developmental data. OBJECTIVE: The objective of the study was to analyze second-trimester human testicular morphology and function. DESIGN: This was an observational study of second-trimester testis development. SETTING: The study was conducted at the Universities of Glasgow and Aberdeen. PATIENTS/PARTICIPANTS: Testes were collected from 57 morphologically normal fetuses of women undergoing elective termination of normally progressing pregnancies (11-19 wk gestation). MAIN OUTCOME MEASURE(S): Testicular morphology, cell numbers, and quantitative expression of 22 key testicular genes were determined. RESULTS: Sertoli cell and germ cell number increased exponentially throughout the second trimester. Leydig cell number initially increased exponentially but slowed toward 19 wk. Transcripts encoding Sertoli (KITL, FGF9, SOX9, FSHR, WT1) and germ (CKIT, TFAP2C) cell-specific products increased per testis through the second trimester, but expression per cell was static apart from TFAP2C, which declined. Leydig cell transcripts (HSD17B3, CYP11A1, PTC1, CYP17, LHR, INSL3) also remained static per cell. Testicular expression of adrenal transcripts MC2R, CYP11B1, and CYP21 was detectable but unchanged. Expression of other transcripts known or postulated to be involved in testicular development (GATA4, GATA6, CXORF6, WNT2B, WNT4, WNT5A) increased significantly per testis during the second trimester. CONCLUSIONS: The second trimester is essential for the establishment of Sertoli and germ cell numbers. Sertoli and Leydig cells are active throughout the period, but there is no evidence of changing transcript levels.

Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells.

Information on the regulation of steroid hormone receptors and their distinct functions within the human endometrial epithelium is largely unavailable. We have immortalized human primary endometrial epithelial cells (EECs) isolated from a normal proliferative phase endometrium by stably transfecting the catalytic subunit (hTERT) of the human telomerase complex and cultured these hTERT-EECs now for over 350 population doublings. Active hTERT was detected in hTERT-EECs employing the telomerase repeat amplification assay protocol. hTERT-EECs revealed a polarized, non-invasive epithelial phenotype with apical microvilli and production of a basal lamina when grown on a three-dimensional collagen-fibroblast lattice. Employing atomic force microscopy, living hTERT-EECs were shown to produce extracellular matrix (ECM) components and ECM secretion was modified by estrogen and progesterone (P4). hTERT-EECs expressed inducible and functional endogenous estrogen receptor-alpha (ER-alpha) as demonstrated by estrogen response element reporter assays and induction of P4 receptor (PR). P4 treatment down-regulated PR expression, induced MUC-1 gene activity and resulted in increased ER-beta transcriptional activity. Gene activities of cytokines and their receptors interleukin (IL)-6, leukemia inhibitory factor (LIF), IL-11 and IL-6 receptor (IL6-R), LIF receptor and gp130 relevant to implantation revealed a 17 beta-estradiol (E2)-mediated up-regulation of IL-6 and an E2- and P4-mediated up-regulation of IL6-R in hTERT-EECs. Thus, hTERT-EECs may be regarded as a novel in vitro model to investigate the role of human EECs in steroid hormone-dependent normal physiology and pathologies, including implantation failure, endometriosis and endometrial cancer.

Demonstration of a non-steroidal, non-inhibin factor in the ovine corpus luteum of pregnancy that reduces pituitary responsiveness to GnRH-induced LH secretion in vitro.

The decline in pulsatile LH secretion and pituitary responsiveness to GnRH as pregnancy advances may be due to non-steroidal factors secreted by the ovine corpus luteum of pregnancy. Corpora lutea from ten ewes on days 70-80 of gestation were homogenized, charcoal-treated and, together with charcoal-treated follicular fluid from superovulated women, were subjected to inhibin immunoaffinity chromatography, reducing dimeric inhibin A and B by >90% and abolishing inhibin bioactivity. These preparations were investigated using cultures of rat pituitary cells. GnRH-induced LH and FSH secretion in vitro was reduced by ovine corpus luteum extract and human follicular fluid by 47+/-5% and 42+/-5% of control LH and by 37+/-5% and 50+/-10% of control FSH, respectively (P<0.001). Extracts prepared from corpora lutea and placentae that were collected on days 50, 90 and 120 of pregnancy (five ewes per stage of pregnancy) showed increased GnRH-induced LH-suppressing bioactivity, particularly in the case of the placental extracts, with a threefold increase in activity. When partially purified by pseudochromatofocusing, GnRH-induced LH-suppressing bioactivity in extracts of ovine corpora lutea was identified at pH 5.40 and 5.77. Although these values are similar to published gonadotrophin surge-attenuating factor (GnSAF) bioactivity pI values, a GnSAF-blocking antiserum had no consistent effect on ovine corpus luteum extract GnRH-induced LH-suppressing bioactivity. It was concluded that the ovine corpus luteum of pregnancy contains a non-steroidal, non-inhibin factor, probably not GnSAF, that has the ability to reduce pituitary responsiveness to GnRH in vitro.

Effects of pregnancy on pulsatile secretion of LH and gonadotrophin-releasing hormone-induced LH release in sheep: a longitudinal study.

Pulsatile LH secretion and its control throughout pregnancy have not been fully determined in sheep. Expt 1 determined the patterns of LH secretion in five ewes on days 10, 20, 60 and 120 of pregnancy and on day 10 postpartum, compared with those on day 10 of the oestrous cycle. Mean (+/- SEM) concentrations of LH declined steadily throughout pregnancy (ANOVA, P < 0.01) and were lower (P < 0.01) on day 60 (0.19 +/- 0.3 ng ml(-1)) and on day 120 (0.18 +/- 0.4 ng ml(-1)) of pregnancy than on day 10 of the oestrous cycle (0.55 +/- 0.04 ng ml(-1)). This decrease was due to a significant reduction in the number and the amplitude of LH pulses. Only on day 120 of pregnancy were progesterone concentrations higher (P < 0.01) than on day 10 of the oestrous cycle. Although concentrations of progesterone on day 10 postpartum were barely detectable, mean LH concentration (0.45 +/- 0.09 ng ml(-1)) was not different from that on day 10 of the oestrous cycle. Expt 2 examined the LH responses in a separate group of four ewes to a physiological dose of GnRH (0.2 microg) on days 10, 20, 60 and 120 of pregnancy and on day 10 postpartum, compared with those on day 10 of the oestrous cycle. The area under the LH response curve and the maximum LH concentrations induced by GnRH declined steadily throughout pregnancy (ANOVA, P < 0.01) and were lower (P < 0.01) on days 60 and 120 of pregnancy than on day 10 of the oestrous cycle, but these parameters were not different between day 10 postpartum and day 10 of the oestrous cycle. Expt 3 examined the LH responses in a separate group of four ewes to a potent GnRH agonist, buserelin (0.5 microg), on days 10, 60 and 120 of pregnancy. The area under the LH response curve and the maximum LH concentrations induced by GnRH were lower (P < 0.01) on days 60 and 120 than on day 10 of pregnancy, but were not different between days 60 and 120. This longitudinal study demonstrates that the pulsatile LH release and pituitary responsiveness to GnRH decreases progressively as pregnancy advances, but does not support the hypothesis that high concentrations of progesterone are solely responsible for the inhibition of pulsatile LH secretion and GnRH-induced LH release during pregnancy in sheep.

Effect of maternal undernutrition on fetal testicular steroidogenesis during the CNS androgen-responsive period in male sheep fetuses.

The aim of this study was to determine the effects of maternal undernutrition, applied during physiologically relevant stages of development of the reproductive system, on reproductive development in male sheep fetuses. Groups of ewes (n = 11-19) were fed rations providing either 100% (high; H) or 50% (low; L) of metabolizable energy requirements for live weight maintenance during selected 'windows', bounded by days 0, 30, 50, 65 and 110 after mating. Ewes of control groups (HH (Expts 1 and 2) and HHH (Expt 3)) were fed the H ration from mating until they were killed at day 50 (Expt 1), day 65 (Expt 2) or day 110 (Expt 3) of gestation, whereas ewes of other groups were fed the L ration for the periods days 0-30 of gestation (LH and LHH), days 31-50 or days 31-65 of gestation (HL and HLH), days 65-110 of gestation (HHL), or day 0 to day 50, day 65 or day 110 of gestation (LL and LLL) when the animals were killed. At day 50 of gestation, there was no effect of nutritional treatment on mean fetal mass or fetal testicular mass, but there was increased expression of mRNA for steroidogenic acute regulatory protein (StAR) in the testes of LL animals (P < 0.05) compared with HH controls. Compared with HH animals, the mean plasma testosterone concentrations of LL fetuses tended to be higher, but this result did not reach significance. At day 65 of gestation there were no significant differences between treatments in mean fetal masses, testicular masses, mean plasma testosterone concentrations or StAR mRNA content. At day 110 of gestation, fetal masses in the LLL group were lower (P < 0.01) than those of control fetuses, although no differences in testicular size or fetal plasma testosterone concentrations were recorded. It is concluded that the effects of undernutrition on reproductive development of male sheep fetuses are dependent on the timing of the period of undernutrition.

Endocrine disrupting chemicals: effects on human male reproductive health.

There is now considerable evidence that male reproductive function is declining in human and wildlife populations. This is coincident with the increasing use and prevalence of man-made chemicals in the environment over the last fifty years. Certain chemicals have subsequently been shown to disturb the developing fetal endocrine system of laboratory animals in utero. In these experiments, treatment caused similar male reproductive problems in offspring as those already observed in wildlife and human populations. In addition, both the human DES data and rodent studies have shown that there are specific windows of gestation when the developing fetal gonad is highly sensitive to small endocrine changes. Animal in vivo and human in vitro studies have identified EDC sensitive genes. Consequently, hypotheses are being generated concerning mechanism of action e.g. disturbed testicular apoptosis and altered hepatic biotransformation of steroids. While animal studies provide us with valuable insights into the range of effects that can be attributed to in utero EDC exposure, varying maternal doses employed by different research groups make relation of the results to human observations difficult. The EDC concentration representative of fetal exposure levels is uncertain. Confounding factors include: (a) the vast number of chemicals termed EDCs, (b) the ability of chemicals to bioaccumulate in body lipid, (c) the metabolism of body lipid during pregnancy releasing the mothers lifetime EDC legacy into circulation and (d) the poorly understood kinetics of EDC transfer across the placenta. Thus, the level of fetal exposure can only be crudely estimated at present. This highlights the need for large animal models of EDC in utero exposure where the partitioning of EDCs between the mother and fetus and transfer across the placenta can be studied in detail. Despite considerable effort the mechanisms by which these endocrine disrupting chemicals exert their effects are still largely unknown. Further studies of the mechanism of action, and consequences, of EDCs in fetal development must be done in order to elucidate how EDCs exert their effects. This can only be achieved using a combined approach whereby animal models are used in combination with in vitro human studies. In conclusion however, there are now sufficient animal model data to prove that EDCs can adversely affect reproductive development and function in the male. Our further understanding of the mechanisms involved may allow intervention strategies whereby we can at least prevent a further decline in male as well as female reproductive health.