Resveratrol supports and alpha-naphthoflavone disrupts growth of human ovarian follicles in an in vitro tissue culture model.

Infertility is a global health problem with an estimated incidence of 15%. Exposure to chemicals is a potential causal factor, and there is a lack of studies examining the effects on female germ cells. Here, we have studied the impact of different aryl hydrocarbon receptor (AHR) modulators on human ovarian follicles using a human ovarian tissue culture model. Expression of AHR was analyzed in tissue samples, and effects of the selected ligands resveratrol (RSVL), 6-formylindolo(3,2-b)carbazole (FICZ), and alpha-naphthoflavone (aNF) on AHR transactivation studied in a granulosa cell tumor line. Cortical human ovarian tissue containing preantral follicles was exposed to the ligands or vehicle (dimethylsulfoxide, DMSO) for seven days in vitro. Follicle growth was assessed by counting and measuring follicles from serial tissue sections, cell death quantified using in situ Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, and steroid hormone production measured using a newly developed ultra-performance liquid chromatography method. AHR was expressed in all donated ovarian tissue samples. FICZ induced AHR transactivation in the granulosa cell line while aNF antagonised it. Compared to DMSO control, FICZ had no effect on follicles in culture, RSVL increased the proportion of growing follicles, and aNF increased cell death, disrupted growth of secondary follicles, increased testosterone, and reduced estradiol levels. We conclude that RSVL supports and aNF disrupts growth of human ovarian follicles in culture. We further conclude that the human ovarian tissue culture model is suitable for studying effects of chemicals on follicular biology.

In utero Programming of Allergic Susceptibility.

BACKGROUND: Around 30-40% of the world's population will experience allergy, the most common and earliest-onset noncommunicable disease. With a steady rise in the incidence of allergic disease over recent decades, up to 18% of children will suffer a respiratory, food or skin allergy before their 18th birthday. There is compelling evidence that the risk of developing allergy is influenced by early life events and particularly in utero exposures. METHODS: A comprehensive literature review was undertaken which outlines prenatal risk factors and potential mechanisms underlying the development of allergy in childhood. RESULTS: Exposures including maternal cigarette smoking, preterm birth and Caesarean delivery are implicated in predisposing infants to the later development of allergy. In contrast, restricted growth in utero, a healthy maternal diet and a larger family size are protective, but the mechanisms here are unclear and require further investigation. CONCLUSION: To ameliorate the allergy pandemic in young children, we must define prenatal mechanisms that alter the programming of the fetal immune system and also identify specific targets for antenatal interventions.

Environmental chemicals change extracellular lipidome of mature human white adipocytes.

Certain environmental chemicals affect the body's energy balance and are known as metabolism disrupting chemicals (MDCs). MDCs have been implicated in the development of metabolic diseases, such as obesity and type 2 diabetes. In contrast to their well-known impact on developing adipocytes, MDC effects leading to altered energy balance and development of insulin resistance in mature white adipocytes, constituents of adult adipose tissue, are largely unclear. Here, we investigated the effects of six well-established environmental MDCs (bisphenol A (BPA), perfluorooctanoic acid (PFOA), triclosan (TCS), p,p-dichlorodiphenyl-dichloroethylene (ppDDE), tributyltin chloride (TBT) and triphenyl phosphate (TPP)) on mature human white adipocytes derived from mesenchymal stem cells in vitro. We aimed to identify biomarkers and sensitive endpoints of their metabolism disrupting effects. While most of the tested exposures had no effect on adipocyte glucose consumption, lipid storage and assessed gene expression endpoints, the highest concentration of triclosan affected the total lipid storage and adipocyte size, as well as glucose consumption and mRNA expression of the glucose transporter GLUT1, leptin and adiponectin. Additionally, an increased expression of adiponectin was observed with TPP and the positive control PPARγ agonist rosiglitazone. In contrast, the lipidomic analysis of the cell culture medium after a 3-day exposure was extremely sensitive and revealed concentration-dependent changes in the extracellular lipidome of adipocytes exposed to nearly all studied chemicals. While some of the extracellular lipidome changes were specific for the MDC used, some effects were found common to several tested chemicals and included increases in lysophosphatidylcholines, glycerophospholipids and ceramides and a decrease in fatty acids, with possible implications in inflammation, lipid and glucose uptake. This study points to early signs of metabolic disruption and likely systemic effects of mature adipocyte exposure to environmental chemicals, as well as to the need to include lipidomic endpoints in the assessment of adverse effects of MDCs.

Human placental androgen receptor variants: Potential regulators of male fetal growth.

INTRODUCTION: Numerous studies show that males have increased intrauterine growth compared to females, and that pregnancy complications may further these growth differences, but the regulatory mechanisms underlying these differences remain unknown. We propose that these growth outcomes may be due to sex-specific differences in androgen sensitivity - giving rise to altered growth signalling pathways - mediated by the differential expression of placental androgen receptor (AR) variants. METHODS: Placental protein and mRNA were used to identify AR protein variant levels and AR-downstream target gene expression, and were then analysed against neonatal measurements. Dihydrotestosterone (DHT)-induced AR protein variant expression and downstream growth factors were examined in vitro. RESULTS: Four known AR variants (AR-FL, AR-V1, AR-V7, and AR-45), and three unknown proteins (120, 90 and 55 kDa) immunoreactive to the anti-AR antibody were identified in human placentae. Male placentae from controlled asthmatic pregnancies had increased AR-45 and decreased AR-V1 and AR-V7 nuclear expression. Increased nuclear AR-45 expression was associated with increased insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), and IGF-binding protein 5 (IGFBP-5) mRNA expression and normal male growth. AR-45 mRNA and protein did not change in the presence of uncontrolled maternal asthma and associated with an increase in small for gestational (SGA) male fetuses. In vitro DHT stimulation increased AR-45 protein and IGF-1R and IGFBP-5 mRNA expression. CONCLUSIONS: Collectively, our data shows altered AR protein expression and downstream signalling targets may contribute to sex-specific fetal growth outcomes in response to an adverse environment, and that AR-45 appears central in mediating these changes.

Gestational exposure to an epidemiologically defined mixture of phthalates leads to gonadal dysfunction in mouse offspring of both sexes.

The increasing concern for the reproductive toxicity of abundantly used phthalates requires reliable tools for exposure risk assessment to mixtures of chemicals, based on real life human exposure and disorder-associated epidemiological evidence. We herein used a mixture of four phthalate monoesters (33% mono-butyl phthalate, 16% mono-benzyl phthalate, 21% mono-ethyl hexyl phthalate, and 30% mono-isononyl phthalate), detected in 1st trimester urine of 194 pregnant women and identified as bad actors for a shorter anogenital distance (AGD) in their baby boys. Mice were treated with 0, 0.26, 2.6 and 13 mg/kg/d of the mixture, corresponding to 0x, 10x, 100x, 500x levels detected in the pregnant women. Adverse outcomes detected in the reproductive system of the offspring in pre-puberty and adulthood included reduced AGD index and gonadal weight, changes in gonadal histology and altered expression of key regulators of gonadal growth and steroidogenesis. Most aberrations were apparent in both sexes, though more pronounced in males, and exhibited a non-monotonic pattern. The phthalate mixture directly affected expression of steroidogenesis as demonstrated in a relevant in vitro model. The detected adversities at exposures close to the levels detected in pregnant women, raise concern on the existing safety limits for early-life human exposures and emphasizes the need for re-evaluation of the exposure risk.

Expression and localisation of c-kit and KITL in the adult human ovary.

BACKGROUND: The c-kit/kit ligand (KITL) signalling axis is an essential component of ovarian folliculogenesis in mammals, but little is known about expression and localisation of its key components in the ovaries of reproductive age women. This study aimed to characterise mRNA expression of c-kit and KITL isoforms and the localisation of c-kit and KITL proteins in adult human premenopausal ovaries. METHODS: This study utilised granulosa cells obtained from the preovulatory follicles of women undergoing assisted reproduction, pieces of ovarian tissue obtained from premenopausal women undergoing gynaecological surgeries and archival paraffin-embedded premenopausal ovarian tissues. Methodology included PCR for gene expression and Western blot or immunohistochemistry for protein expression. RESULTS: Both c-kit mRNA isoforms, known as GNNK+ and GNNK-, were detected in human ovarian cortex, while KITL protein isoforms (KITL1 and KITL2) were present in ovarian cortex and human granulosa cells. Immunohistochemistry showed expression of KITL and c-kit protein in multiple cell types within follicles throughout development, from primordial follicles to large antral follicles, in addition to atretic follicles. Oocytes of all follicle stages expressed c-kit protein exclusively. Interestingly, unlike animal models, expression of both proteins displayed a less cell-type specific distribution with immunostaining present in granulosa, theca and stromal cells, suggesting that autocrine signalling occurs within the human ovary. CONCLUSION: The results of this study indicate that c-kit/KITL signalling also occurs in the human ovary, as established in various animal models, and may involve previously unknown autocrine signalling.

Mouse GDF9 decreases KITL gene expression in human granulosa cells.

Kit ligand (KITL) is an important granulosa cell-derived growth factor in ovarian folliculogenesis, but its expression and function in human granulosa cells are currently poorly understood. Based on studies performed in animal models, it was hypothesised that KITL gene expression in human granulosa cells is regulated by androgens and/or growth differentiation factor 9 (GDF9). We utilised two models of human granulosa cells, the KGN granulosa tumour cell line and cumulus granulosa cells obtained from preovulatory follicles of women undergoing assisted reproduction. Cells were treated with combinations of 5α-dihydrotestosterone (DHT), recombinant mouse GDF9, and the ALK4/5/7 inhibitor SB431542. KITL mRNA levels were measured by quantitative real-time PCR. No change in KITL mRNA expression was observed after DHT treatment under any experimental conditions, but GDF9 treatment resulted in a significant decrease in KITL mRNA levels in both KGN and cumulus cells. The effect of GDF9 was abolished by the addition of SB431542. These results indicate that KITL is not directly regulated by androgen signalling in human granulosa cells. Moreover, this study provides the first evidence that GDF9 negatively regulates KITL gene expression in human granulosa cells providing new information on the regulation of these important growth factors in the human ovary.