Neonicotinoids differentially modulate nicotinic acetylcholine receptors in immature and antral follicles in the mouse ovary†.

Neonicotinoids are the most widely used insecticides in the world. They are synthetic nicotine derivatives that act as nicotinic acetylcholine receptor agonists. Although parent neonicotinoids have low affinity for the mammalian nicotinic acetylcholine receptor, they can be activated in the environment and the body to positively charged metabolites with high affinity for the mammalian nicotinic acetylcholine receptor. Imidacloprid, the most popular neonicotinoid, and its bioactive metabolite desnitro-imidacloprid differentially interfere with ovarian antral follicle physiology in vitro, but their effects on ovarian nicotinic acetylcholine receptor subunit expression are unknown. Furthermore, ovarian nicotinic acetylcholine receptor subtypes have yet to be characterized in the ovary. Thus, this work tested the hypothesis that ovarian follicles express nicotinic acetylcholine receptors and their expression is differentially modulated by imidacloprid and desnitro-imidacloprid in vitro. We used polymerase chain reaction, RNA in situ hybridization, and immunohistochemistry to identify and localize nicotinic acetylcholine receptor subunits (α2, 4, 5, 6, 7 and ß1, 2, 4) expressed in neonatal ovaries (NO) and antral follicles. Chrnb1 was expressed equally in NO and antral follicles. Chrna2 and Chrnb2 expression was higher in antral follicles compared to NO and Chrna4, Chrna5, Chrna6, Chrna7, and Chrnb4 expression was higher in NO compared to antral follicles. The α subunits were detected throughout the ovary, especially in oocytes and granulosa cells. Imidacloprid and desnitro-imidacloprid dysregulated the expression of multiple nicotinic acetylcholine receptor subunits in NO, but only dysregulated one subunit in antral follicles. These data indicate that mammalian ovaries contain nicotinic acetylcholine receptors, and their susceptibility to imidacloprid and desnitro-imidacloprid exposure varies with the stage of follicle maturity.

The role of the aryl hydrocarbon receptor in mediating the effects of mono(2-ethylhexyl) phthalate in mouse ovarian antral follicles†.

Di(2-ethylhexyl) phthalate (DEHP) is a pervasive environmental toxicant used in the manufacturing of numerous consumer products, medical supplies, and building materials. DEHP is metabolized to mono(2-ethylhexyl) phthalate (MEHP). MEHP is an endocrine disruptor that adversely affects folliculogenesis and steroidogenesis in the ovary, but its mechanism of action is not fully understood. Thus, we tested the hypothesis that the aryl hydrocarbon receptor (AHR) plays a functional role in MEHP-mediated disruption of folliculogenesis and steroidogenesis. CD-1 mouse antral follicles were isolated and cultured with MEHP (0-400 µM) in the presence or absence of the AHR antagonist CH223191 (1 µM). MEHP treatment reduced follicle growth over a 96-h period, and this effect was partially rescued by co-culture with CH223191. MEHP exposure alone increased expression of known AHR targets, cytochrome P450 (CYP) enzymes Cyp1a1 and Cyp1b1, and this induction was blocked by CH223191. MEHP reduced media concentrations of estrone and estradiol compared to control. This effect was mitigated by co-culture with CH223191. Moreover, MEHP reduced the expression of the estrogen-sensitive genes progesterone receptor (Pgr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr) and co-treatment with CH223191 blocked this effect. Collectively, these data indicate that MEHP activates the AHR to impair follicle growth and reduce estrogen production and signaling in ovarian antral follicles.

Ovarian antral follicles metabolize imidacloprid in vitro.

Neonicotinoid insecticides are synthetic nicotine derivatives that have high affinity for invertebrate nicotine receptors and low affinity for mammalian nicotine receptors. However, imidacloprid (IMI), the most commonly used neonicotinoid, can be bioactivated by the liver in mammals to desnitro-imidacloprid, an intermediate metabolite that effectively binds and activates mammalian receptors. However, it is not known if other tissues such as the ovaries can metabolize IMI. Thus, the present study tested the hypothesis that ovarian antral follicles metabolize and bioactivate IMI. Antral follicles were dissected from the ovaries of CD-1 mice and cultured in media containing dimethyl sulfoxide or IMI (0.2-200 µg/ml) for 48 and 96 h. Media were subjected to liquid chromatography-mass spectrometry for detection of phase I IMI metabolites. Follicles from the cultures were used for gene expression analysis of metabolic enzymes associated with IMI metabolism. All IMI metabolites were detected at 48 and 96 h. Oxidized IMI intermediates were detected in media from cultured follicles, but not environmental controls. Reduced IMI intermediates were detected in media from cultured follicles and the environmental controls. At 48 h, IMI did not affect expression of any metabolic enzymes compared with control. At 96 h, IMI induced Cyp2e1 and Cyp4f18 compared with control. These data indicate that mouse ovarian follicles metabolize IMI and that IMI induces ovarian Cyp expression over time.

Imidacloprid and Its Bioactive Metabolite, Desnitro-Imidacloprid, Differentially Affect Ovarian Antral Follicle Growth, Morphology, and Hormone Synthesis In Vitro.

Imidacloprid is a neonicotinoid pesticide used in large-scale agricultural systems, home gardens, and veterinary pharmaceuticals. Imidacloprid is a small molecule that is more water-soluble than other insecticides, increasing the likelihood of large-scale environmental accumulation and chronic exposure of non-targeted species. Imidacloprid can be converted to the bioactive metabolite desnitro-imidacloprid in the environment and body. Little is known about the mechanisms by which imidacloprid and desnitro-imidacloprid induce ovarian toxicity. Thus, we tested the hypothesis that imidacloprid and desnitro-imidacloprid differentially affect antral follicle growth and steroidogenesis in vitro. Antral follicles were dissected from the ovaries of CD-1 mice and cultured in media containing vehicle control or 0.2 µg/mL-200 µg/mL of imidacloprid or desnitro-imidacloprid for 96 h. Follicle morphology was monitored, and follicle size was measured every 24 h. At the end of the culture periods, media were used to quantify follicular hormone levels, and follicles were used for gene expression analysis of steroidogenic regulators, hormone receptors, and apoptotic factors. Imidacloprid did not affect follicle growth or morphology compared to the control. Desnitro-imidacloprid inhibited follicle growth and caused follicles to rupture in culture compared to the control. Imidacloprid increased progesterone, whereas desnitro-imidacloprid decreased testosterone and progesterone compared to the control. Desnitro-imidacloprid also changed estradiol compared to the control. At 48 h, IMI decreased the expression of Star, Cyp17a1, Hsd17b1, Cyp19a1, and Esr2 and increased the expression of Cyp11a1, Cyp19a1, Bax, and Bcl2 compared to the control. IMI also changed the expression of Esr1 compared to the control. At 48 h, DNI decreased the expression of Cyp11a1, Cyp17a1, Hsd3b1, Cyp19a1, and Esr1 and increased the expression of Cyp11a1, Hsd3b1, and Bax compared to the control. At 72 h of culture, IMI significantly decreased the expression of Cyp19a1 and increased the expression of Star and Hsd17b1 compared to the control. At 72 h, DNI significantly decreased the expression of Cyp11a1, Cyp17a1, Hsd3b1, and Bax and increased the expression of Esr1 and Esr2. At 96 h, IMI decreased the expression of Hsd3b1, Cyp19a1, Esr1, Bax, and Bcl2 compared to the control. At 96 h, DNI decreased the expression of Cyp17a1, Bax, and Bcl2 and increased the expression of Cyp11a1, Hsd3b1, and Bax compared to the control. Together, these data suggest mouse antral follicles are targets of neonicotinoid toxicity, and the mechanisms of toxicity differ between parent compounds and metabolites.

The effects of inhaled pollutants on reproduction in marginalized communities: a contemporary review.

Important differences in health that are closely linked with social disadvantage exist within and between countries. According to the World Health Organization, life expectancy and good health continue to increase in many parts of the world, but fail to improve in other parts of the world, indicating that differences in life expectancy and health arise due to the circumstances in which people grow, live, work, and age, and the systems put in place to deal with illness. Marginalized communities experience higher rates of certain diseases and more deaths compared to the general population, indicating a profound disparity in health status. Although several factors place marginalized communities at high risk for poor health outcomes, one important factor is exposure to air pollutants. Marginalized communities and minorities are exposed to higher levels of air pollutants than the majority population. Interestingly, a link exists between air pollutant exposure and adverse reproductive outcomes, suggesting that marginalized communities may have increased reproductive disorders due to increased exposure to air pollutants compared to the general population. This review summarizes different studies showing that marginalized communities have higher exposure to air pollutants, the types of air pollutants present in our environment, and the associations between air pollution and adverse reproductive outcomes, focusing on marginalized communities.

Correction: Fletcher et al. Effects of Phthalate Mixtures on Ovarian Folliculogenesis and Steroidogenesis. Toxics 2022, 10, 251.

The authors wish to make the following corrections to this paper [...].

Iodoacetic acid exposure alters the transcriptome in mouse ovarian antral follicles.

Iodoacetic acid (IAA) is an unregulated water disinfection byproduct that is an ovarian toxicant. However, the mechanisms of action underlying IAA toxicity in ovarian follicles remain unclear. Thus, we determined whether IAA alters gene expression in ovarian follicles in mice. Adult female mice were dosed with water or IAA (10 or 500 mg/L) in the water for 35-40 days. Antral follicles were collected for RNA-sequencing analysis and sera were collected to measure estradiol. RNA-sequencing analysis identified 1063 differentially expressed genes (DEGs) in the 10 and 500 mg/L IAA groups (false discovery rate FDR < 0.1), respectively, compared to controls. Gene Ontology Enrichment analysis showed that DEGs were involved with RNA processing and regulation of angiogenesis (10 mg/L) and the cell cycle and cell division (500 mg/L). Pathway Enrichment analysis showed that DEGs were involved in the phosphatidylinositol 3-kinase and protein kinase B (PI3K-Akt), gonadotropin-releasing hormone (GnRH), estrogen, and insulin signaling pathways (10 mg/L). Pathway Enrichment analysis showed that DEGs were involved in the oocyte meiosis, GnRH, and oxytocin signaling pathways (500 mg/L). RNA-sequencing analysis identified 809 DEGs when comparing the 500 and 10 mg/L IAA groups (FDR < 0.1). DEGs were related to ribosome, translation, mRNA processing, oxidative phosphorylation, chromosome, cell cycle, cell division, protein folding, and the oxytocin signaling pathway. Moreover, IAA exposure significantly decreased estradiol levels (500 mg/L) compared to control. This study identified key candidate genes and pathways involved in IAA toxicity and can help to further understand the molecular mechanisms of IAA toxicity in ovarian follicles.

Effects of Phthalate Mixtures on Ovarian Folliculogenesis and Steroidogenesis.

The female reproductive system is dependent upon the health of the ovaries. The ovaries are responsible for regulating reproduction and endocrine function. Throughout a female's reproductive lifespan, the ovaries undergo continual structural changes that are crucial for the maturation of ovarian follicles and the production of sex steroid hormones. Phthalates are known to target the ovaries at critical time points and to disrupt normal reproductive function. The US population is constantly exposed to measurable levels of phthalates. Phthalates can also pass placental barriers and affect the developing offspring. Phthalates are frequently prevalent as mixtures; however, most previous studies have focused on the effects of single phthalates on the ovary and female reproduction. Thus, the effects of exposure to phthalate mixtures on ovarian function and the female reproductive system remain unclear. Following a brief introduction to the ovary and its major roles, this review covers what is currently known about the effects of phthalate mixtures on the ovary, focusing primarily on their effects on folliculogenesis and steroidogenesis. Furthermore, this review focuses on the effects of phthalate mixtures on female reproductive outcomes. Finally, this review emphasizes the need for future research on the effects of environmentally relevant phthalate mixtures on the ovary and female reproduction.

REPRODUCTIVE TOXICOLOGY: Effects of chemical mixtures on the ovary.

The ovaries play a critical role in female reproductive health because they are the site of oocyte maturation and sex steroid hormone production. The unique cellular processes that take place within the ovary make it a susceptible target for chemical mixtures. Herein, we review the available data regarding the effects of chemical mixtures on the ovary, focusing on development, folliculogenesis, and steroidogenesis. The chemical mixtures discussed include those to which women are exposed to environmentally, occupationally, and medically. Following a brief introduction to chemical mixture components, we describe the effects of chemical mixtures on ovarian development, folliculogenesis, and steroidogenesis. Further, we discuss the effects of chemical mixtures on corpora lutea and transgenerational outcomes. Identifying the effects of chemical mixtures on the ovaries is paramount to preventing and treating mixture-inducing toxicity of the ovary that has long-term consequences such as infertility and ovarian disease.

Endocrine Disruptors in Water and Their Effects on the Reproductive System.

Anthropogenic contaminants in water can impose risks to reproductive health. Most of these compounds are known to be endocrine disrupting chemicals (EDCs). EDCs can impact the endocrine system and subsequently impair the development and fertility of non-human animals and humans. The source of chemical contamination in water is diverse, originating from byproducts formed during water disinfection processes, release from industry and livestock activity, or therapeutic drugs released into sewage. This review discusses the occurrence of EDCs in water such as disinfection byproducts, fluorinated compounds, bisphenol A, phthalates, pesticides, and estrogens, and it outlines their adverse reproductive effects in non-human animals and humans.

Mechanisms of action of agrochemicals acting as endocrine disrupting chemicals.

Agrochemicals represent a significant class of endocrine disrupting chemicals that humans and animals around the world are exposed to constantly. Agrochemicals can act as endocrine disrupting chemicals through a variety of mechanisms. Recent studies have shown that several mechanisms of action involve the ability of agrochemicals to mimic the interaction of endogenous hormones with nuclear receptors such as estrogen receptors, androgen receptors, peroxisome proliferator activated receptors, the aryl hydrocarbon receptor, and thyroid hormone receptors. Further, studies indicate that agrochemicals can exert toxicity through non-nuclear receptor-mediated mechanisms of action. Such non-genomic mechanisms of action include interference with peptide, steroid, or amino acid hormone response, synthesis and degradation as well as epigenetic changes (DNA methylation and histone modifications). This review summarizes the major mechanisms of action by which agrochemicals target the endocrine system.