Status of single-cell RNA sequencing for reproductive toxicology in zebrafish and the transcriptomic trade-off.

The utilization of transcriptomic studies identifying profiles of gene expression, especially in toxicogenomics, has catapulted next-generation sequencing to the forefront of reproductive toxicology. An innovative yet underutilized RNA sequencing technique emerging into this field is single-cell RNA sequencing (scRNA-seq), which provides sequencing at the individual cellular level of gonad tissue. ScRNA-seq provides a novel and unique perspective for identifying distinct cellular profiles, including identification of rare cell subtypes. The specificity of scRNA-seq is a powerful tool for reproductive toxicity research, especially for translational animal models including zebrafish. Studies to date not only have focused on 'tissue atlassing' or characterizing what cell types make up different tissues but have also begun to include toxicant exposure as a factor that this review aims to explore. Future scRNA-seq studies will contribute to understanding exposure-induced outcomes; however, the trade-offs with traditional methods need to be considered.

Occupational exposure limits for reproductive toxicants - A comparative analysis.

We investigated the level of protection of reproductive and developmental toxicity offered through occupational exposure limits (OELs) and Derived No-Effect Levels for workers' inhalation exposure (wDNELs). We compared coverage of substances that have a harmonised classification as reproductive toxicant 1 A or 1B (Repr.1 A/B), numerical values and scientific basis of 12 lists of OELs and wDNELs from REACH Registrants' and the Committee for Risk Assessment. Across the 14 sources of OELs and wDNELs, 53 % of the Repr1A/B-substances had at least one exposure limit (counting groups of metals as one entry). Registrants' wDNELs covered the largest share, 40 %. The numerical values could be highly variable for the same substance across the lists. How often reproductive toxicity is identified as the critical effect varies between the examined lists, both due to different assessments of the same substance and different substance coverage. Reviewing the margin of safety to reproductive toxicity cited in the documents, we found that 15 % of safety margins were lower to reproductive toxicity than the critical effect. To conclude, neither the REACH nor work environment legislation supply wDNELs or OELs for a substantial share of known reproductive toxicants. EU OELs cover among the fewest substances in the range, and in many cases national OELs or wDNELs are set at more conservative levels.

An in vitro testicular organoid model for the study of testis morphogenesis, somatic cell maturation, endocrine function, and toxicological assessment of endocrine disruptors.

Male reproductive capacity has fallen considerably in recent decades; in addition, the incidence of testicular cancer has increased in many developed countries. The cause of this phenomenon is unknown, but environmental toxicants are considered a major contributing factor. To study potential reproductive toxicants, robust in vitro testis models are needed. We have recently established a porcine testis organoid system with a high resemblance to the architectures of innate testis tissue. Here, we further investigated the testis morphogenesis, cell maturation, and endocrine function of the testis organoids. We also challenged this system with abiraterone, a steroidogenic inhibitor, to validate its suitability as an in vitro platform for endocrine toxicology tests. Our results showed that the testis cells in the organoids reorganize into testis cordal structures, and the cordal relative areas increase in the organoids over time of culture. Moreover, the diameters and cell numbers per cross-section of the cordal structures increased over time. Interestingly, Sertoli cells in the organoids gradually underwent maturational changes by showing increased expression of androgen receptors, decreased expression of the anti-müllerian hormone, and formation of the blood-testis barrier. Next, we confirmed that the organoids respond to hormonal stimulation and release multiple sex hormones, including testosterone, estradiol, and progesterone. Finally, we showed that the production of testosterone and estradiol in this system can be inhibited in response to the steroidogenic inhibitor. Taken together, our organoid system provides a promising in vitro platform for male reproductive toxicology studies on testis morphogenesis, somatic cell maturation, and endocrine production.

Neonicotinoids differentially modulate nicotinic acetylcholine receptors in immature and antral follicles in the mouse ovary1.

Neonicotinoids are the most widely used insecticides in the world. They are synthetic nicotine derivatives that act as nicotinic acetylcholine receptor (nAChR) agonists. Although parent neonicotinoids have low affinity for the mammalian nAChR, they can be activated in the environment and the body to positively charged metabolites with high affinity for the mammalian nAChR. Imidacloprid (IMI), the most popular neonicotinoid, and its bioactive metabolite desnitro-imidacloprid (DNI) differentially interfere with ovarian antral follicle physiology in vitro, but their effects on ovarian nAChR subunit expression are unknown. Furthermore, ovarian nAChR subtypes have yet to be characterized in the ovary. Thus, this work tested the hypothesis that ovarian follicles express nAChRs and their expression is differentially modulated by IMI and DNI in vitro. We used PCR, RNA in situ hybridization, and immunohistochemistry to identify and localize nAChR subunits (α2, 4, 5, 6, 7 and ß1, 2, 4) expressed in neonatal ovaries and antral follicles. Chrnb1 was expressed equally in neonatal ovaries and antral follicles. Chrna2 and Chrnb2 expression was higher in antral follicles compared to neonatal ovaries and Chrna4, Chrna5, Chrna6, Chrna7 and Chrnb4 expression was higher in neonatal ovaries compared to antral follicles. The α subunits were detected throughout the ovary, especially in oocytes and granulosa cells. IMI and DNI dysregulated expression of multiple nAChR subunits in neonatal ovaries, but only dysregulated one subunit in antral follicles. These data indicate that mammalian ovaries contain nAChRs, and their susceptibility to IMI and DNI exposure varies with the stage of follicle maturity.

Maternal PFOS exposure affects offspring development in Nrf2-dependent and independent ways in zebrafish (Danio rerio).

Perfluorooctanesulfonic acid (PFOS) is a ubiquitous legacy environmental contaminant detected broadly in human samples and water supplies. PFOS can cross the placenta and has been detected in cord blood and breastmilk samples, underscoring the importance of understanding the impacts of maternal PFOS exposure during early development. This study aimed to investigate the effects of a preconception exposure to PFOS on developmental endpoints in offspring, as well as examine the role of the transcription factor Nuclear factor erythroid-2-related factor (Nrf2a) in mediating these effects. This transcription factor regulates the expression of several genes that protect cells against oxidative stress including during embryonic development. Adult female zebrafish were exposed to 0.02, 0.08 or 0.14 mg/L PFOS for 1 week (duration of one cycle of oocyte maturation) and then paired with unexposed males from Nrf2a mutant or wildtype strains. Embryos were collected for two weeks or until completion of 5 breeding events. PFOS was maternally transferred to offspring independent of genotype throughout all breeding events in a dose-dependent manner, ranging from 2.77 to 23.72 ng/embryo in Nrf2a wildtype and 2.40 to 15.80 ng/embryo in Nrf2a mutants. Although embryo viability at collection was not impacted by maternal PFOS exposure, developmental effects related to nutrient uptake, growth and pancreatic ß-cell morphology were observed and differed based on genotype. Triglyceride levels were increased in Nrf2a wildtype eggs from the highest PFOS group. In Nrf2a wildtype larvae there was a decrease in yolk sac uptake while in Nrf2a mutants there was an increase. Additionally, there was a significant decrease in pancreatic ß-cell (islet) area in wildtype larvae from the 0.14 mg/L PFOS accompanied by an increase in the prevalence of abnormal islet morphologies compared to controls. Abnormal morphology was also observed in the 0.02 and 0.08 mg/L PFOS groups. Interestingly, in Nrf2a mutants there was a significant increase in the pancreatic ß-cell area in the 0.02 and 0.08 mg/L PFOS groups and no changes in the prevalence of abnormal islet morphologies. These results suggest that the regulation of processes like nutrient consumption, growth and pancreatic ß-cell development are at least partially modulated by the presence of a functional Nrf2a transcriptomic response. Overall, preconception exposure to environmental pollutants, such as PFOS, may impact the maturing oocyte and cause subtle changes that can ultimately impact offspring health and development.

Effects of superparamagnetic iron oxide nanoparticles (SPIONS) testicular injection on Leydig cell function and sperm production in a murine model.

In the domain of medical advancement, nanotechnology plays a pivotal role, especially in the synthesis of biocompatible materials for therapeutic use. Superparamagnetic Iron Oxide Nanoparticles (SPIONs), known for their magnetic properties and low toxicity, stand at the forefront of this innovation. This study explored the reproductive toxicological effects of Sodium Citrate-functionalized SPIONs (Cit_SPIONs) in adult male mice, an area of research that holds significant potential yet remains largely unknown. Our findings reveal that Cit_SPIONs induce notable morphological changes in interstitial cells and the seminiferous epithelium when introduced via intratesticular injection. This observation is critical in understanding the interactions of nanomaterials within reproductive biological systems. A striking feature of this study is the rapid localization of Cit_SPIONs in Leydig cells post-injection, a factor that appears to be closely linked with the observed decrease in steroidogenic activity and testosterone levels. This data suggests a possible application in developing nanostructured therapies targeting androgen-related processes. Over 56 days, these nanoparticles exhibited remarkable biological distribution in testis parenchyma, infiltrating various cells within the tubular and intertubular compartments. While the duration of spermatogenesis remained unchanged, there were many Tunel-positive germ cells, a notable reduction in daily sperm production, and reduced progressive sperm motility in the treated group. These insights not only shed light on the intricate mechanisms of Cit_SPIONs interaction with the male reproductive system but also highlight the potential of nanotechnology in developing advanced biomedical applications.

Network toxicology, molecular docking technology, and experimental verification revealed the mechanism of cantharidin-induced testicular injury in mice.

As a protein kinase inhibitor, cantharidin (CTD) exhibits antitumor activities. However, CTD is highly toxic, thereby limiting clinical applications. Moreover, relatively few studies have investigated CTD-induced reproductive toxicity, thus the underlying mechanism remains unclear. In this study, the toxic effects of CTD on mouse testis were confirmed in vivo and the potential mechanism was predicted by network toxicology (NT) and molecular docking technology. Proteins involved in the signaling pathways and core targets were verified. The results showed that different concentrations of CTD induced weight loss increased the testicular coefficient, and caused obvious pathological damage to testicular cells. The NT results showed that the main targets of CTD-induced testicular injury (TI) included AKT1, Caspase 3, Bcl-2, and Bax. The results of pathway enrichment analysis showed that CTD-induced TI was closely related to apoptosis and the PI3K/AKT and HIF-1 signaling pathways. Molecular docking methods confirmed high affinity between CTD and key targets. Western blot analysis showed that CTD inhibited expression of PI3K, AKT, and the anti-apoptotic protein Bcl-2, while promoting expression of the pro-apoptotic proteins Bax and Caspase 3. These results suggest that CTD-induced TI involves multiple targets and pathways, and the underlying mechanism was associated with inhibition of the apoptosis-related PI3K/AKT signaling pathway.

PFAS Modulate Osmotic Signaling Independent of Gravimetric Changes in the Rat Uterus.

Various PFAS have been identified as potential endocrine-disrupting chemicals due to estrogen receptor activation, impacts on puberty timing, or impacts on hormonally sensitive endpoints in fish. This study screened multiple PFAS in the rat uterotrophic assay to determine potential estrogenic effects on the uterus with PFAS exposure. This study also explored PFAS-dependent uterine signaling with an osmotic stress mRNA gene expression array. Briefly, Sprague-Dawley rats (26-39 days old) were ovariectomized, and uterine tissue was allowed to regress for a 3-week period of recovery. Animals were then exposed daily via oral gavage to PFAS for 4 days, and then uterine weight was determined. In contrast to the positive control estrogens, the PFAS tested (4:2, 6:2, and 8:2FTOH; perfluorooctanesulfonamide (PFOSA), perfluorononanoic acid (PFNA), perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS), nafion byproduct 2 (NBP2), 1H,1H,8H,8H-perfluorooctane-1,8-diol (FC8-diol) and 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol)) caused no significant changes in the uterine weight. Hormonally active compounds can act as carcinogens, and because earlier rodent work has demonstrated that chronic PFOA exposure is associated with increased risk of uterine cancer, uterine mRNA gene expression was explored with an osmotic stress RT-qPCR array. PFAS exposure significantly upregulated multiple genes across the array, with PFOSA being the compound most similar to the reference estrogens (estradiol benzoate and ethinyl estradiol) in its expression pattern. Also, across all PFAS, pathway analysis revealed that the paxillin pathway, a pathway important in tumor suppressor gene SHP-2 signaling, was significantly upregulated with PFAS exposure. These results demonstrate that in vitro estrogen screens or impacts in fish may show different responses from direct impacts on mammalian uterine weight as assessed with the uterotrophic assay. This study also builds out new mechanisms that may contribute to understanding of carcinogenic changes seen in the uterus after PFAS exposure.

Effect of dexpanthenol on cyclophosphamide-induced ovarian toxicity: a histological and molecular study in rats.

RESEARCH QUESTION: Does dexpanthenol work as an effective therapeutic agent against cyclophosphamide (CYC)-induced premature ovarian failure (POF) in rats? DESIGN: A total of 28 female Wistar Albino rats were randomly divided into four groups (n = 7 per group). The POF and POF plus dexpanthenol groups were intraperitoneally administered CYC at an initial dose of 50 mg/kg, followed by 8 mg/kg for 14 days. The dexpanthenol and POF plus dexpanthenol groups were both intraperitoneally administered dexpanthenol at a dose of 500 mg/kg/day for 15 days. RESULTS: In the group administered CYC, the following was observed: a decrease in the ovarian index; a decrease in the numbers of primordial, primary, secondary and antral follicles; an increase in the number of corpus luteum and atretic follicles; a decrease in proliferation cell nuclear antigen immunoreactivity; a significant reduction in anti-Müllerian hormone and oestradiol levels; and an increase in serum FSH levels compared with controls. Dexpanthenol, on the other hand, reversed these effects. Quantitative reverse transcription polymerase chain reaction analyses showed that dexpanthenol increased Bcl-2, Akt1, mTOR, Nrf2 and HO-1 in CYC-induced ovarian tissues, but decreased Bax, Cas3, Hsp27, Hsp70, and Hsp90. Dexpanthenol treatment has a potential for inhibiting the intrinsic apoptotic pathway and oxidative stress levels in ovarian tissues via the downregulation of the mRNA expression of heat shock proteins and the activation of Nrf2/HO-1 pathways. CONCLUSIONS: Our findings demonstrated that dexpanthenol is an effective agent against POF caused by CYC; however, further experimental and clinical data are needed to use it effectively.

The flame retardant triphenyl phosphate alters the epigenome of embryonic cells in an aquatic in vitro model.

Triphenyl phosphate (TPhP) is an organophosphate flame retardant and plasticizer that is added to a wide variety of consumer and industrial products. It is also a ubiquitous environmental pollutant. Exposure to TPhP has been shown to alter gene expression in metabolic and estrogenic signaling pathways in in vitro and in vivo models of a variety of species, and as such, is considered to be an endocrine disrupting chemical. Exposure to endocrine disrupting chemicals is increasingly being associated with changes to the epigenome, especially during embryonic development. The aim of this study was to evaluate whether TPhP exposure in aquatic ecosystems has the ability to alter the epigenome in two immortal cell lines derived from trout (Oncorhynchus mykiss). This study assessed whether 24 h exposure to TPhP resulted in changes to histone modification and DNA methylation profiles in steelhead trout embryonic cells and rainbow trout gill epithelial cells. Results show that several epigenetic modifications on histone H3 and DNA methylation are altered in the embryonic cells following TPhP exposure, but not in the gill epithelial cells. Specifically, histone H3 acetylation, histone H3 mono-methylation and global DNA methylation were found to be reduced. The alterations of these epigenetic modification profiles in the embryonic cells suggest that exposure to TPhP during fetal development may alter gene expression in the developing embryo, likely in metabolic and estrogenic pathways. The impacts to the epigenome determined in this study may even carry multigenerational detrimental effects on human and ecosystem health, which requires further investigation.

HESI workshop summary: Interpretation of developmental and reproductive toxicity endpoints and the impact on data interpretation of adverse events.

The Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (HESI-DART) group held a hybrid in-person and virtual workshop in Washington, DC, in 2022. The workshop was entitled, "Interpretation of DART in Regulatory Contexts and Frameworks." There were 154 participants (37 in person and 117 virtual) across 9 countries. The purpose of the workshop was to capture key consensus approaches used to assess DART risks associated with chemical product exposure when a nonclinical finding is identified. The decision-making process for determining whether a DART endpoint is considered adverse is critical because the outcome may have downstream implications (e.g., increased animal usage, modifications to reproductive classification and pregnancy labeling, impact on enrollment in clinical trials and value chains). The workshop included a series of webinar modules to train and engage in discussions with federal and international regulators, clinicians, academic investigators, nongovernmental organizations, contract research organization scientists, and private sector scientists on the best practices and principles of interpreting DART and new approach methodologies in the context of regulatory requirements and processes. Despite the differences in regulatory frameworks between the chemical and pharmaceutical sectors, the same foundational principles for data interpretation should be applied. The discussions led to the categorization of principles, which offer guidance for the systematic interpretation of data. Step 1 entails identifying any hazard by closely analyzing the data at the study endpoint level, while Step 2 involves assessing risk using weight of evidence. These guiding principles were derived from the collective outcomes of the workshop deliberations.

Placental transfer of tofacitinib in the ex vivo dual-side human placenta perfusion model.

Tofacitinib is a small molecule Janus kinase (JAK) inhibitor, introduced to the European market in 2017, for the treatment of rheumatoid arthritis, psoriatic arthritis and ulcerative colitis. In the treatment of women with autoimmune diseases, pregnancy is a relevant issue, as such diseases typically affect women in their reproductive years. Currently, there is limited data on the use of tofacitinib during pregnancy. To estimate the extent of placental transfer in the absence of clinical data, we conducted ex vivo dual-side perfused human placental cotyledon perfusions. Term placentas were perfused for 180 min with tofacitinib (100 nM, added to the maternal circuit) in a closed-closed configuration. At the end of the perfusions, drug concentrations in the maternal and fetal reservoirs were near equilibrium, at 35.6 ± 5.5 and 24.8 ± 4.7 nM, respectively. Transfer of tofacitinib was similar to that observed for the passive diffusion marker antipyrine (100 µg/mL, added to the maternal reservoir). Final antipyrine maternal and fetal concentrations amounted to 36.9 ± 3.0 and 36.7 ± 1.3 µg/mL, respectively. In conclusion, in the ex vivo perfused placenta tofacitinib traverses the placental barrier rapidly and extensively. This suggests that substantial fetal tofacitinib exposure will take place after maternal drug dosing.

Impact of bisphenol A on cell viability and inflammatory cytokine production in human cervical epithelial cells.

PROBLEM: An intact cervix is a barrier that prevents pathogenic bacteria from invading the uterine and amniotic cavity during pregnancy. Its disruption is associated with ascending infection and adverse pregnancy outcomes. This study analyzed the effects of bisphenol A (BPA), a chemical used in plastics manufacturing, on cell death and inflammation in cervical epithelial cells. METHODS: Ectocervical epithelial (ecto) and endocervical epithelial (endo) cells were treated with 100 ng/mL and 300 ng/mL of BPA for 48 h. The cells were subjected to flow cytometry using annexin V and propidium iodide to determine apoptosis and necrosis, cell cycle analysis, and ELISA to determine the levels of inflammatory cytokines (IL-6, IL-8, and IL-10). RESULTS: Low-dose and high-dose BPA significantly increased the live ecto cell population dose-dependently. BPA did not have any noticeable effect on cell cycle progression in either cell type. BPA treatment also decreased the apoptotic ecto and endo cell population dose-dependently. Lastly, high dose BPA significantly increased IL-6 in ecto and endo cells. However, IL-8 and IL-10 were not affected by BPA treatments. CONCLUSION: Chemical exposure damage to the cervix can lead to adverse pregnancy outcomes. Our study showed that the BPA concentrations reported in pregnant subjects do not induce cervical cell toxicity . The decrease in apoptosis and increase in live cells may be a compensatory mechanism to preserve the integrity of the cervical epithelial layer.

Insights from the maternal lineage of the F2 generation after exposure to an environmentally relevant dose of benzo(a)pyrene in the male rats of F0 generation.

Benzo(a)pyrene (BaP) is a substance with the potential to induce endocrine disruption in the F0 generation and cause adverse multigenerational effects (F1 generation) for reproductive parameters in rats. The objective of this study was to investigate the occurrence of transgenerational inheritance in the reproductive aspects of male and female rats belonging to the F2 generation (MF2). This investigation was conducted following the exposure of male rats from the F0 generation to BaP to assess potential effects on subsequent generation from the maternal lineage (F1). For that, juvenile male Wistar rats (F0) were orally exposed to BaP (0.1 µg/kg/day) for 31 consecutive days. In adulthood, they were mated with untreated females to obtain female offspring (F1), which later produced the MF2. In the MF2 generation, both males and females exhibited increased body weight on postnatal day (PND) 1. In MF2 males, we observed delayed preputial separation, altered pup weight, reduced levels of follicle-stimulating hormone (FSH), increased intratesticular testosterone levels, decreased type A sperm, epididymal disturbances, reduced 5 α-reductase activity, increased testicular proliferation, and alterations in testicular antioxidant enzymes. In MF2 females, we noted morphological uterine enlargement, reduced sexual activity, and decreased progesterone levels. The findings suggest that the alterations observed in both MF2 males and females can be attributed to modifications in the sperm from F0 generation, which were subsequently transmitted to F1 females and MF2 generation due to BaP exposure.

Reproductive toxicity following in utero and lactational exposure to a human-relevant phthalate mixture in rats.

This rodent (Wistar rats) study examined reproductive effects of in utero/lactational exposure to a mixture of 6 antiandrogenic phthalates (PMix): diisobutyl phthalate, di-n-butyl phthalate, diisopentyl phthalate, butylbenzyl phthalate, di-2-ethylhexyl phthalate, and diisononyl phthalate. The PMix was defined based on exposure data from pregnant women in Brazil. Experimental groups were established by extrapolating the estimated human dose to rats (0.1 mg/kg/day), followed by up to 3 additional doses corresponding to 5, 1000, and 5000 times the starting rat dose: 0 (control), 0.1, 0.5, 100, and 500 mg/kg/day. The fetal experiment assessed gestational exposure effects on fetal gonads, whereas the postnatal experiment evaluated reproductive parameters in males and females after in utero and lactational exposure. Prenatal exposure decreased fetal testicular testosterone production at 0.5 and 500 mg/kg/day. PMix 500 also reduced mRNA expression of steroidogenesis-related genes, upregulated transcript expression of the retinoic acid-degrading enzyme Cyp26b1, and increased multinucleated gonocytes incidence in fetal testes. Postnatal assessment revealed antiandrogenic effects at the highest dose, including reduced anogenital distance, nipple retention, and decreased weight of reproductive organs. Early puberty onset (preputial separation) was observed at the lowest dose in males. In contrast, females did not show significant changes in fetal and adult endpoints. Overall, the PMix recapitulated early and late male rat phthalate syndrome phenotypes at the highest dose, but also induced some subtle changes at lower doses, which warrant confirmation and mechanistic assessments. Our data support the use of epidemiologically defined mixtures for exposure risk assessments over traditional toxicological approaches.

An optimized radioimmunoassay for quantification of total serum thyroxine (T4) in fetal, neonatal, and pregnant rats.

Identifying xenobiotics that interrupt the thyroid axis has significant public health implications, given that thyroid hormones are required for brain development. As such, some developmental and reproductive toxicology (DART) studies now require or recommend serum total thyroxine (T4) measurements in pregnant, lactating, and developing rats. However, serum T4 concentrations are normally low in the fetus and pup which makes quantification difficult. These challenges can be circumvented by technologies like mass spectrometry, but these approaches are expensive and not always widely available. To demonstrate the feasibility of measuring T4 using a commercially available assay, we examine technical replicates of rat serum samples measured both by liquid chromatography mass spectrometry (LC/MS/MS) and radioimmunoassay (RIA). These samples were obtained from rats on gestational day 20 (dams and fetuses) or postnatal day 5 (pups), following maternal exposure to the goitrogen propylthiouracil (0-3 ppm) to incrementally decrease T4. We show that with assay modification, it is possible to measure serum T4 using low sample volumes (25-50 µL) by an RIA, including in the GD20 fetus exposed to propylthiouracil. This proof-of-concept study demonstrates the technical feasibility of measuring serum T4 in DART studies.

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.

Adverse effects of microplastics and nanoplastics on the reproductive system: A comprehensive review of fertility and potential harmful interactions.

In recent years, microplastics (MPs) and nanoplastics (NPs) have caused ubiquitous environmental pollution and raised widespread concern about their potential toxicity to human health, especially in the reproductive system. Moreover, infertility affects >15 % of couples worldwide, and the birth rate is decreasing. Environmental factors are some of the most important causes of infertility. However, little is known about the effects of MPs and NPs on the testes and ovaries. These particles can enter the body primarily via ingestion, inhalation, and skin contact, target the reproductive system in a size-dependent manner and disturb germ cell and other somatic cell development. Our study systematically reviewed the adverse effects of plastic particles on reproductive function and offers valuable insights into the different stages of germ cells and the potential mechanisms. Moreover, the synergistic reproductive toxicity of these particles and carried contaminants was summarized. Given the limited research scale, a shift toward innovative technologies and the adoption of multiple omics are recommended for advancing related studies. Further study is needed to explore the reproductive toxicity of MPs and NPs based on their size, polymer type, shape, and carried toxins, establish effective protective measures, and develop precision medicine for targeted reproductive damage.

GRPR down-regulation inhibits spermatogenesis through Ca2+ mediated by PLCß/IP3R signaling pathway in long-term formaldehyde-exposed rats.

Formaldehyde (FA), which is known as an air pollutant, has been proven to induce male infertility. However, the underlying mechanism of FA-induced male infertility remains elusive. In this study, 24 male SD rats were exposed to different levels of FA (0, 0.5, 2.46, and 5 mg/m3) for eight consecutive weeks. Through HE staining and sperm smear, we observed that FA exposure resulted in spermatogenic injury and the sperm quality decreased in rats. The qRT-PCR and Western blot analysis further revealed that GRPR was down-regulated in testicular tissues of FA-exposed rats as well as primary spermatogenic cells. Meanwhile, ZDOCK uncovered an interaction between GRPR and PLCß. In addition, the CCK8, Fluo 3-AM and Flow cytometry results showed that FA exposure suppressed the expression of GRPR, PLCß and IP3R, consequently reducing the Ca2+ concentration in spermatogenic cells, inducing apoptosis and inhibiting proliferation of spermatogenic cells. Moreover, rescue experiments confirmed that promoting GRPR could improve intracellular Ca2+ concentration by upregulating PLCß and IP3R, partially reducing the apoptosis and promoting the proliferation of FA-treated spermatogenic cells. These findings revealed that GRPR participates in spermatogenesis through Ca2+ mediated by the PLCß/IP3R signaling pathway in FA-exposed rats.

2,4-dichlorophenoxyacetic acid (2,4-D) exposure during postnatal development alters the effects of western diet on mouse prostate.

Western diet (WD), abundant in saturated fats and simple carbohydrates, has been associated with the development of prostate diseases. In addition, 2,4-dichlorophenoxyacetic acid (2,4-D), an herbicide used in agricultural and non-agricultural settings, may interfere with the endocrine system impacting reproductive health. The association of both factors is something common in everyday life, however, there are no relevant studies associating them as possible modulators of prostatic diseases. This study evaluated the action of the herbicide 2,4-D on the postnatal development of the prostate in mice fed with WD. Male C57Bl/6J mice received simultaneously a WD and 2,4-D at doses of 0.02, 2.0, or 20.0 mg/kg b.w./day for 6 months. The prolongated WD intake induced obesity and glucose intolerance, increasing body weight and fat. WD induced morphological changes and increased PCNA-positive epithelial cells in prostate. Additionally, the WD increased gene expression of AR, antioxidant targets, inflammation-related cytokines, cell repair and turnover, and targets related to methylation and miRNAs biosynthesis compared to the counterpart (basal diet). 2,4-D (0.02 and 2.0) changed prostate morphology and gene expression evoked by WD. In contrast, the WD group exposed to 20 mg/kg of 2,4-D reduced feed intake and body weight, and increased expression of androgen receptor and genes related to cell repair and DNA methylation compared to the negative control. Our results showed that 2,4-D was able to modulate the effects caused by WD, mainly at lower doses. However, further studies are needed to elucidate the mechanisms of 2,4-D on the obesogenic environment caused by the WD.