The new biocompatible material for mouse ovarian follicle development in three-dimensional in vitro culture systems.
Kim Eun Jung,Yang Chungmo,Lee Jaewang,Youm Hye Won,Lee Jung Ryeol,Suh Chang Suk,Kim Seok Hyun
Theriogenology
To establish a protocol of optimized three-dimensional (3D) culture of ovarian follicles, various biomaterials have been investigated with regard to their properties and functions on in vitro follicle growth. The present study aims to compare the new biomaterial, extracellular matrix-derived soft hydrogel (ES-hydrogel) and alginate, and evaluate the effects of biomaterials on further in vitro 3D culture growth of ovarian follicle and oocyte maturation. The isolated follicles from mouse ovaries were randomly divided into two-dimensional (2D) culture, alginate and ES-hydrogel, and just seeded on culture wells (2D culture) or encapsulated with alginate or ES-hydrogel (3D culture). Culture media from each group were collected on days 4, 8 and 10 or 11 for 17ß-oestradiol (E2) and progesterone (P4) measurement. On day 10 of in vitro culture, follicular survival and pseudo-antrum formation rate were examined, and oocyte maturation was induced by adding human chorionic gonadotropin and epidermal growth factor. After 17 h, ovulated mature oocytes collected and analyzed for oocyte diameter, normal spindle and chromosome alignment configuration, reactive oxygen species (ROS) level, and mitochondrial membrane potential level. To compare mechanical properties of two biomaterials, storage modulus was measured with the advanced rheometric expansion system. Our results showed that follicles cultured in ES-hydrogel, were significantly superior to those cultured 2D or alginate in the pseudo-antrum formation rate, cumulus-oocyte complexes (COCs) rate, MII oocyte rate, normal spindle rate, and E2 production. The ES-hydrogel and alginate groups were not significantly different in follicle survival rate, oocyte diameter, P4 production, ROS, and mitochondrial membrane potential levels. The storage modulus of ES-hydrogel was lower than that of alginate, suggesting that the improved follicular physiology and oocyte maturation in the ES-hydrogel group was due to better hormone exchange through a less stiff encapsulating material. This study shows that 3D culture system using ES-hydrogel effectively improve the outcome of in vitro ovarian follicle culture, supporting follicle morphology and growth and enhancing oocyte maturation. It means one of the most important factors for 3D culture of ovarian follicle was the selection of appropriate and effective biomaterial that can preserve the structure and morphology of ovarian follicle and facilitate nutrition and hormone exchange.
10.1016/j.theriogenology.2019.12.009
RGD-modified dextran hydrogel promotes follicle growth in three-dimensional ovarian tissue culture in mice.
Theriogenology
In vitro follicle growth is a promising technology to preserve fertility for cancer patients. We previously developed a three-dimensional (3-D) ovarian tissue culture system supported by mouse tumor cell-derived Matrigel. When murine ovarian tissues at 14 days old were cultured in Matrigel drops, antrum formation and oocyte competence were significantly enhanced compared with those cultured without Matrigel. In this study, we tested whether nonanimal-derived dextran hydrogels can support a 3-D ovarian tissue culture. We employed chemically defined dextran hydrogels consisting of dextran polymers crosslinked with polyethylene glycol (PEG)-based cell-degradable crosslinker. To determine the optimal gel elasticity for the 3-D tissue culture, we measured Young's modulus of dextran hydrogels at four concentrations (1.75, 2.25, 2.75, and 3.25 mmol/L), and cultured ovarian tissues in these gels for 7 days. As a result, 2.25 mmol/L dextran hydrogel with Young's modulus of 224 Pa was appropriate to provide physical support as well as to promote follicle expansion in the 3-D system. To mimic the natural extracellular matrix (ECM) environment, we modified the dextran hydrogels with two bioactive factors: ECM-derived Arg-Gly-Asp (RGD) peptides as a cell-adhesive factor, and activin A. The ovarian tissues were cultured in 2.25 mmol/L dextran hydrogels under four different conditions: Activin-/RGD- (A-R-), A + R-, A-R+, and A + R+. On Day 7 of culture, follicle and oocyte sizes were significantly increased in the RGD-modified conditions compared with those without RGD. The RGD-modified hydrogels also promoted mRNA levels of steroidogenic-related genes and estradiol production in the 3-D ovarian tissue culture. In vitro maturation and developmental competence of follicular oocytes were remarkably improved in the presence of RGD. In particular, blastocyst embryos were obtained only from A-R+ or A+R+ conditions after in vitro fertilization. We also determined synergistic effects of the RGD peptides and activin A on follicle growth and oocyte development in the 3-D tissue culture. In conclusion, our results suggest that RGD-modified dextran hydrogels provide an ECM-mimetic bioactive environment to support folliculogenesis in a 3-D ovarian tissue culture system.
10.1016/j.theriogenology.2022.02.009
A Novel Three-Dimensional Follicle Culture System Decreases Oxidative Stress and Promotes the Prolonged Culture of Human Granulosa Cells.
ACS applied materials & interfaces
Tissue engineering advancements have made it possible to modify biomaterials to reconstruct a similar three-dimensional structure of the extracellular matrix (ECM) for follicle development and to supply the required biological signals. We postulated that an artificial polysaccharide hydrogel modified with an ECM mimetic peptide may produce efficient irritation signals by binding to specific integrins providing a suitable environment for follicular development and influencing the behavior of human granulosa cells (hGCs). Laminin, an important component of the extracellular matrix, can modulate hGCs and oocyte growth. Specifically, follicles of mice were randomly divided into two-dimensional (2D) and three-dimensional (3D) culture systems established by a hydrogel modified with RGD or laminin mimetic peptides (IKVAV and YIGSR) and RGD (IYR). Our results showed that 3D cultured systems significantly improved follicle survival, growth, and viability. IYR peptides enhanced the oocyte meiosis competence. Additionally, we explored the effect of 3D culture on hGCs, which improved hGCs viability, increased the proportion of S- and G2/M-phase cells, and inhibited cell apoptosis of hGCs. On days 1 and 2, the secretion of progesterone was reduced in 3D-cultured hGCs. Notably, 3D-cultured hGCs exhibited delayed senescence, decreased oxidative stress, and elevated mitochondrial membrane potential. Moreover, the expression levels of cumulus expansion-related genes (COX2, HAS2, and PTX3) and integrin α6β1 were upregulated in 3D-cultured hGCs. In conclusion, a 3D culture utilizing hydrogels modified with Laminin-mimetic peptides can provide a durable physical environment suitable for follicular development. The laminin-mimetic peptides may regulate the biological activity of hGCs by attaching to the integrin α6β1.
10.1021/acsami.2c18734
Regulated cell death in neutrophils: From apoptosis to NETosis and pyroptosis.
Seminars in immunology
Neutrophils are among the most abundant immune cells, representing about 50%- 70% of all circulating leukocytes in humans. Neutrophils rapidly infiltrate inflamed tissues and play an essential role in host defense against infections. They exert microbicidal activity through a variety of specialized effector mechanisms, including phagocytosis, production of reactive oxygen species, degranulation and release of secretory vesicles containing broad-spectrum antimicrobial factors. In addition to their homeostatic turnover by apoptosis, recent studies have revealed the mechanisms by which neutrophils undergo various forms of regulated cell death. In this review, we will discuss the different modes of regulated cell death that have been described in neutrophils, with a particular emphasis on the current understanding of neutrophil pyroptosis and its role in infections and autoinflammation.
10.1016/j.smim.2023.101849
Co-exposure of bisphenol A and selenium deficiency induces pyroptosis via ROS/NLRP3 pathway in chicken spleen.
Poultry science
Bisphenol A (BPA) is widely applied in plastic products, which will produce immunotoxicity to organisms after spilling in the environment, and become a kind of endocrine disruptor. Selenium (Se) is an essential trace element and plays an important role in maintaining redox homeostasis and immune function. BPA exposure and Se deficiency often occur together in livestock and poultry farming, however, studies on the effects of joint exposure on chicken immunotoxins have not been reported. Therefore, this study established a chicken spleen and MDCC-MSB1 cell model under the combined effects of BPA exposure or/and Se deficiency. Transcriptomic analysis showed that BPA exposure and/or Se deficiency induced differential enrichment of positive regulatory pathways such as NLRP3 inflammatory complex assembly, inflammatory response and cellular oxidative stress response. In the -Se+BPA group, pathological damage was significantly increased, Se content decreased, BPA accumulation, oxidative stress and pyroptosis. Meanwhile, the roles and mechanisms of oxidative stress and pyroptosis in BPA exposure or/and Se deficiency-induced splenic tissue injury were investigated by using IF and qRT-PCR methods. The results showed that joint BPA exposure with Se deficiency resulted in more significant changes in the above outcomes than 1 of them. The oxidative stress inhibitor NAC effectually reduced Se deficiency and BPA-induced oxidative stress and pyroptosis, further suggests that oxidative stress mediated Se deficiency or/and BPA-induced pyroptosis. This study revealed that BPA exposure and Se deficiency induced spleen pyroptosis in chickens via the ROS/NLRP3 pathway. These results provide the theoretical basis for the toxicity of BPA in poultry and enrich the toxicological mechanism of combined exposure of Se deficiency and environmental toxins.
10.1016/j.psj.2024.104150
Bisphenol A induces miR-708-5p through an ER stress-mediated mechanism altering neuronatin and neuropeptide Y expression in hypothalamic neuronal models.
McIlwraith Emma K,Lieu Calvin V,Belsham Denise D
Molecular and cellular endocrinology
Bisphenol A (BPA) is an endocrine disrupting chemical that promotes obesity. It acts on the hypothalamus by increasing expression of the orexigenic neuropeptides, Npy and Agrp. Exactly how BPA dysregulates energy homeostasis is not completely clear. Since microRNAs (miRNA) have emerged as crucial weight regulators, the question of whether BPA could alter hypothalamic miRNA profiles was examined. Treatment of the mHypoA-59 cell line with 100 μM BPA altered a specific subset of miRNAs, and the most upregulated was miR-708-5p. BPA was found to increase the levels of miR-708-5p, and its parent gene Odz4, through the ER stress-related protein Chop. Overexpression of an miR-708-5p mimic resulted in a reduction of neuronatin, a proteolipid whose loss of expression is associated with obesity, and an increase in orexigenic Npy expression, thus potentially increasing feeding through converging regulatory pathways. Therefore, hypothalamic exposure to BPA can increase miR-708-5p that controls neuropeptides directly linked to obesity.
10.1016/j.mce.2021.111480
Modulatory effects of bisphenol A on the hepatic immune response.
Environmental pollution (Barking, Essex : 1987)
The liver is a primary line of defense for protection from external substances next to the intestinal barrier. As a result, the hepatic immune system plays a central role in liver pathophysiology. Bisphenol A (BPA) is one of the most common endocrine disrupting chemicals and is primarily metabolized in the liver. Due to its ability to bind to estrogen receptors, BPA is well known to possess estrogenic activity and disrupt reproductive functions. The phase I and Phase II metabolism reactions of BPA mainly occur in the liver with the help of enzymes including cytochrome P450 (CYP), uridine 5'-diphospho-glucuronosyltransferase-glucuronosyltransferases, sulfotransferases, and glutathione-S-transferases. Although the majority of BPA is excreted after conjugation by these enzymes, untransformed BPA induces the production of reactive oxygen species through disruption of the enzymatic complex CYP, lipid accumulation, mitochondrial dysfunction, endoplasmic reticulum stress and inflammatory injury in the liver. Moreover, it has been proposed to possess a potential immunomodulatory effect. Indeed, several in vivo and in vitro studies have reported that low doses of BPA increase the population of T cells with type 1 T helper (Th1), Th2, and Th17 cells. Although the current literature lacks clear evidence on the mechanisms by which BPA is involved in T cell mediated immune responses, recent multi-omics studies suggest that it may directly interact with the antigen processing and presentation pathways. In this review, we first discuss the metabolism of BPA in the liver, before exploring currently available data on its effects on liver injury. Finally, we review its modulatory effects on the hepatic immune response, as well as potential mechanisms. By conducting this review, we aim to improve understanding on the relationship between BPA exposure and immune-related liver injury, with a focus on the antigen processing and presentation pathway and T cell-mediated response in the liver.
10.1016/j.envpol.2023.122430
Enhanced endoplasmic reticulum stress signaling disrupts porcine sertoli cell function in response to Bisphenol A exposure.
Journal of environmental management
Bisphenol A (BPA), a pervasive substance in our daily lives and livestock excreta, poses significant threats due to its infiltration into foods and water sources. BPA has adverse impacts on male reproductive function, particularly affecting the critical Sertoli (ST) cells that play a pivotal role in the process of spermatogonia differentiating into spermatozoa. In this study, we examined the prevalence of BPA within the pig industry and delved into the impact of BPA exposure on the motility of boar sperm, the function of pig ST cells, as well as the underlying molecular mechanisms involved. This study revealed spatial disparities in the global distribution of BPA and its analogue contamination, utilizing data compiled from 130 comprehensive studies. The average concentration of BPA found in pig feed ranges from 9.7 to 47.9 μg/kg, while in serum, it averages between 55.1 and 75.6 ng/L. The BPA concentration in feed exhibits a negative correlation with sperm viability and the percentage of progressive motile spermatozoa. Exposure to BPA reduced sperm motility in boar and ST cell activity at both 6 and 24 h. The transcriptome analysis revealed that, compared to untreated control cells, endoplasmic reticulum stress (ERS)-related genes were upregulated in ST cells exposed to BPA at 6 and 24 h. This activation of ERS in ST cells was mediated by receptor protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring protein-1α (IRE1α), and activating transcription factor 6 (ATF6). Additionally, BPA exposure triggered oxidative stress and a proinflammatory response mediated by the transcription factor NF-κB, accompanied by an increase in downstream proinflammatory cytokines. BPA exposure also led to apoptosis in ST cells and upregulated the expression levels of pro-apoptosis proteins. However, inhibiting ERS activity with 4-PBA attenuated the BPA-induced inflammatory response and apoptosis in ST cells. Our findings suggest that BPA induced apoptosis and inflammatory response in porcine ST cells through persistent activation of ERS, thereby compromising the normal function of these cells.
10.1016/j.jenvman.2024.122908
Roles of ERK/Akt signals in mitochondria-dependent and endoplasmic reticulum stress-triggered neuronal cell apoptosis induced by 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, a major active metabolite of bisphenol A.
Huang Chun-Fa,Liu Shing-Hwa,Su Chin-Chuan,Fang Kai-Min,Yen Cheng-Chieh,Yang Ching-Yao,Tang Feng-Cheng,Liu Jui-Ming,Wu Chin-Ching,Lee Kuan-I,Chen Ya-Wen
Toxicology
Bisphenol A (BPA) is recognized as a harmful pollutant in the worldwide. Growing studies have reported that BPA can cause adverse effects and diseases in human, and link to a potential risk factor for development of neurodegenerative diseases (NDs). 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which generated in the mammalian liver after BPA exposure, is a major active metabolite of BPA. MBP has been suggested to exert greater toxicity than BPA. However, the molecular mechanism of MBP on the neuronal cytotoxicity remains unclear. In this study, MBP exposure significantly reduced Neuro-2a cell viability and induced apoptotic events that MBP (5-15 μM) exhibited greater neuronal cytotoxicity than BPA (50-100 μM). The mitochondria-dependent apoptotic signals including the decrease in mitochondrial membrane potential (MMP) and the increase in cytosolic apoptosis-induced factor (AIF), cytochrome c release, and Bax protein expression were involved in MBP (10 μM)-induced Neuro-2a cell death. Exposure of Neuro-2a cells to MBP (10 μM) also triggered endoplasmic reticulum (ER) stress through the induction of several key molecules including glucose-regulated protein (GRP)78, C/EBP homologous protein (CHOP), X-box binding protein (XBP)-1, protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme(IRE)-1, activation transcription factor(AFT)4 and ATF6, and caspase-12. Pretreatment with 4-PBA (an ER stress inhibitor) and specific siRNAs for GRP78, CHOP, and XBP-1 significantly suppressed the expression of these ER stress-related proteins and the activation of caspase-12/-3/-7 in MBP-exposed Neuro-2a cells. Furthermore, MBP (10 μM) exposure dramatically increased the activation of extracellular regulated protein (ERK)1/2 and decreased Akt phosphorylation. Pretreatment with PD98059 (an ERK1/2 inhibitor) and transfection with the overexpression of activation of Akt1 (myr-Akt1) effectively suppressed MBP-induced apoptotic and ER stress-related signals. Collectively, these results demonstrate that MBP exposure exerts neuronal cytotoxicity via the interplay of ERK activation and Akt inactivation-regulated mitochondria-dependent and ER stress-triggered apoptotic pathway, which ultimately leads to neuronal cell death.
10.1016/j.tox.2021.152764
Effects of single or combined exposure to bisphenol A and mono(2-ethylhexyl)phthalate on oxidant/antioxidant status, endoplasmic reticulum stress, and apoptosis in HepG2 cell line.
Environmental science and pollution research international
Endocrine disrupting chemicals (EDCs) may affect many biological processes like growth and stress response. Bisphenol A (BPA) is a plasticizer that is used to harden plastics and polycarbonates. Phthalates are used to add flexibility to polyvinyl chloride containing plastics. The main metabolite of di(2-ethylhexyl) phthalate (DEHP) is mono(2-ethylhexyl) phthalate (MEHP) and it is even more toxic than the parent compound. Humans are usually exposed to these chemicals in mixtures by different routes starting from fetal period. However, there are not many studies in literature that investigate the combined effects of these chemicals. The aim of this study is to investigate toxic effects of BPA and/or MEHP on HepG2 cell line. We have evaluated cytotoxicity, cytomorphological, apoptotic changes, oxidative stress, oxidant/antioxidant status alterations, and endoplasmic reticulum (ER) stress. Combined exposure to BPA and MEHP caused alterations in oxidant/antioxidant status and ER stress marker proteins in both cytoplasmic and nuclear cellular fractions. We can suggest that combined exposure to EDCs may cause serious toxicological outcomes and more mechanistic studies are needed to determine the combined toxic effects.
10.1007/s11356-022-22937-6
Bisphenols A, F, S and AF trigger apoptosis and/or endoplasmic reticulum stress in human endometrial stromal cells.
Toxicology
Disruption of non-differentiated endometrial stromal cells could have noxious consequences in female reproduction, impairing endometrial remodelling and implantation. Following the classification of bisphenol A (BPA) as an endocrine disrupting chemical, it started to be gradually withdrawn from the market, being substituted by structural analogues, whose effects in human health are not fully understood. This work used a telomerase-immortalized human endometrial stromal cell line (St-T1b) to study the effects of BPA and its three most commercialized structural analogues (ranked: bisphenols S, F and AF) on endometrial stromal cells to understand their effects on female reproductive function. Bisphenols showed dissimilar effects. All four compounds generated endoplasmic reticulum (ER) stress. In addition, bisphenols A, F and AF induced apoptosis through different mechanisms, with bisphenol AF causing cell cycle arrest at G/M phase. Bisphenol AF decreased mitochondrial transmembrane potential and bisphenols A, F and AF produced oxidative stress.
10.1016/j.tox.2022.153282
Fluorene-9-bisphenol exposure induces cytotoxicity in mouse oocytes and causes ovarian damage.
Jia Zhenzhen,Wang Hongyu,Feng Zeyang,Zhang Shaozhi,Wang Lining,Zhang Jingwen,Liu Qianqian,Zhao Xin,Feng Daofu,Feng Xizeng
Ecotoxicology and environmental safety
Fluorene-9-bisphenol (BHPF), a substitute for bisphenol A, is a chemical component of plastics for industrial production. There is evidence that BHPF exerts an antioestrogenic effect on mice, induces endometrial atrophy and leads to adverse pregnancy outcomes. However, the effects of BHPF on oocyte maturation and ovary development as well as its possible mechanisms remain unclear. The objective of this study was to investigate the toxicity and mechanism of BHPF exposure in mouse oocytes in vitro and in vivo. Our results showed that BHPF could inhibit the maturation of oocytes in vitro by reducing the protein level of p-MAPK and destroying the meiotic spindle. We found that in vitro, BHPF-treated oocytes showed increased ROS levels, DNA damage, mitochondrial dysfunction, and expression of apoptosis- and autophagy-related genes, such as Bax, cleaved-caspase 3, LC 3 and Atg 12. In addition, in vivo experiments showed that BHPF exposure could induce the expression of oxidative stress genes (Cat, Gpx 3 and Sod 2) and apoptosis genes (Bax, Bcl-2 and Cleaved-caspase 3) and increase the number of atresia follicles in the ovaries. Our data showed that BHPF exposure affected the first polar body extrusion of oocytes, increased oxidative stress, destroyed spindle assembly, caused DNA damage, altered mitochondrial membrane potentials, induced apoptosis and autophagy, and affected ovarian development.
10.1016/j.ecoenv.2019.05.019
Bisphenol A promotes autophagy in ovarian granulosa cells by inducing AMPK/mTOR/ULK1 signalling pathway.
Environment international
BACKGROUND:Bisphenol A (BPA) is a widespread endocrine-disrupting chemical with estrogen like effects, which could interfere with the human reproductive system by disrupting the normal function of granulosa cells (GCs) leading to abnormal ovarian function. However, the mechanism of its toxicity on human GCs has not been clearly described thus far. METHODS:106 normogonadotropic infertile women undergoing their first in-vitro fertilization-embryo transfer (IVF-ET) cycle were recruited. Urinary BPA level and the early outcomes of IVF-ET were analysed. Patients were divided to low and high BPA exposure groups using the median urinary BPA concentration as the cut-off value. In-vivo and in-vitro studies were conducted using mice and human granulosa cell line (KGN cells). Female Kunming mice approximately 6-8 weeks of age were poisoned with BPA at different dosages (1, 10 or 100 μg/kg) by oral gavage once daily for 2 weeks, while KGN cells were exposed to BPA at the concentration of 1, 10 or 100 nM for 24 h, 48 h or 72 h. BPA-induced ovarian morphologic changes were analysed by histopathology investigation. Cell viability and apoptosis were evaluated using CCK-8, TUNEL and flowcytometric, respectively. Hormone levels were determined using ELISA and the molecular mechanism studies were conducted using immunofluorescence, RT-PCR and western blots. RESULTS:The oocyte retrieval rate, maturation rate and embryo implantation rate significantly decreased with the higher level of urinary BPA concentration. Peak E2 level was lower in high BPA group, but no statistical significance could be observed. In BPA treated mice, cystic dilation of the follicles with a decreased number of GCs could be observed histopathologically. Decreased E2, P4 and AMH level and GCs autophagy could be detected both in-vivo and in-vitro with the activation of AMPK/mTOR/ULK1 signalling pathway. As being confirmed in KGN cells, phosphorylated AMPK and ULK1 increased while phosphorylated mTOR decreased, and by inhibition autophagy using knockdown of AMPK or 3-MA, adverse effects of BPA exposure in-vitro could be reversed. CONCLUSION:BPA exposure might abnormally influence human ovarian functions leading to abnormal folliculogenesis by activation of autophagy in GCs through AMPK/mTOR/ULK1 pathway.
10.1016/j.envint.2020.106298