Exposure to a phthalate mixture disrupts ovulatory progesterone receptor signaling in human granulosa cells in vitro

• Published in: Biology of reproduction 2023-10, Vol.109 (4), p.552-565
• Main Authors: Hannon, Patrick R., Akin, James W., Curry, Thomas E.
• Format: Article
• Language: English
• Subjects: ADAMTS-1 protein; Cell culture; Cells, Cultured; Chemokine receptors; Chorionic gonadotropin; Chorionic Gonadotropin - metabolism; Chorionic Gonadotropin - pharmacology; CXCR4 protein; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases - metabolism; Female; fertility; Gonadotropins; Granulosa cells; Granulosa Cells - metabolism; Humans; In vitro fertilization; Kinases; Luteinizing hormone; Metalloproteinase; mixture; mRNA; ovary; Ovulation; Pentraxins; Phthalate esters; phthalates; Pituitary (anterior); Progesterone; Progesterone - pharmacology; progesterone receptor; Progestin; Progestins - pharmacology; Protein kinase A; Proteins; Receptors, Progesterone - metabolism; Reproductive status; rescue; RESEARCH ARTICLE; RNA, Messenger - metabolism; Steroidogenesis; Supplements; Thrombospondin; Toxicity
• ISSN: 0006-3363; 1529-7268; 1529-7268
• DOI: 10.1093/biolre/ioad091

Exposure to phthalates disrupts ovarian function. However, limited studies have investigated the effects of phthalate mixtures on ovulation, especially in women. Human granulosa cells were used to test the hypothesis that exposure to a phthalate mixture (PHTmix) disrupts progesterone (P4)/progesterone receptor (PGR) signaling, which is a crucial pathway for ovulation. In addition, progestin and cyclic adenosine 3′, 5′-monophosphate (cAMP) supplementation were tested as methods to circumvent phthalate toxicity. Granulosa cells from women undergoing in vitro fertilization were acclimated in culture to regain responsiveness to human chorionic gonadotropin (hCG; clinical luteinizing hormone analogue). Granulosa cells were treated with or without hCG, and with or without PHTmix (1–500 µg/ml; dimethylsulfoxide = vehicle control) for 0.5–36 h. In the supplementation experiments, cells were treated with or without R5020 (stable progestin), and with or without 8-Br-cAMP (stable cAMP analogue). Exposure to hCG + PHTmix decreased P4 levels and mRNA levels of steroidogenic factors when compared to hCG. This was accompanied by decreased mRNA levels of PGR and downstream P4/PGR ovulatory mediators (ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), C-X-C motif chemokine receptor 4 (CXCR4), pentraxin 3 (PTX3), and regulator of G protein signaling 2 (RGS2)) in the hCG + PHTmix groups compared to hCG. Exposure to hCG + PHTmix 500 µg/ml decreased cAMP levels and protein kinase A activity compared to hCG. Supplementation with progestin in the hCG + PHTmix 500 µg/ml group did not rescue toxicity, while supplementation with cAMP restored PGR levels and downstream P4/PGR mediator levels to hCG levels. These findings suggest that phthalate mixture exposure inhibits P4/PGR signaling in human granulosa cells via decreased steroidogenesis, cAMP levels, and protein kinase A activity. Restored P4/PGR signaling with cAMP supplementation provides a potential cellular target for intervention of phthalate-induced ovulatory dysfunction in women. Summary Sentence Exposure to a phthalate mixture decreased progesterone production and inhibited PGR signaling in human granulosa cells, and PGR signaling was restored with cAMP supplementation. Graphical Abstract