Evaluation of ezrin and fascin 1 in the PFOS treated Sertoli cell culture: An in vitro study

Year 2020, Volume: 4 Issue: 1, 16 - 20, 02.03.2020

Nazlı Ece Gungor-ordueri

https://doi.org/10.30565/medalanya.573983

Abstract

Aim: Depending on the findings resulting from the knock-downing of ezrin and of fascin 1 in vivo, we aim to show the defects or disruption of the blood-testis barrier (BTB) structure and F-actin bundling after Perfluorooctanesulfonate (PFOS) treatment in primary Sertoli cell culture. 

Methods: Study Design: Primary Sertoli cell isolation was occurred with control and PFOS-treated (20M) groups. Sertoli cells were prepared for both experiments as 0.5 x 106 cell/ml. Method: Dual-labeled immunofluorescence analysis to assess co-localization of fascin 1 with ezrin both in Sertoli cells was performed, and Co-IP, by using lysates of seminiferous tubules, was performed using actin and ezrin proteins to identify specific protein-protein interaction with fascin 1. 

Results: Firstly, we showed that ezrin and fascin 1, which were components of the ectoplasmic specialization were co-localized in the Sertoli cells and also they were interacted each other. Secondly, we indicated that they were dislocated in the PFOS-treated Sertoli cells in vitro. Because of PFOS (20M), the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory proteins at the cell-cell interface necessary to maintain localization of actin-regulatory at the BTB.

Conclusion: In summary, these findings suggest that ezrin and fascin 1 can work together to preserve BTB integrity by regulating F-actin organization in the PFOS-mediated Sertoli cell disruption. 

Keywords

Blood-testis barrier, ectoplasmic specialization, F-actin, fascin-1, PFOS

Supporting Institution

The Rockefeller University, The Population Council

Thanks

Dr. C. Yan Cheng

PFOS ile tedavi edilen Sertoli hücre kültüründe ezrin ve fascin 1'in araştırılması: İn vitro bir çalışma

Abstract

Amaç: Bu çalışmada, Ezrin ve Fascin 1’in in vivo olarak baskılanması sonucundaki bulgularla birlikte, Kan testis bariyeri (KTB) yapısının bozulması ve primer Sertoli hücre kültüründe, Perflorooktansülfonat (PFOS) muamelesinden sonra F-aktin demetlenmesinin gösterilmesi amaçlanmaktadır.

Yöntem: Çalışma Tasarımı: Primer Sertoli hücre izolasyonu, kontrol ve PFOS ile muamele edilen (20μM) gruplarla yapıldı. Sertoli hücre konsantrasyonu her iki deney için 0.5 x 106 hücre/ml olacak şekilde hazırlandı. Metod: Hem Fascin 1'in Sertoli hücrelerinde Ezrin ile birlikte lokalizasyonunu değerlendirmek üzere çift etiketli immünofloresan analizi yapıldı, hem de seminifer tübül lizatlarından aktin ve Ezrin proteinlerinin Fascin 1 proteini ile spesifik protein-protein etkileşimini tanımlamak için Co-IP deneyi uygulandı.

Bulgular: İlk olarak, ektoplazma özelleşmesi bileşenleri olan Ezrin ve Fascin 1 in Sertoli hücrelerinde ko-lokalize olduğu ve birbirleri ile etkileşime girdiği gösterilmiştir. İkinci olarak ise PFOS ile muamele edilen Sertoli hücrelerinde Ezrin ve Fascin 1 dislokasyonu gösterilmiştir. 20μM PFOS uygulandığında aktin tabanlı hücre iskeleti KTB’deki hüce-hücre bağlantı bölgelerindeki aktin düzenleyici proteinlerin yayılmasını ve/veya lokalizasyonunu destekleyememiştir.

Tartışma: Sonuç olarak bu bulgular, PFOS aracılı Sertoli hücre bozulmasında, Ezrin ve Fascin 1'in KTB bütünlüğünü korumak için F-aktin organizasyonunu düzenleyerek birlikte çalışabileceğini desteklemektedir.

Keywords

Kan-testis bariyeri, ektoplazma özelleşmesi, F-aktin, Ezrin, Fascin-1, PFOS

References

• 1. Wen Q, Tang EI, Gao Y, Jesus TT, Chu DS, Lee WM, et al. Signaling pathways regulating blood-tissue barriers - Lesson from the testis. Biochim Biophys Acta Biomembr. 2018;1860(1):141-53. PMID: 28450047
• 2. Xiao X, Yang WX. Actin-based dynamics during spermatogenesis and its significance. J Zhejiang Univ Sci B. 2007;8(7):498-506. PMID: 17610330
• 3. Song HW, Wilkinson MF. In vitro spermatogenesis: A long journey to get tails. Spermatogenesis. 2012;2(4):238-44. PMID: 23248764
• 4. Fehon RG, McClatchey AI, Bretscher A. Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol. 2010;11(4):276-87. PMID: 20308985
• 5. McClatchey AI. Merlin and ERM proteins: unappreciated roles in cancer development? Nat Rev Cancer. 2003;3(11):877-83. PMID: 14668818
• 6. Gungor-Ordueri NE, Tang EI, Celik-Ozenci C, Cheng CY. Ezrin is an actin binding protein that regulates sertoli cell and spermatid adhesion during spermatogenesis. Endocrinology. 2014;155(10):3981-95. PMID: 25051438
• 7. Edwards RA, Bryan J. Fascins, a family of actin bundling proteins. Cell Motil Cytoskeleton. 1995;32(1):1-9. PMID: 8674129
• 8. Yamashiro-Matsumura S, Matsumura F. Intracellular localization of the 55-kD actin-bundling protein in cultured cells: spatial relationships with actin, alpha-actinin, tropomyosin, and fimbrin. J Cell Biol. 1986;103(2):631-40. PMID: 3525578
• 9. Paul AG, Jones KC, Sweetman AJ. A first global production, emission, and environmental inventory for perfluorooctane sulfonate. Environ Sci Technol. 2009;43(2):386-92. PMID: 19238969
• 10. Betts KS. Perfluoroalkyl acids: what is the evidence telling us? Environ Health Perspect. 2007;115(5):A250-6. PMID: 17520044
• 11. Peden-Adams MM, Stuckey JE, Gaworecki KM, Berger-Ritchie J, Bryant K, Jodice PG, et al. Developmental toxicity in white leghorn chickens following in ovo exposure to perfluorooctane sulfonate (PFOS). Reprod Toxicol. 2009;27(3-4):307-18. PMID: 19071210
• 12. Hoffman K, Webster TF, Weisskopf MG, Weinberg J, Vieira VM. Exposure to polyfluoroalkyl chemicals and attention deficit/hyperactivity disorder in U.S. children 12-15 years of age. Environ Health Perspect. 2010;118(12):1762-7. PMID: 20551004
• 13. Gao Y, Chen H, Xiao X, Lui WY, Lee WM, Mruk DD, et al. Erratum: Perfluorooctanesulfonate (PFOS)-induced Sertoli cell injury through a disruption of F-actin and microtubule organization is mediated by Akt1/2. Sci Rep. 2017;7(1):6950. PMID: 28747671
• 14. Wan HT, Zhao YG, Wei X, Hui KY, Giesy JP, Wong CK. PFOS-induced hepatic steatosis, the mechanistic actions on β-oxidation and lipid transport. Biochim Biophys Acta. 2012;1820(7):1092-101. PMID: 22484034
• 15. Wan HT, Mruk DD, Wong CK, Cheng CY. Perfluorooctanesulfonate (PFOS) perturbs male rat Sertoli cell blood-testis barrier function by affecting F-actin organization via p-FAK-Tyr(407): an in vitro study. Endocrinology. 2014;155(1):249-62. PMID: 24169556
• 16. Mruk DD, Cheng CY. Enhanced chemiluminescence (ECL) for routine immunoblotting: An inexpensive alternative to commercially available kits. Spermatogenesis. 2011;1(2):121-2. PMID: 22319660
• 17. Su L, Mruk DD, Cheng CY. Regulation of the blood-testis barrier by coxsackievirus and adenovirus receptor. Am J Physiol Cell Physiol. 2012;303(8):C843-53.PMID: 22875787
• 18. Melzer D, Rice N, Depledge MH, Henley WE, Galloway TS. Association between serum perfluorooctanoic acid (PFOA) and thyroid disease in the U.S. National Health and Nutrition Examination Survey. Environ Health Perspect. 2010;118(5):686-92. PMID: 20089479
• 19. Song M, Kim YJ, Park YK, Ryu JC. Changes in thyroid peroxidase activity in response to various chemicals. J Environ Monit. 2012;14(8):2121-6. PMID: 22699773
• 20. Zhang J, Liang J, Zhu H, Li C, Wu Q. PFOS and PCB 153 have direct adverse effects on neonatal testis modeled using a coculture of primary gonocyte and sertoli cells. Environ Toxicol. 2013;28(6):322-31. PMID: 21544924
• 21. Qiu L, Zhang X, Zhang X, Zhang Y, Gu J, Chen M, et al. Sertoli cell is a potential target for perfluorooctane sulfonate-induced reproductive dysfunction in male mice. Toxicol Sci. 2013;135(1):229-40. PMID: 23761298
• 22. Liu L, Zhang Y, Chang X, Li R, Wu C, Tang L, et al. Fluorochloridone perturbs blood-testis barrier/Sertoli cell barrier function through Arp3-mediated F-actin disruption. Toxicol Lett. 2018;295:277-87. PMID: 29981920
• 23. Cheng CY, Mruk DD. The blood-testis barrier and its implications for male contraception. Pharmacol Rev. 2012;64(1):16-64. PMID: 22039149
• 24. Li N, Mruk DD, Mok KW, Li MW, Wong CK, Lee WM, et al. Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption. FASEB J. 2016;30(4):1436-52. PMID: 26678449
• 25. Li N, Mruk DD, Lee WM, Wong CK, Cheng CY. Is toxicant-induced Sertoli cell injury in vitro a useful model to study molecular mechanisms in spermatogenesis? Semin Cell Dev Biol. 2016;59:141-56. PMID: 26779951