Ubiquitous Halogenated Flame Retardant Toxicants Impair Spermatogenesis in a Human Stem Cell Model Open Access

Abstract

Sperm counts have rapidly declined in the Western male over the past four decades. This rapid decline remains largely unexplained. Exposure to environmental toxicants provides one potential explanation for this decline. Flame retardants are highly prevalent and persistent in the environment, but many have not been assessed for their effects on human spermatogenesis. Using a human stem cell-based model of spermatogenesis, we evaluated four major flame retardants, TDCPP, TDBPP, HBCDD, and TBBPA, under acute conditions simulating occupational-level exposures. Here we show that TDCPP, TDBPP, HBCDD, and TBBPA are human male reproductive toxicants in vitro. While not specifically impacting the survival of haploid spermatids, these toxicants affect spermatogonia and primary spermatocytes through mitochondrial membrane potential perturbation and ROS generation, ultimately causing apoptosis. These results are in stark contrast to persistent per- and polyfluoroalkyl substance exposure, where sub-cellular processes such as spermatogenic cell marker expression are perturbed upon exposure to PFOS, PFOA, and PFNA, but cell viability is ultimately unaffected. Additionally, tentative results looking at persistent exposure to halogenated flame retardants in distinctively different genetic backgrounds suggest different mechanisms of action for acute versus persistent conditions and also highlights how genetics may play a vital role in mediating the reproductive toxicity of these compounds. Taken together, these results show that halogenated flame retardants affect human spermatogenesis in vitro and potentially implicate this highly prevalent class of toxicants in the decline of Western males’ sperm counts.

Table of Contents

Distribution Agreement i

Approval Sheet ii

Abstract Cover Page. iii

Abstract iv

Acknowledgments. vi

List of Abbreviations. xii

List of Figures. xv

Chapter 1: Introduction.. 1

1.1 Chemical exposure has been linked to declining male fertility. 1

1.2 Halogenated flame retardants have been linked to human disease phenotypes. 2

1.3 Halogenated flame retardants and male infertility. 5

1.4 Tris(1,3-dichloro-2-propyl)phosphate (TDCPP) 7

1.5 Tris(2,3-dibromopropyl)phosphate (TDBPP) 8

1.6 Hexabromocyclododecane (HBCDD) 9

1.7 Tetrabromobisphenal A (TBBPA) 10

1.8 Halogenated flame retardant exposure and male sex hormone disruption.. 13

1.8.1 Human epidemiological studies. 13

1.8.2 In vitro studies. 15

1.8.3 In vivo animal studies. 16

1.9 Halogenated flame retardant exposure and female sex hormone disruption.. 16

1.9.1 Human epidemiological studies. 16

1.9.2 In vitro studies. 17

1.9.3 Animal studies. 18

1.10 Halogenated flame retardant exposure and spermatogenesis. 18

1.10.1 Human epidemiological studies. 18

1.10.2 In vitro studies. 18

1.10.3 Animal studies. 19

1.11 Halogenated flame retardant exposure and oogenesis. 20

1.11.1 Human epidemiological studies. 20

1.11.2 In vitro studies. 20

1.11.3 Animal studies. 20

1.12 Halogenated flame retardant exposure and birth outcomes. 21

1.12.1 Human epidemiological studies. 21

1.12.2 Animal studies. 23

1.13 Halogenated flame retardant exposure and birth defects. 23

1.13.1 Human epidemiological studies. 23

1.13.2 In vitro studies. 24

1.13.3 Animal studies. 24

1.14 Halogenated flame retardant exposure and potential intergenerational impacts. 25

1.14.1 Human epidemiological studies. 25

1.14.2 In vitro studies. 26

1.14.3 Animal studies. 26

1.15 Occupationally exposed workers are at highest risk for chemical exposure and reproductive toxicity. 27

1.16 The future of reproductive toxicant research.. 27

Chapter 2: Ubiquitous Halogenated Flame Retardant Toxicants Impair Spermatogenesis in a Human Stem Cell Model 32

2.1 Author’s Contribution and Acknowledgement of Reproduction.. 33

2.2 Abstract 35

2.3 Introduction.. 36

2.4 Methods. 45

2.4.1 Cell culture of H1 ESCs. 45

2.4.2 Differentiation of ESCs. 46

2.4.3 Chemical treatment of SSCs – Acute flame retardant exposure. 47

2.4.4 Chemical treatment of SSCs – Persistent PFAS exposure. 48

2.4.5 Chemical treatment of SSCs – Persistent flame retardant exposure. 48

2.4.6 Cell viability and apoptosis. 48

2.4.7 Mitochondrial membrane potential 49

2.4.8 Reactive oxygen species (ROS) generation.. 50

2.4.9 l-Sulforaphane Rescue. 51

2.4.10 Haploid cell production and cell cycle progression.. 52

2.4.11 Spermatogonial cell lineage markers. 53

2.4.12 Quantification and Statistical Analysis. 54

2.5 Results. 56

2.5.1 Acute TDCPP, TDBPP, HBCDD, and TBBPA exposure induce apoptosis in in vitro spermatogenic cells derived from H1 ESCs, but persistent PFAS exposure does not decrease cell viability. 56

2.5.2 Acute TDCPP, TDBPP, HBCDD, and TBBPA and persistent PFOS, PFOA, and PFNA exposure decreases the viability of spermatogonia derived from H1 ESCs. 110

2.5.3 Acute TDCPP, TDBPP, HBCDD, and TBBPA and persistent PFOS, PFOA, and PFNA exposure decreases the viability of primary spermatocytes derived from H1 ESCs. 170

. 202

2.5.4 Acute TDCCP, TDBPP, HBCDD, and TBBPA exposure impairs cell cycle progression in in vitro cultures but does not affect haploid sperm viability, and persistent PFAS exposure does not impact cell cycle or haploid cells. 227

2.5.5 Acute TDCPP, TDBPP, TBBPA, and HBCDD exposure decreases GSH/GSSG ratios while TBBPA exposure increases reactive oxygen species levels in in vitro spermatogenesis, but persistent PFAS exposure decreases ROS production.. 264

2.6 Discussion.. 351

Chapter 3: General Discussion and Future Directions. 359

3.1 Introduction.. 359

3.2 Methods. 362

3.3 Results. 362

3.4 Discussion.. 364

Chapter 4: References 415

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Cell Health Sciences, Toxicology
Keyword	male infertility halogenated flame retardant reproductive toxicity tetrabromobisphenol A (TBBPA) tris(2,3-dibromopropyl)phosphate (TDBPP) tris(1,3-dichloro-2-propyl)phosphate (TDCPP) in vitro spermatogenesis perfluorooctanesulfonic acid (PFOS) perfluorononanoic acid (PFNA) perfluorooctanoic acid (PFOA) hexabromocyclododecane (HBCDD) spermatogenesis per- and polyfluoroalkyl substance human embryonic stem cell
Committee Chair / Thesis Advisor	Dr. Charles A. Easley IV, University of Georgia Dr. Anthony W.S. Chan, Emory University
Committee Members	Dr. David J. Katz, Emory University Dr. Michele Marcus, Emory University Dr. Alicia K. Smith, Emory University Dr. W. Michael Caudle , Emory University

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