Lifestage-Specific Modeling Platform for Adverse Outcome Pathways of

Male Reproductive and Developmental Processes

Rachel Shaffer

University of Washington Seattle

School of Public Health

May 18, 2017

Driving Factors in Alternative Model Development for Male Reproductive System

• Environmental Exposures: Effects of chemicals on male reproductive endpoints are significant public health concern

– Testicular dysgenesis syndrome: origin in early fetal exposure to environmental chemicals (Skakkebaek, et al. 2001)

– Decline in semen quality observed in some populations (Nordcap, et al. 2012)

• Drug Development: Testicular toxicity is often discovered late in process, leading to substantial cost and delay in production – Male reproductive development is one of the most sensitive toxicological endpoints

– No reliable clinical biomarkers of testicular toxicity (Saldutti et al, 2013)

• High Cost of Existing Systems: Current methods are expensive, time-consuming, and animal-intensive (ex: OECD guidelines) – Reproductive and developmental toxicity testing under Europe’s REACH (Registration,

Evaluation, Authorization and Restriction of Chemicals) program are estimated to cost ~$9 billion (~7 billion Euros) and require ~49 million animals (Rovida, et al. 2009; Scialli, et al. 2008)

Challenges in Developmental and Reproductive Toxicity Assessment

• Complexity of development

• Phases of susceptibility

• Toxicity is context-dependent

• Complex toxicokinetics & dynamics

Key Aspects Needed in Enhanced Toxicity Testing Platforms

for Male Reproductive Systems

Long term viability and maintenance of Germ, Sertoli and Leydig cells in an in vivo like niche-evidenced by selective staining over 21 days Testosterone production by Leydig cells

Interaction between Sertoli cells and Germ cells

Early germ cell meiosis

Changes in gene expression profiles in response to toxicant exposure

Saldutti et al, 2013

Faustman Lab Approach

Rat in vitro co-culture

• Co-culture optimized in 2005 (Yu et al. 2005). Characterized normal dynamics as well as response to toxicants (Yu et al. 2008, Yu et al. 2011, Harris et al. 2016).

Rat in vivo transcriptomic data

• Global gene expression dynamics PND 6-10 (Wegner et al. in progress).

Mouse in vivo transcriptomic data

• Global gene expression dynamics throughput testicular development and spermatogenesis GD11-PND56 (Wegner et al. 2015).

Mouse in vitro co-culture

• Preliminary optimization and characterization of the mouse co-culture system; toxicant dose response; developmental timeline. (Wilder et al. in progress).

Systems Biology

Framework

Project Aims

1. Develop a systems biology platform for integrating normal and adverse responses across testis development in rodents in vivo and in vitro

2. Establish a new mouse co-culture system based on a previous rat co-culture system and quantify baseline characteristics of the mouse culture

3. Use the mouse co-culture to evaluate effects of cadmium treatment during critical windows of susceptibility

Aim 1: Systems Biology Platform for Integration of In Vivo & In Vitro Responses

CS Wilder et al, In Process

Aim 2 &3: Mouse Co-Culture System Baseline & Cadmium Response

Baseline Results: Testosterone Production

Baseline Results: Protein Analysis by Western Blot

Marker Cell Type

3-B Hydroxysteroid

dehydrogenase (3B-

HSD)

Leydig Cells

C-kit Germ and Leydig cells

Proliferating cell

nuclear antigen (PCNA) Proliferating cells

Anti-Mullerian

hormone (AMH) Sertoli cell

RNA Binding Motif

(RBM) Germ cell

Cadmium Exposure Results: Cytoxicity (LDH)

Cadmium Exposure Results: Testosterone Production

Cadmium Exposure Results: Protein Analysis by Western Blot

Marker Cell Type

3-B Hydroxysteroid

dehydrogenase (3B-

HSD)

Leydig Cells

C-kit Germ and Leydig cells

Proliferating cell

nuclear antigen (PCNA) Proliferating cells

Anti-Mullerian

hormone (AMH) Sertoli cell

RNA Binding Motif

(RBM) Germ cell

DIV3

control 3BHSD (green)

AMH (red)

Cd10 RBM (green)

AMH (red)

DIV7

3BHSD (green)

AMH (red)

RBM (green)

AMH (red)

Preliminary Results: Protein Analysis by Immunoflourescence

DIV16

Next Steps

• IF optimization

• Pilot testing of “testes-on-a-chip” microfluidics system

– Part of UW’s EPA-funded Predictive Toxicology Center

Nortisbio.com

Conclusion (1) • Systems Biology Platform

• Anchoring system to developmental timeline is crucial in ensuring

translation from in vitro to in vivo systems

• Similarities/differences in critical windows of development

• Important for human health risk assessment

• Baseline characterization

• System supports the healthy growth of Sertoli cells, Leydig cells, and

germ cells for up to 16 days in culture.

• Co-culture captures relevant windows of susceptibility (based on

systems biology platform)

• Cadmium exposure

• Dose-dependent cytotoxicity and differential susceptibility based on

timepoint.

• 3D mouse testis co-culture system reflects endpoints relevant to

reproductive and developmental toxicants.

Conclusion (2)

2007 2017

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Acknowledgements

• Elaine Faustman

• Sungwoo Hong

• Carly Strecker Wilder

• Collin White

• Tomomi Workman

• Bill Griffith

• Susanna Wegner

• Sean Harris

• Julie Park

This research was funded by the U.S.

EPA – Science to Achieve Results (STAR)

Program Grant #83573801 and NIEHS

award T32ES015459.

*Its contents are solely the

responsibility of the grantee and do not

necessarily represent the official views

of the funding agencies