# Presentation: 684857

- Research Triangle Park,  North Carolina

## Optimization of the Sliced Testis Steroidogenesis Assay

- Carol S. Sloan, Amanda B. Goodman,
-  Susan W. Pearce and Rochelle W. Tyl
- RTI International
- August 2003
- This study was performed under a subcontract to Battelle Laboratories, Columbus, Ohio for prime contract No. 68-W-01-023 for the U.S. Environmental Protection Agency

## Optimization of the Sliced Testis Assay

- Implemented in Two Phases

## Basic Sliced Testis Assay

- At first timepoint –designated baseline – media is removed and discarded
- Fresh media is added and an aliquot is collected
- Half of the samples are challenged with a stimulant, such as hCG
- Aliquots of media are collected at 1, 2, 3 and 4 hours post-challenge
- Medium samples are analyzed for testosterone concentration in a RIA assay
- Medium samples are analyzed for Lactate Dehydrogenase (LDH) as a measure of cell viability in a spectrophotometric assay

**Notes:**
hCG = 0.1 IU/mL
Samples are 0.5 mL
Samples can be frozen at –70oC for up to one month

##  Technical Flow Illustration of Sliced Testis Assay

## Phase I Design

## Results of Phase I Optimizations

- Medium-199 without phenol red
- Gaseous atmosphere of 5% CO2 / 95% O2
- Rats that were 11 weeks of age showed results similar to those that were 15 weeks of age, therefore rats 11-15 weeks can be used in the assay; 22 week old rats exhibit reduced testosterone production

## Phase II: Primary Experimental Phase

- Factors that may affect assay performance were tested
- These factors were divided into four sections where each section was composed of factors that might produce interactions

## Phase II Design

## Phase II

- To be tested:
- Incubation Conditions
- Incubation Temperature
- Incubation Vessel Type
- Incubation Shaker Speed
- Incubation Media Volume
- hCG Concentrations
- Testes Preparation Conditions
- Testicular Fragment Size
- Time Delay before starting the assay
- Organ Preparation Technique
- Sample Aliquot Volume

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## Predicted values for Specified Optimum Factor Combinations for Models

-  with Baseline Concentration Removed

- *Values in parentheses are standard errors

## Conclusions from Incubation Factors Experiments

- Optimal temperature for incubation was approximately 36oC
- Optimal vessel for incubation was the scintillation vial
- Optimal Shaker speed was between 135 and 200 rpm, approximately 175 rpm
- Optimal media volume was approximately 4-5 mL
- Optimal hCG concentration for stimulation was 0.08 to 0.1 IU/mL

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##  Predicted Values for Specified Optimum Factor Combinations

-  (Models with Baseline Concentration Removed)

- *Values in parentheses are standard errors

## Conclusions from Testes Preparation Experiments

- Optimal fragment size varied from 100-200 mg with stimulated versus non-stimulated assays
- Time delay before the start of the incubation should be no more than 1 hour from the time of testicular tissue removal from the male
- The solution that the testes are collected in may be either cold DPBS or cold M-199
- The sample aliquot size removed for testing should be around 0.5 mL

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## Summary

- 11-15 week old rats should be used
- Media 199 without phenol red is the preferred media
- Gaseous atmosphere of 5% CO2 / 95% O2
- Optimal temperature for incubation was approximately 36oC
- Optimal vessel for incubation was the scintillation vial
- Optimal Shaker speed was approximately 175 rpm
- Optimal media volume was approximately 4-5 mL
- Optimal hCG concentration for stimulation was 0.08 to 0.1 IU/mL
- Optimal fragment size varied from 100-200 mg
- Time delay before the start of the incubation should be no more than 1 hour from the time of testicular tissue removal
- The solution that the testes are collected in may be either cold DPBS or cold M-199
- The sample aliquot size removed for testing should be around 0.5 mL

## Points to Ponder

- All of these assays were performed on control rat testes
- No assays included any test chemicals except for possible vehicles that may be used in the pre-validation and validation assays
- It may be necessary to measure specific cell viability ( LDH is not cell specific)
- Use of beta-HSD (an enzyme more specific to Leydig cells) staining

## (RTI International)
Ms. L. McDonald
Mr.J. Gray
Ms. V.  Wilson
Ms. N. M. Kuney
Ms. K. Vick 
Ms. A. Parham 
Mr. M. Crews
Ms. L. Pelletier
Ms. S. Parker
Mr. C. Andrew Clayton
Ms. Margaret Zeller Byron
Ms. Bonnie Hamby
Mr. F. N. Ali
Dr. D. B. Feldman, DVM

- Ms. L. McDonald
- Mr.J. Gray
- Ms. V.  Wilson
- Ms. N. M. Kuney
- Ms. K. Vick
- Ms. A. Parham
- Mr. M. Crews
- Ms. L. Pelletier
- Ms. S. Parker
- Mr. C. Andrew Clayton
- Ms. Margaret Zeller Byron
- Ms. Bonnie Hamby
- Mr. F. N. Ali
- Dr. D. B. Feldman, DVM

**Quality Assurance**
- Ms. M. D. Phillips (RTI)
- Mr. R. Patterson, QA Consultant
- Ms. K. Cummings (ILS)
**Battelle**
- Dr. Jerry D. Johnson
- Dr. David P. Houchens
- Dr. Paul Feder
**EPA**
- Mr. Gary Timm
- Dr. Jerome Goldman

**ACKNOWLEDGEMENTS**