# Presentation: 509364

## Optimization of the Sliced Testis Steroidogenesis Assay

- Carol S. Sloan, Amanda B. Goodman, Rochelle Tyl
- RTI International
- June 2003

## Background

- The DRP named several possible procedures to study steroidogenesis and its’ ability to characterize the endocrine effects of various environmental contaminants, industrial substances and pesticides
- Tissue culture
- Purified cell preparations
- Cell lines
- Ovarian *in vitro* steroidogenesis assay
- Testicular *in vitro* steroidogenesis assay

## Why Sliced Testis Assay?

- Minimal Cost
- Quick
- Uses Standard Laboratory Equipment
- Basic Laboratory Training
- Stable Preparation
- Uses Reduced Number of Animals
- Can be Standardized
- Well-defined endpoint in testosterone concentration
- Can be modified to use intermediate hormonal endpoints

## Optimization of the Sliced Testis Assay

- This was planned to be Implemented in Two Phases

## Prototypical Sliced Testis Assay

- Testes are weighed and placed in DPBS buffer
- Each testis is sliced along the longitudinal axis into slices of proper weight
- Slices are placed with 5mL of media
- Incubated at 34oC in 5%CO2 and 95% air on a shaker

**Notes:**
Media is M-199 with sodium bicarbonate, HEPES, BSA and soybean trypsin inhiditor and is adjusted to a pH of 7.4
Time from removal to slicing is under 1 hr. / 135rpm

## Sliced Testis Assay

- At first time-point –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 are collected at 1, 2, 3 and 4 hours post-challenge
- Samples are analyzed for testosterone concentration in a RIA 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 1: Preliminary Experimental Phase

- Establish whether a given level of each factor affects assay performance
- The factors are unlikely to have an interaction or at best a minimal interaction with another experimental factor
- An effect of one of these factors would require additional verification experiments after sensitivity analysis

## Phase I Design

## 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

## Testosterone Radioimmunoassay	

- RIA
- Used to measure testosterone concentration of the aliquots taken at  various time-points
- Commercial kit
- Utilizes 125 I-testosterone and a testosterone-specific antibody affixed to tubes

## Testosterone RIA 

- Verification of the assay using Media 199 without phenol red
- Determination of assay accuracy, sensitivity, precision and parallelism

## Slide 14

## Testosterone RIA Validation with M-199

## Lactate Dehydrogenase (LDH)Assay 

- Validation of the assay using Media-199 without phenol red
- Measuring the assay accuracy, sensitivity, and precision
- Performed at Laboratory Corporation of America

## Slide 17

## Phase I	

- Tested:
- Media Type
- Eagles MEM
- RPMI-1640
- Medium-199
- Gaseous Atmosphere
- 5% CO2 / 95% air
- 5% CO2 / 95% O2
- Air
- Rat Age
- 11 weeks
- 15 weeks
- 22 weeks

## PHASE I: Experimental Design

## Slide 20

## 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

## Phase II

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

## Slide 23

## Slide 24

##  Technical Flow of Sliced Testis Assay

## Phase II Optimization

- Currently Being Analyzed