[Frontiers in Bioscience E3, 1209-1220, June 1, 2011]

Developmental patterns of PPAR and RXR gene expression during spermatogenesis

Kelwyn Thomas1,2, Dae-Yong Sung1,2, Xing Chen1,2, Winston Thompson2,3, Y. Eugene Chen4, John McCarrey5, William Walker6, Michael Griswold7

1Department of Anatomy and Neurobiology, Morehouse School of Medicine, Atlanta, GA, 30310-1495, 2Cooperative Reproductive Science Research Center, Morehouse School of Medicine, Atlanta, GA, 30310-1495, 3Department of Obstetrics and Gynecology, Morehouse School of Medicine, Atlanta, GA, 30310-1495, 4 Cardiovascular Center, University of Michigan Medical Center, Ann Arbor, MI 48105, 5 Department of Biology, University of Texas, San Antonio, TX 78249, 6Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261, 7School of Molecular Biosciences, Washington State University, Pulliman, WA 99164-4060

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Isolation of seminiferous epithelium and spermatogenic cells
3.2. Isolation of primary rat Sertoli cells
3.3. RNA isolation and quantitative real-time PCR (qRT-PCR) Assay
3.4. PPAR adenoviral infection of primary Sertoli cells
3.5. Microarray Analysis
3.6. Microarray Statistical Analysis
3.7. Two dimensional SDS-PAGE analysis of lysate from Sertoli cells
4. Results
5. Discussions
6. Acknowledgement
7. References

1. ABSTRACT

Members of the family of nuclear receptors that include peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are important mediators of selective gene activation during development and cellular differentiation. In this study, developmentally-specific PPAR and RXR patterns of expression that occur in somatic and germ cell populations in the testis were determined using quantative real-time PCR (qRT-PCR) studies on RNAs that were isolated from StaPut purified mouse germ cells and primary rat Sertoli cells. These qRT-PCR studies indicate that transcripts encoding the PPAR-Alpha (α), -Beta (β), and -Gamma (γ) and RXR -Alpha (α), -Beta (β), and -Gamma (γ) are developmentally expressed in both differentiating germ and Sertoli cells. In further experiments aimed at deciphering the physiological role that PPAR-Gamma (γ) plays in Sertoli cells, 15-day primary rat Sertoli cells were infected with recombinant adenoviral vectors containing PPAR-Gamma (γ) cDNA and PPAR-Gamma (γ) RNAi constructs. Affymetrix microarray analysis and qRT-PCR validation studies using total RNA isolated from these transfected cells indicated that PPAR-Gamma (γ) regulates the pattern of expression of key lipid metabolic genes in Sertoli cells.