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Stage-Specific Expression of Genes Associated with Rat Spermatogenesis: Characterization by Laser-Capture Microdissection and Real-Time Polymerase Chain Reaction¹

^a Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia 3168 ^b Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia 3800

Spermatogenesis in the rat consists of 14 unique morphologic cellular associations between Sertoli cells and developing germ cells within the seminiferous epithelium. The complexity of the cellular associations leads to difficulty in the isolation of individual cells at a defined stage of development for the study of their unique patterns of gene or protein expression. Thus, laser-capture microdissection is an ideal technique to permit such analysis. This study used laser-capture microdissection and real-time reverse transcription-polymerase chain reaction (RT-PCR) to quantitate the stage-specific expression of a series of genes of functional significance in hormonal regulation and cell-cell interactions in spermatogenesis, including cathepsin-L, CREM-, transition protein-1, androgen receptor, ß1-integrin, N-cadherin, and hypoxanthine phosphoribosyltransferase (HPRT). Frozen sections (10 µm) were obtained from normal adult rat testes. Laser-capture microdissection (LCM) was used to capture all cells in cross-sections of seminiferous tubules that were grouped into stages I–V, VII–VIII, and IX–XIII. Transition protein-1 expression was lowest during stages I–V and increased 5.9-fold during stages VII–VIII and IX–XIII (P < 0.01). Cathepsin-L expression was highest during stages I–V and VII–VIII, falling 4.9-fold during stages IX–XIII (P < 0.05). Similarly, CREM- expression was highest during stages I–V and VII–VIII, falling 1.6-fold during stages IX–XIII (P < 0.05). A novel CREM- isoform lacking the phosphorylation domain was also characterized but was not stage-specific. ß1-Integrin, N-cadherin, and androgen receptor expression did not change between the spermatogenic stages examined. HPRT housekeeper expression was lowest during stages I–V but increased 1.5-fold during stages VII–VIII and IX–XIII (P < 0.05). This study is the first to apply LCM and real-time RT-PCR analysis to quantitate stage-specific changes in the expression of multiple genes in the seminiferous epithelium.

First decision: 18 March 2002.

¹ National Health and Medical Research Council of Australia Program Grant Regkey No. 983212.

² Correspondence: Peter Stanton, Prince Henry's Institute of Medical Research, P.O. Box 5152, Clayton 3168, VIC, Australia. FAX: 61 3 9594 6125; peter.stanton{at}med.monash.edu.au

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