Testicular Macrophage Activity Correlates with Leydig Cell Apoptosis following Ethylene Dimethane Sulfonate (EDS) Treatment of Mature Male Sprague‐Dawley Rats

Abstract

Tumor necrosis factor alpha (Tnfα), a cytokine secreted by macrophages is known to initiate cell apoptosis. Other laboratories have reported that macrophages are directly associated with Leydig cells in testicular interstitium. Using reverse transcription of total RNA (RT) and real‐time quantifiable polymerase chain reaction (qPCR) with quantification of expression using the (−) ΔΔCt method, we found increased Tnfα mRNA expression within 24 hr of EDS treatment. We also found that CD68 mRNA (a macrophage/monocyte/dendritic cell marker) increased within 24 hr of EDS treatment. Thus, not only are macrophages present in testes but macrophages may also be increasing in cell number/testis following EDS treatment. Leydig cells are lost via apoptosis following EDS treatment and analysis of the Leydig cell markers, Lhr and Insl‐3 mRNA levels declined more than 80%. Since these genes are expressed primarily in Leydig cells, this suggested an 80% loss of Leydig cells by 24 hr. Using the TUNEL assay that detects DNA fragmentation, we confirmed that Leydig cell apoptosis is time dependent and is increased with increased time following EDS exposure. We suggest a potential paracrine action of macrophages through secretion of TNFα to induce Leydig cell apoptosis.