In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH.
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abstract
In vitro culture systems are valuable tools for investigating reproductive mechanisms in the testis. Here, we report the use of the precision-cut in vitro system using equine testicular slices. Testes were collected from immature light breed stallions (n=3) and cut into slices (mean slice weight= 13.85 0.20 mg; mean slice thickness=515.00 2.33 m) using the precision-cut tissue-slicing method. Four tissue slices were placed on a grid floating on medium in individual vials. After a 1-h preincubation, they were exposed to medium containing ovine luteinizing hormone (oLH) at concentrations of 0, 5, 50, and 500 ng/ml for 6 h at 32 C. Viability of the tissue was maintained based on histological integrity and lack of appreciable lactate dehydrogenase in the medium. The production and release of testosterone (T) and estradiol-17 (E2) into the medium was measured following in vitro culture. The addition of oLH increased T and E2 at least 400% and 120%, respectively, over the 0-ng oLH control cultures. Testicular gene expression was assessed with in situ hybridization methodology for steroidogenic acute regulatory protein (StAR protein), phosphodiesterase 3B (PDE3B), and outer dense fiber of sperm tails 2 (ODF2) mRNAs. In situ hybridization revealed an oLH concentration-dependent increase in the concentration of StAR protein mRNA in Leydig cells. No differences were observed for the expression of PDE3B or ODF2 genes in seminiferous tubules among treatment groups as expected. These results demonstrate the value of in vitro culture of the precision-cut tissue slices for studies of the regulation of steroidogenesis and gene expression in the stallion testes.
