In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH
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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 µm) 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-17β (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.
KeywordsOrgan culture system Stallion Testis StAR protein Luteinizing hormone
Financial support for this project was provided by the H. Patricia Link Endowment Equine Research Fund, Texas A&M University. The authors are grateful for the gifts of plasmids containing cDNAs for ODF2 from Dr. Frans van der Hoorn (University of Calgary, Alberta, Canada) and PDE3B from Dr. Vincent Manganiello (National Institutes of Health, Bethesda, MD). In addition, the authors would like to thank Linda Love and Beltex, Inc. for assistance in obtaining the testis tissues, Dr. Robert Burghardt (Texas A&M University, College Station, TX) for providing microscopy facilities and Cindy Balog Alvarez, Pat Chen and Dr. John Nelson for technical assistance.
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