(T428) IN VITRO SPERMATOGONIAL DIFFERENTIATION IN JSD MOUSE TESTES IS STIMULATED BY ELEVATED TEMPERATURE.
Porter, Karen1, Meistrich, Marvin1, 1 UT M.D. Anderson Cancer Center, Houston, TX
ABSTRACT- Male mice homozygous for the jsd mutation undergo an initial wave of spermatogenesis, but within a few weeks, spermatogenesis stops and tubules contain primarily Sertoli cells and A spermatogonia. We have determined that cryptorchidism will restore spermatogenesis in jsd mice despite high intratesticular testosterone levels, indicating that spermatogenesis in jsd testes is only sensitive to increased testosterone at reduced scrotal temperatures in vivo. In this study we cultured jsd mouse testis explants at scrotal (32.5°C) and abdominal (37°C) temperatures. Jsd mouse testes were harvested at 10 weeks of age, cut into pieces, placed on Corning Transwell inserts with DMEM/F12 based media with stem cell factor (SCF), and incubated at 32.5°C or 37°C for 9 days. At the end of incubation, the explants were fixed in Bouin's solution and processed for histology. Differentiation was determined by counting the number of leptotene and later spermatocytes per 1,000 Sertoli cells. We found that 100% of the samples cultured with SCF at 37°C exhibited differentiation to spermatocytes, compared to 15% to spermatocytes for cultures with SCF at 32.5°C. At time zero, samples averaged 0.4±0.3 spermatocytes per 1,000 Sertoli cells and samples cultured for 9 days at 37°C increased to 9.9±2.6 spermatocytes per 1,000 Sertoli cells, compared to samples cultured at 32.5°C, which at 0.1±0.02 spermatocytes per 1,000 Sertoli cells, showed no increase from time zero counts. These data indicate that incubation of jsd mouse testis explants at 37°C enhances spermatogonial differentiation as seen in cryptorchid mice in vivo. We are planning experiments using media with no additives to determine whether SCF is required for differentiation and with flutamide, to determine the effect of androgen blockade on differentiation.
KEY WORDS: spermatogenesis, jsd mice, testis, culture