Quantitative evaluation of the maintenance and development of spermatocytes and round spermatids in cultured tubule fragments from immature rat testis.

Maintenance and development of spermatocytes and round spermatids was studied in an in-vitro incubation system. This system consisted of open tubule fragments from 26-day-old rat testes, obtained after collagenase treatment. The tubule fragments contained Sertoli cells and spermatogenic cells up to and including a small number of early round spermatids. The number of primary spermatocytes and round spermatids in the tubule fragments was estimated using flow-cytometric analysis, immediately after isolation and after 72 h of incubation. In addition, the activity of LDH-C4 in the tubule fragments was measured. After 72 h of incubation, the percentage of spermatocytes was reduced by 70-80%, but the percentage of spermatids was doubled. The total LDH-C4 activity per well was increased 2-3-fold during 72 h of incubation of the fragments. A modest improvement of the culture results was observed when a combination of FSH, insulin, retinol and testosterone was added to the medium. LDH-C4 activity was investigated to see whether it could be used as a quantitative marker of isolated and cultured spermatocytes and spermatids. It was observed that LDH-C4 activity per cell was decreased when spermatocytes and spermatids were isolated and/or incubated at 4 degrees C. However, the cellular enzyme activity returned to control values during subsequent incubation of the cells at 32 degrees C, either in the absence or presence of a protein synthesis inhibitor. Cellular LDH-C4 activity may be influenced not only by temperature, but possibly also by other cell isolation conditions. It is concluded that LDH-C4 activity may not be a reliable quantitative marker for the presence of spermatocytes and spermatids in culture, but should be used in combination with other analytical methods such as DNA estimation and DNA flow cytometry.