Culture of Principal Cells From the Rat Caput Epididymidis1

Abstract

Studies of epididymal function would be facilitated if principal or basal cells could be cultured. Principal cells from the caput epididymidis were isolated by elutriation and suspended (4 × 10⁶ /ml) in culture medium supplemented with serum. Cells were cultured at 34°C within a floating collagen matrix prepared by mixing equal volumes of the principal cell suspension and an ice-cold collagen solution when plating the cells in the culture dish. After 20–24 h, serum-free medium was used. Cultures contained both isolated cells and clusters of cells surrounded by collagen fibers, but cell division was not seen. Based on observations by transmission electron microscopy, 82% of the cells in Day 5 cultures were principal cells and 75% of these cells were morphologically normal. Fine structure of principal cells on Days 1, 3 or 5 was similar to that of cells in situ. Metabolism of [ ³H] testosterone was evaluated over Days 1–3 and 3–5. Virtually all of the ³H recovered from the culture medium was present as 5α-reduced metabolites (70% dihydrotestosterone). ³H-protein produced during 48 h of incubation with [ ³H] L-leucine and released into the culture medium was evaluated by 3 procedures. Immunoelectrophoresis against anti-rat cauda epididymal plasma consistently produced 1 precipitin arc. Filtration of native lyophilized protein on Sephadex G-100 revealed 3 ³H-proteins (107,000, 65,000 and 26,000 daltons). The middle peak was concentrated and gave a single peak (62,000 daltons) on Sephadex G-75. SDS-gel electrophoresis revealed numerous ³H-polypeptides. Based on cell morphology, testosterone metabolism, and protein synthesis and release, principal cells cultured within a collagen matrix retained their integrity and function for at least 5 days.