IN VITRO-CULTURED BOVINE GRANULOSA AND OVIDUCTAL CELLS SECRETE SPERM MOTILITY-MAINTAINING FACTOR(S)

Since bovine cumulus oophorous and oviductal cell cultures are known t o support and maintain frozen-thawed bovine sperm viability and motili ty for extended time periods, we investigated whether granulosa cell ( GC)- and oviductal cell (OC)-conditioned media have similar effects. G C and OC were cultured for 3 days in TCM-199 medium supplemented with 10% fetal calf serum. At that time, the supernatant was discarded from GC and the monolayers were covered with Sp-TALP medium containing 6 m g/ml bovine serum albumin, while the OC were recovered by centrifugati on and transferred to culture bottles containing Sp-TALP. Two days lat er, GC-conditioned and OC-conditioned Sp-TALP were recovered and dialy zed, and their retentates were lyophilized. Bovine follicular fluid (B FF) was also dialyzed, and its retentate was lyophilized. When sperm w ere incubated in GC- or OC-conditioned media, motility remained above 62% and 42% at 6 hr and 30 hr, respectively, and motility was higher t han that of the control both at 6 hr (39%; P<0.001) and at 30 hr (9%; P<0.0001). Similarly, when sperm were incubated in the lyophilized ret entates of GC- and OC-conditioned media and in BFF at a dose of 0.1, 0 .5, or 1.0 mg/ml, the motility rates were higher both at 6 hr (P<0.05) and at 30 hr (P<0.01) compared to the control. The increase in motili ty was dose dependent; a 1.0 mg/ml dose improved (P<0.05) motility com pared to a 0.1 mg mg/ml dose. Heat treatment of the retentates of GC, OC, and BFF at 55 degrees C for 30 min did not destroy their ability t o support and maintain motility. However, heating at 100 degrees C for 5 min destroyed their ability to support motility. Molecular sieving of retentates on Sephacryl S-300 yielded fractions that were highly ef fective (P<0.01) in enhancing and maintaining motility compared to the other fractions. In conclusion, GC and OC secrete nondialyzable, heat -labile factor(s), which support and maintain sperm viability and moti lity for up to 30 hr. (C) 1994 Wiley-Liss, Inc.