Characterization of porcine oviductal epithelial cells, cumulus cells and granulosa cells-conditioned media and their ability to induce acrosome reaction on frozen-thawed bovine spermatozoa

The mammalian female reproductive tract cells have been wildely used in in vitro fertilization. We studied the secretory proteins of porcine oviductal epithelial cells (POEC), and cumulus cells (CC) co-cultured with granulosa cells (GC) in conditioned media (CM), and their ability in inducing acrosome reaction (AR) on frozen-thawed bovine spermatozoa. POEC and CC + GC were cultured in M 199 medium for 48, 96 and 144 h prior to investigation of protein secretion. The results from SDS-PAGE showed secretory proteins sizes of about 17, 22 and >220 kDa in both CM from POEC and CC + GC. To test the ability of CM from POEC and CC + GC in inducing AR, the CM was frozen for 1–3 months before incubating with frozen-thawed bovine spermatozoa. Percentages of the spermatozoa with AR were determined under an inverted microscope and it was found that the tested group 1 (incubated with fresh and frozen CM from POEC for 1–3 months) were 78.44 ± 7.25, 75.78 ± 4.41, 65.22 ± 5.59 and 50.56 ± 6.25, respectively. The tested group 2 (incubated with fresh and frozen CM from CC + GC for 1–3 months) had the percentages of the spermatozoa with AR at 88.67 ± 4.03, 82.22 ± 3.46, 71.00 ± 3.16, and 58.56 ± 4.69, respectively. Statistical analysis of all group percentages indicated that they were significantly different (P < 0.05). Transmission electron microscope studies demonstrated that there were morphological changes on the sperm heads which resulted in the leakage of acrosin and subsequent AR. We concluded that the CM from both POEC and CC + GC efficiently increased in vitro AR on frozen-thawed bovine spermatozoa. Identification and functions of these secretory proteins are currently under investigation which may lead us to a better understanding of other beneficial effects in the utilization of both CM conditions.