Using in vitro epithelial cell culture to mimic the in vivo conditions in the oviduct. Adoption of bovine oviduct epithelium cultivation on permeable support for study of sperm binding capacity in relation to bull fertility.
MetadataShow full item record
In mammalian, fertilization is the origin to life. Researchers have found that the oviduct is the site in the female reproductive tract where capacitation of spermatozoa, fertilization and early embryonic development occurs. Fertilization-competent sperm cells that manage to reach the oviduct will interact with the oviduct epithelial cells, forming a sperm reservoir, and release themself at ovulation. An in vitro cell model system is needed to adopt increased knowledge about this interaction. In this study the main aim was to establish an in vitro bovine oviduct epithelial cell (BOEC) culture system that mimics the in vivo conditions in the oviduct. Therefore, BOECs were cultivated on membrane support and the cells were characterized by immunostaining of cell specific marker proteins and real time PCR (RT-PCR) analysis of OVGP1 gene expression. The cultivated BOECs were further used to evaluate sperm binding capacity in semen from high and low fertile NRF bulls. The statement that capacitated sperm cells are unable to bind BOECs, led to the adoption of a flow cytometry Ca2+ analysis protocol, as capacitated cells have a high level of Ca2+. Additionally, the semen used in the sperm binding capacity assay was evaluated for viability, acrosomal integrity, capacitation and DNA fragmentation. Results revealed that cultivated BOECs were a pure oviduct epithelial cell line. They grew in an increased cell height, had the ability to stay viable 5 days post-confluence and were able to bind spermatozoa. However, the OVGP1 gene expression was lost during cultivation time. The sperm binding capacity results did not show any significant differences between the bulls with high and low fertility. These findings show that the cultivation of BOECs on membrane was successfully achieved and the cells mimic the in vivo to a greater extent than BOECs on plastic. However, further optimization of the sperm binding assay is needed. The adopted protocol for Ca2+ analysis revealed a significant higher Ca2+ level in bulls with high fertility than the low fertility group. From this result it can be speculated that capacitation ability of sperm cells may have an effect on the oviduct-sperm release capacity and thus fertilization competence.
Mastergradsoppgave i næringsrettet bioteknologi, Avdeling for lærerutdanning og naturvitenskap, Høgskolen i Hedmark, 2013. Master of applied and commercial biotechnology.