Neurons essential for reproductive functions are distributed in hypothalamus. The precise functions of these neurons are not fully analyzed at a cellular level, especially in domestic animals. To analyze cellular mechanisms that control ruminant reproduction, we generated neuronal cell lines by immortalizing fetal goat hypothalamus.
The hypothalamic arcuate nucleus was collected from a female Shiba goat at E118. The tissue was dispersed and plated. Twenty-four hours after the primary culture, lentiviral vector containing SV40 large T antigen gene (T-Ag) was added, then cells were selected by G418. We performed cell cloning from the obtained cell population, and analyzed gene expressions of each clone by RT-PCR. The expression of neuronal marker (neuron specific enolase (NSE)), glial marker (glial fibrillary acidic protein (GFAP)), T-Ag, and GAPDH were examined.
The primary-cultured cells acquired strong proliferative activity after the viral infection. All clones expressed T-Ag, suggesting that these cells were immortalized by the insertion of T-Ag. We obtained 61 clones from the immortalized population. Thirty-six out of 61 clones were NSE-positive and GFAP-negative, which were defined to be neuron-derived cell lines.
In summary, we established a number of neuronal cell lines derived from goat hypothalamus. Further analysis of expression of neuropeptides or receptors related to reproduction is required to determine neuronal cell lines appropriate for examining central mechanism regulating reproduction in vitro.
This work was supported in part by the Research Program on Innovative Technologies for Animal Breeding, Reproduction, and Vaccine Development from the Agriculture, Forestry and Fisheries Research Council, Japan (REP2005).
02 - 04 Sep 2014
World Congress of Reproductive Biology