Purification And Identification Of Inhibin From Porcine Follicular Fluid And Immunohistochemical Localization Of Inhibin-α Subunit In Nerve Center Of Rat

1. Purification and identification of crude inhibin from porcine follicular fluidThe porcine follicular fluid was abstracted by column chromatography with Sephadex G-200 firstly and then with Sephadex G-100. The effect of the crude inhibin on development of genitals induced by HCG and inhibition to oviduct's dropsy of mice was observed. The result showed that peak one remarkably inhibited the augment weight of ovary and the whole genitals, and oviduct's dropsy, which exhibited inhibitory activities in peak one.2. Detection of crude inhibin by Western BlotPorcine follicular fluid, abstracts from peak one and peak two were analysed by SDS-PAGE, and then by Western Blot with monoantibody of inhibin a subunit. The positive sites were seen in 18KD band in electrophoretic melod of follicular fluid and peak one, confirming the existence of inhibin in porcine follicular fluid and abstracts from peak one.3. Immunohistochemical localization of inhibin a subunit in hypothalamus and pituitary of the ratIn order to study the mechanism of the transport of inhibin through the blood-brain barrier, the normal mature rats and the rats of ovariotomy were injected with inhibin a subunit and crude inhibin. Then immunohistochemical localization of inhibin was performed. The result show that positive sites were detected in the hypothalamus and pituitary of normal mature rats, but no positive sites were seen in rats after 10 days of ovariotomy. No positive sites were found in the same position of the rat injected with inhibin a subuit at a dose of 0.1 ug at different time, but positive sites were detected in the hypothalamus with 0.2 ug after 4h but not in pituitary. Positive sites were detected both in the hypothalamus and pituitary with 0.5 ug after 2h and with crude inhibin of 0.2 ml. Theresults showed that inhibin a subunit exsited in hypothalamus and pituitary of the normal rats but not in the rats after 10 days of ovariotomy; inhibin a subunit could arrive at hypothalamus and pituitary, with a relation with dose and time.