Influence of the assay method used on the selection of the most active forms of FSH from the human pituitary.

The biological properties of different forms of human pituitary FSH, varying in their molecular charge, were investigated. FSH in two individual human pituitaries and a pool of 30 human pituitaries was extracted and subjected to electrophoresis. From each electrophoresis 14 consecutive fractions with the highest RIA activity were examined with in vitro and in vivo bioassays. The in vitro assay was based upon the estimation of oestradiol produced by cultured Sertoli cells from 10 day old rats. The in vivo bioassay was an hCG augmented test using immature female mice injected on 3 consecutive days. The increase in ovarian weight was the index of response. Both in the individual and in the pooled pituitary material the less negatively charged forms had the highest activity in the in vitro bioassay. In contrast, the more negatively charged forms had the highest activity in the in vivo bioassay. Forms of FSH from each of the two individual pituitary extracts were pooled according to their migration rate and injected iv into mice. The amount of FSH remaining in the circulation of the mouse after 1 h was related to the molecular charge. The highest value was obtained with the pool containing the more negatively charged forms of the hormone. The results indicate that the disappearance rate of the FSH molecule is a dominant factor in the in vivo bioassay. A consequence of these observations will be that the assay method chosen to monitor the purification of FSH will have a major influence on the biological properties of the final preparation.