Abstract. The charge and charge heterogeneity of FSH, LH and TSH in 63 extracts of individual human pituitaries were investigated by use of zone electrophoresis in 0.17% agarose suspension in veronal buffer at pH 8.6 followed by hormone analyses by radioimmunoassay. The migration velocities are similar to those in free solution and directly proportional to differences in charge in the buffer. The technique gave highly reproducible results and the recovery of hormone activity after electrophoresis was about 100%. Methods for estimation of median charge, expressed as median migration rate, and of degree of charge heterogeneity are described. Each pituitary contained a sequence of different forms, estimated at 20 or more, of each of the three hormones, with minor differences in charge. The degree of charge heterogeneity within the pituitary was similar for FSH, LH and TSH. The distribution curves of radioimmunological activity in relation to migration rate were close to normality for the three glycoprotein hormones. This was in contrast to that found for Prl and GH. The merits of the electrophoretic technique are discussed.
The use of a reaction between compounds in a two phase system – a solid phase and a liquid phase – has simplified the technical procedure of the radioimmunoassays. Antigens or antibodies can be coupled covalently to insoluble polymers to form stable conjugates with retention of immunological binding capacity. Such immunosorbents can be used in radioimmunoassays in many different ways and with the solid phase in different forms. Some of these techniques seem to be universally applicable for the assay of antigens or antibodies. In one of these, antigens or antibodies are chemically coupled to cyanogen bromide activated insoluble polysaccharides in a particle form. This method which has several advantages when compared with most other radioimmunological methods is discussed in particular. Finally, a detailed description of the preparation of an immunosorbent and its use in such a radioimmunoassay system is given.
Abstract. The biological activity of FSH in vitro (B-vitro) was compared with its radioimmunological (RIA) activity in 38 pituitary extracts from men and women. The B-vitro method was based upon the estimation of oestradiol produced by cultured Sertoli cells from 10-day-old rats. The mean B-vitro/RIA ratios for FSH of men and young and elderly women were almost identical. The mean values of median charge of the forms of FSH differed significantly between the three groups of human adults. The B-vitro/RIA ratio of different forms of FSH, separated by electrophoresis of 14 individual pituitary extracts, was higher for less negatively charged than for more negatively charged forms. Some forms of FSH with the same charge and separated from different pituitary extracts, e.g. from a young and an elderly woman, had significantly different B-vitro/RIA ratios. This ratio, thus, was not related to the charge per se of the hormone. However, the relation between the B-vitro/RIA ratio and the charge of FSH was similar for individual pituitary extracts when charge was expressed in relation to median charge. The results suggest that different molecular structural variations of FSH are involved in the polymorphisms observed within and between individual pituitaries. In human adults, the variation of FSH between the pituitaries mainly affects the metabolic clearance rate (MCR) of the hormone, whereas the variation within the individual pituitary affects both the MCR of the hormone and its biological effect at the target cell.
Abstract. The relationship between charge on human FSH (hFSH) molecules and their metabolic clearance rate (MCR) was investigated. The median charge of the hFSH molecules was expressed as their median mobility at electrophoresis in 0.075 m sodium veronal buffer, pH 8.6. MCR was estimated after single iv injection in mice of unfractionated and fractionated extracts of human pituitaries. There was a highly significant (P < 0.001) correlation between charge and MCR both for forms of FSH present within the individual pituitary and for FSH in different pituitaries. After the iv injection there was a gradual change to a more negative median charge of hFSH in plasma. This was explained by the more rapid clearance of the less negatively charged forms of hFSH and thus selective survival of different forms in the circulation. This is a most likely explanation for the differences in median charge between FSH in pituitaries and in sera of men and women of corresponding age. The results suggest that MCR of human FSH is controlled by a gonadal-pituitary feed-back mechanism which involves changes in the structure of the FSH produced by the pituitary. It is suggested that charge is one factor involved in the regulation of the survival of FSH in the circulation and that this is of physiological importance for the control of the ovarian function during the menstrual cycle.
Abstract. The median charge and the degree of charge heterogeneity of FSH, LH and TSH in extracts from pituitaries of 63 individuals were determined by electrophoresis in 0.17% agarose suspension at pH 8.6 followed by hormone analyses by radioimmunoassay. Charge was expressed as migration rate in relation to that of human serum albumin. FSH, LH and TSH showed similar degrees of charge heterogeneity within the pituitary, and this degree of heterogeneity was not related to sex or age. There was a sex- and age-related variation in median charge of FSH and LH but not of TSH. The forms of FSH and LH were more acidic at higher ages and the most acidic forms were found in elderly women. Women 32 to 47 years old had more acidic forms of FSH than those 14 to 20 years of age. The median charge of LH was similar in young men and women, while that of FSH was more basic in young women than in young men. These results indicate that the effect of gonadal feedback on the molecular charge is different for FSH and LH. Studies with neuraminidase-treated pituitary extracts indicated that the sex- and age-dependent variations of the charge on FSH were due to differences in the sialic acid content. The amount of FSH in the pituitary was significantly smaller in men than in women. The contents of TSH and LH in the pituitary were lower in the higher age groups. No such decrease with age was found for FSH.
Bruce Hobson and Leif Wide
When human chorionic gonadotrophin in the urine of women with chorioadenoma and men and women with choriocarcinoma, was assayed both biologically (B) and immunologically (I) the mean ratio, B/I, between the groups was significantly different (P < 0.001). When individual results in the two groups were compared it was not possible to distinguish between a case of chorioadenoma from one of choriocarcinoma. There was a distinct difference between the B/I ratio of urine specimens from women who were still alive and those who died subsequently. A high B/I ratio was associated with a good prognosis and a low B/I ratio with a poor one.
Leif Wide and Bruce Hobson
Abstract. 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.
Berndt Kjessler and Leif Wide
The relation between gonadotrophin secretion and average gametic output has been investigated in 643 healthy male partners of barren couples. FSH and ICSH excretion in urine were determined by means of bioassay and radioimmunoassay respectively, and FSH in serum was determined by radioimmunoassay.
The mean sperm counts were calculated for each patient from serial seminal analyses over a 12 week observation period.
The subjects were grouped according to their average gametic output and statistically significant differences were found between all mean excretion values for FSH, and for all but one pair of ICSH mean excretion values, when groups of patients with decreasing gametic output were compared. Individual assay results were, however, widely spread within each subgroup with extensive overlapping between the groups.
A general trend towards an increase in FSH as well as of ICSH secretion with decreasing gametic output was found. Thus, the mean excretion of FSH was found to be increased by 2.7 times, and the mean excretion of ICSH by 1.7 times, when azoospermic males were compared with males with a mean sperm count of 20 mill, per ml or more.
There was no evidence of accumulation of males with low or absent gonadotrophin secretion in any subgroup, and this may indicate that only few, if any, of the ordinary healthy male partners in the present couples suffered from a primary pituitary failure to secrete FSH or ICSH.
About 50 per cent of the males with increased FSH secretion and low or absent gametic output may have a selective spermatogenic failure, while the remainders are likely to suffer from a more general gonadal deficiency, since they also have a concomitant rise in ICSH.