Studies on the biological and immunological properties of human follitropin: profiles of two international reference preparations and of an aqueous extract of pituitary glands after electrofocusing

Abstract

Abstract. Two international reference preparations, the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH/ICSH) for Bioassay (identified hereafter by its code no.: 69/104) and the First International Standard of Urinary FSH and LH (ICSH) for Bioassay (hereafter: hMG 1st IS) and an aqueous extract of human pituitary glands (hPE) were fractionated in triplicate by isoelectric focusing on sucrose density gradient (Ampholine, pH range 2.5–10.0). The FSH activity was monitored in each fraction by an in vitro bioassay, a radioimmunoassay and a receptor binding assay. Unfractionated 69/104 was used as standard in each assay system. Compared with the hPE, the activity profiles of the 69/104 and hMG reference preparations were spread over a significantly wider pH range; the biological activity eluted in the pH range of 3.5–5.0 was of the order of 90% for hPE, 72% for 69/104 and less than 60% for hMG. Major variations in biological to immunological (B/I) and biological to receptor binding (B/R) activity ratios were observed in the individual electrofocusing fractions. The B/I ratios ranged from 0.8 to 2.2 (69/104), 1.0–5.7 (hMG) and 1.3–6.0 (hPE), respectively. The variation in the corresponding B/R ratios was: 0.5–1.7 (69/104), 0.58–4.0 (hMG) and 0.55–1.5 (hPE). When equal aliquots from each of the electrofocusing fractions were combined and this 're-constituted' pool was compared with the starting material, significant differences were observed in the B/I ratios: 1.63 instead of 1.0 (69/104), 2.58 vs 2.34 (hMG) and 2.72 vs 1.32 (hPE). There was no significant change in the mean B/R ratios before and after electrofocusing: 1.0 vs 1.02 (69/104), 1.09 vs 1.02 (hMG) and 1.05 vs 1.04 (hPE). The mean recovery of biological activity in each of the three reconstituted pools of the three preparations was 97% for 69/104, 88% for hMG and 98% for hPE, and the corresponding recoveries of receptor binding activities were 95%, 94% and 98%, respectively. In contrast, the mean recovery of immunological reactivity was only 55% (69/104), 80% (hMG) and 47% (hPE), respectively. The reduction in the immunological reactivity of the 69/104 preparation without any apparent loss of biological or receptor binding activities following electrofocusing indicates that its immunoreactivity is unstable under mild experimental conditions, which do not influence its biological and receptor binding activities. Hence, whereas this preparation might possibly be suitable as a standard for in vitro bioassays and receptor binding assays, it is unsuitable as a standard for the radioimmunoassay of hFSH.

Biosynthesis and Secretion of Follicle-Stimulating Hormone*

“…Studies performed on hFSH extracted from AP glands or urine have revealed the presence of several FSH species as disclosed by isoelectric focusing (253,258,262,270). Since the majority of these studies have been performed on pituitary hFSH preparations that have been exposed to chemical manipulation during their partial or total purification, comparisons with studies in other species that utilize crude pituitary extracts are questionable.…”

“…It has recently been demonstrated that gonadotropins exist in multiple forms which exhibit differences in biological, receptor binding and immunological activities (240,242,251,252). Multiple species of gonadotropins have been isolated and characterized from AP glands (72, 86, 238, 252), urine (242,253) and serum (248,251) of rodents (72, 240,252), rabbit (252), pig (252), dog (252), sheep (254), chicken (255), monkey (238,239), and man (251,253). This multiplicity has been disclosed by gel filtration (238,246,247,256), affinity chromatography (72, 86), and isoelectric focusing (242,251,257,258) as well as by kinetic studies performed on pituitary extracts and circulating gonadotropins (243)(244)(245)(246).…”

Naturally Occurring Antihormones: Secretion of FSH Antagonists by Women Treated with a GnRH Analog

“…Because of the lack of a sensitive FSH bioassay, earlier investigators used radioligand receptor assays to assess FSH bioactivity (15). The receptor assay, however, cannot differentiate between agonistic and antagonistic isoforms of FSH.…”

“…Studies with gonadotropins from different species have showvn variations in isohormone activities (11,15,16). Disparate bioto immunoactivities of different acidic FSH isoforms are believed to be due to variations in sialic acid residues that would confer a longer biological half-life and higher in vivo biopotency (3,4,17).…”

Improved in vitro Bioassay of Follitropin

“…Human follitropin preparations (hFSH) purified from pituitary glands or from uri nary extracts have all proved to be heteroge neous in nature [Saxena and Ratnam, 1978: Zaidi et al, 1981, 1982, This heterogeneity may be one of the reasons for the discrepancy between biological activity and immunologi cal reactivity that has been observed for many FSH preparations [ Van Damme el al., 1980;Zaidi el al., 1981Zaidi el al., , 1982, However, it was not settled whether this microhetero geneity was caused by the purification proce dures or was a characteristic of the FSH mol ecules also when present in body fluids. Wide [1981] very recently reported that FSH in sera from women of fertile ages was less acidic than FSH from men or postmeno pausal women, indicating an influence of the sex hormones on the structure of the FSH molecules.…”