Abstract
A highly purified follicle-stimulating hormone (FSH) preparation has been obtained from sheep pituitary glands by extraction with ethanol followed by fractionation with metaphosphoric acid and ammonium sulfate, gel filtration on Sephadex G-150, ion exchange chromatography on carboxymethyl Sephadex, hydroxylapatite chromatography, preparative acrylamide disc gel electrophoresis, and gel filtration on Sephadex G-75. The purified FSH contained 133 units of the National Institutes of Health ovine follicle-stimulating hormone standard (NIH-FSH-S1) per mg of dry weight as judged by augmentation bioassays. This preparation also contained 0.04 unit of the National Institutes of Health ovine luteinizing hormone standard (NIH-LH-S1) per mg of dry weight as determined by ovarian ascorbate depletion bioassays and more than 1 unit of NIH-LH-S1 per mg as determined by the hypophysectomized male rat ventral prostate bioassay. Radioimmunoassay indicated that the purified FSH contained only 3% as much immunoreactive luteinizing hormone as found in the National Institutes of Health ovine FSH standard (NIH-FSH-S4). Administration of low doses of the purified FSH to hypophysectomized female and male rats not only resulted in increased weights of the ovaries and testes but also of the uteri, ventral prostates, and seminal vesicles. Analytical acrylamide disc gel electrophoresis of the purified FSH at pH 8.9 displayed a single broad zone; however, immunodiffusion in agar showed a diffuse precipitin line plus two fine precipitin lines. The molecular weight of sheep FSH was approximately 33,000 as determined by ultracentrifugation. Electrofocusing in carrier ampholytes indicated an isoelectric point of pH 4.6. Amino acid analysis showed that the purified FSH contained a higher content of threonine and half-cystine and a lower content of phenylalanine than sheep FSH preparations previously described.
Highlights
Sephadex G-150, ion exchange chromatography on carboxymethyl Sephadex, hydroxylapatite chromatography, preparative acrylamide disc gel electrophoresis, and gel filtration on
Radioimmunoassay indicated that the purified follicle-stimulating hormone (FSH) contained only 3% as much immtmoreactive luteinizing hormone as found in the National Institutes of Health ovine FSH standard (NIH-FSH-S4)
Administration of low doses of the purified FSH to hypophysectomized female and male rats resulted in increased weights of the ovaries and testes and of the uteri, ventral prostates, and seminal vesicles
Summary
Routine Bioassays-Intact 21-day-old n-ale and female rats were used to monitor FSH and LH activities during the development of the purification procedure. Subcutaneous injections were administered the afternoon that the rats were obtained (Day 21) and twice daily the 4 days. The animals were autopsied on the morning of Day 26. FSH activity was based on an increase in ovarian weight (10). LII activity was based on an increase in seminal vesicle weight (10, 11). Specific FSH activity was determined for the principal FSH fractions by Steelman-Pohley augmentation bioassays (6). Three doses of the FSH reference standard NIH-FSH-S3 and each unknown were administered to immature female rats. Four animals were used for each dose. The results were evaluated by statistical methods for parallel line assays as described by Bliss (12)
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