W. H. McShan

Purification of luteinizing hormone from sheep pituitary glands and evidence for several physicochemically distinguishable active components

Abstract 1. 1. A highly purified preparation of luteinizing hormone has been obtained from sheep pituitary glands by extraction with ethanol, metaphosphoric acid and (NH 4 ) 2 SO 4 fractionations, and ion-exchange chromatography on CM-Sephadex. This luteinizing hormone preparation designated CM3 contained 1.6 and 1.9 units of NIH-LH-S1 per mg dry wt. as measured by ovarian ascorbic acid depletion bioassays and the ventral prostate bioassay, respectively. The level of follicle-stimulating hormone contamination was approx. 0.025% as judged by augmentation bioassay. 2. 2. Three major luteinizing hormone fractions (Fr 1, Fr 2, Fr 3) were obtained when the purified luteinizing hormone preparation CM3 was chromatographed on hydroxylapatite. Ovarian ascorbic acid depletion bioassays and the ventral prostate bioassay indicated that two of the luteinizing hormone fractions (Fr 2, Fr 3) contained 2.0–2.6 units of NIH-LH-S1 per mg dry wt. Augmentation bioassays and hypophysectomized female rat bioassays indicated the low level of follicle-stimulating hormone contamination in the preparation CM3 was concentrated in Fr 1 after hydroxylapatite chromatography. Therefore, the highly active luteinizing hormone fractions (Fr 2, Fr 3) were essentially free of follicle-stimulating hormone contamination. 3. 3. Physicochemical analyses of the three luteinizing hormone fractions, as well as the purified luteinizing hormone preparation CM3, were conducted employing gel filtration, ultracentrifugation, analytical acrylamide disc gel electrophoresis, preparative acrylamide disc gel electrophoresis, electrofocusing, amino acid analysis, carbohydrate analyses, and immunodiffusion in agar gels. Perceivable differences among the three luteinizing hormone fractions could be detected with analytical acrylamide disc gel electrophoresis, preparative acrylamide disc gel electrophoresis, and electrofocusing. Moreover, amino acid and carbohydrate analyses suggested there might be differences in the amino acid and carbohydrate compositions among the luteinizing hormone fractions. Dissimilarity in molecular size and immunological specificity was not detected. In addition to the differences found among the luteinizing hormone fractions, data are presented which show that electrophoretically distinguishable luteinizing hormone components were contained within two fractions (Fr 1, Fr 2), as well as preparation CM3. 4. 4. These results suggest a broad spectrum of physicochemically similar luteinizing hormone components which differ from one another to the degree necessary for chromatographic and electrophoretic resolution.

Relation of stage of cycle and source of luteinizing hormone to superovulation in dairy cattle.

Summary 1. Two series of studies involving a total of 26 heifers and cows are reported. 2. Five heifers superovulated during the luteal phase of the estrual cycle produced 34 eggs none of which were fertilized. Five heifers treated during the follicular phase produced 43 eggs of which 74% had cleaved. All 6 heifers treated during the luteal phase, but none of the 6 follicular heifers, had pyometra. 3. Thirty and 40 g.e. of sheep FSH gave responses about equal but considerably greater than a 20 g.e. dose. 4. USG produced approximately twice as many corpora as HCG in the intravenous injection when they were used in combination with either sheep pituitary FSH or PMS. 5. A combination of PMS and HCG may produce satisfactory superovulation and should be studied further.

Purification of follice-stimulating hormone from horse anterior pituitary glands

Abstract Fresh horse-pituitary glands were extracted with 40% ethanol and the gonadotropins were recovered by increasing the alcohol concentration to 85% followed by drying with acetone. This preparation was further extracted with water at pH 5, and the extract was adjusted to pH 7 and lyophilized. The follicle-stimulating hormone in the pH-5-souluble fraction was purified by zone electrophoresis and resolved into six components by starch-gel electrophoresis. One of these components contained follicle-stimulating hormone which was recovered in the elution cell and the contaminating starch was separated by chro,atography on Dowex- 2 X8. This follicle-stimulating hormone stimulated estrogen production in immature hypophysectomized female rats and was essentially free of luteinizing hormone and other pituitary hormones. It migrated as a single zone in starch-gel electrophoresis and sedimented as a single boundary in the ultracentrifuge.

A METHOD FOR THE PREPARATION OF FOLLICLE-STIMULATING HORMONE FROM SHEEP PITUITARY GLANDS.

Abstract A method is described for the preparation of highly purified follicle-stimulating hormone from fresh sheep pituitary glands. A gonadotropic preparation obtained by a modified Koenig-King procedure is subjected to zone electrophoresis on a vertical column of cellulose using 0.02 M phosphate buffer (pH 7.7) which effects a partial separation of the follicle-stimulating hormone from the luteinizing hormone. The follicle-stimulating hormone fraction is filtered through Sephadex G-25 and is further purified by chromatography on diethylaminoethyl-cellulose equilibrated with 0.006 M Tris buffer (pH 7.7) in 0.001 M versene and 6 M urea. The active fraction obtained was rechromatographed under the same conditions and the product was shown to consist of a major and a minor component by starch-gel electrophoresis at pH 4.0 and 8.6. The follicle-stimulating hormone was recovered in the major zone at pH 8.6. The preparation obtained by chromatography on diethylaminoethyl-cellulose when assayed by the Steelman-Pohley method was shown to be 6.5 times as active as the follicle-stimulating hormone supplied by the Endocrinology Study Section of the National Institutes of Health whereas after starch-gel electrophoresis, it was 9.7 times as active. The follicle-stimulating hormone obtained by chromatography on diethylaminoethyl-cellulose and by starch-gel electrophoresis when assayed in hypophysectomized female rats, stimulated the ovaries and caused estrogen production as indicated by the distension of the uteri with fluid and, when assayed in hypophysectomized male rats, both preparations showed low but essentially the same luteinizing-hormone activities as indicated by small increases in the weights of the ventral prostates. This low level of luteinizing activity is tentatively attributed to an inherent property of the follicle-stimulating hormone.

Gonadotrophic Activity of Granules Isolated from Rat Pituitary Glands.

Summary Approximately 60% of the succinoxidase activity of rat pituitary glands were found to be associated with the large granule fraction, and more than 50% of the gonadotrophin of these glands was associated with two granule fractions isolated from sucrose homogenates by differential centrifu-gation. The supernatant fraction from which the granules were removed contained the remaining gonadotrophic activity. The gonadotrophin can be extracted from the isolated granules with isotonic sodium chloride solution.

Carbohydrate Properties of Pituitary Follicle-Stimulating and Luteinizing Preparations.

Summary The carbohydrate content of our follicle-stimulating and luteinizing preparations is given in terms of glucose calculated from the reducing action of the hydrolysates. Results from enzymatic inactivation and electrodialysis are given which suggest that the follicle-stimulating activity may be associated with a carbohydrate grouping.

Effects of LH-RH on isolated pituitary gonadotropic cells.

Summary Rat anterior pituitary glands were dissociated with Pronase and the cells were separated by velocity sedimentation at unit gravity. After 30 min of incubation of the enriched gonadotropic cells with LH-RH, there was a significant increase in LH and FSH in the incubation medium. LH-RH (100 ng/ml) and 10−3 M cAMP both caused significant increases in LH in the incubation medium after 24 hr of incubation. This investigation was supported by Public Health Service Training Grant IT05-GM 01932-03, The Ford Foundation Grant 630-0505A, and the National Institutes of Health Training Grant 5-T01-HD-00104-08. The authors thank Dr. W. F. White of Abbott Laboratories for the synthetic LH-RH, and NIAMD, NIH for the rat FSH and LH radioimmunoassay kits.

Further Purification of the Follicle-Stimulating Hormone and Its Effect in Normal and Hypophysectomized Rats.

Summary A method is described for the preparation of follicle-stimulating preparations by tryptic digestion and the removal of a fraction which caused reactions in the human being at the site of injection. Preparations made by the tryptic digestion method before and after it was changed to remove the local reacting factor were assayed both in normal and hypophysectomized rats. By macroscopic observation the ovaries from both kinds of rats appeared to contain only follicles but microscopic examination showed that a few contained lutein tissue indicating that not all the preparations were entirely devoid of luteinizing activity.

Shock Produced by the Application of Tourniquets to the Hind Limbs of Rats.

Summary 1. A method for producing tourniquet shock in rats is described. The method yields graded and reproducible results. 2. The percentage survival of rats to tourniquet shock was progressively decreased as the time of tourniquet application was increased. Following 3% hours of tourniquets 100% of the rats survived, while after 4 hours of tourniquets only 3% survived. 3. Rats which had tourniquets applied for 4 hours with periods of release for 1 to 6 hours, followed by a second application of tourniquets, showed 100% in contrast to 3% survival for those without replacement, while 87.5% survived when the tourniquets were replaced after 7 hours. 4. The degree of hemoconcentration developing in rats in tourniquet shock was approximated by hemoglobin determinations. Increases in hemoglobin ranged between 27 and 52%.

Radioimmunoassay Determinations of Gonadotropic Hormone Content in Different Regions of Male and Female Rat Adenohypophyses

Summary Radioimmunoassay of regions of pituitary tissue suggested a tendency for the gonadotropic hormones to be more localized in the dorsomedial and cephalic regions of the gland. However, these hormones were present throughout the gland and whether the localization in particular regions was of biological significance could not be determined due to variability factors. The 2 hormones were generally located in the same regions of the gland, but evidence was not obtained to clarify whether both FSH and LH activities were present in one cell type.