Isolation of the Luteinizing Hormone and Follicle-stimulating Hormone-releasing Hormone from Porcine Hypothalami ”

A new procedure is described for the isolation on a preparative scale of a hypothalamic decapeptide which stimulates the release of both luteiaizing hormone (LH) and folliclestimulating hormone (FSH) from the pituitary. The isolation was achieved mainly by countercurrent distribution. The yield from 240,000 porcine hypothalami was 11.4 mg. Thin layer chromatography and electrophoresis showed the material to be homogeneous. The amino acid analyses showed the presence of 2 residues of glycine and 1 residue each of histidine, arginine, tryptophan, serine, glutamic acid, proline, leucine, and tyrosine. This decapeptide stimulates release of both LH and FSH in vivo as well as in vitro in doses smaller than 1 ng. It is thought to be the hypothalamic hormone which controls secretion of both LH and FSH from the pituitary gland.

SUMMARY A new procedure is described for the isolation on a preparative scale of a hypothalamic decapeptide which stimulates the release of both luteiaizing hormone (LH) and folliclestimulating hormone (FSH) from the pituitary. The isolation was achieved mainly by countercurrent distribution. The yield from 240,000 porcine hypothalami was 11.4 mg. Thin layer chromatography and electrophoresis showed the material to be homogeneous. The amino acid analyses showed the presence of 2 residues of glycine and 1 residue each of histidine, arginine, tryptophan, serine, glutamic acid, proline, leucine, and tyrosine.
This decapeptide stimulates release of both LH and FSH in vivo as well as in vitro in doses smaller than 1 ng. It is thought to be the hypothalamic hormone which controls secretion of both LH and FSH from the pituitary gland.
The release of luteinizing hormone and follicle-stimulating hormone from the anterior pituitary gland is controlled by the hypothalamus (1, 2). Initially, it was thought that two distinct, hypothalamic hormones LH1 releasing hormone and FSH releasing hormone were responsible for stimulating the release of LH and FSH, respectively (2). Recent work in our laboratory (3-7) clearly established that one hypothalamic hormone designated provisionally LH-RH/FSH-RH stimulates secretion of bot,h LH and FSH in laboratory and domestic animals and man. This paper describes a new isolation procedure for LH-RH/ FSH-RH which is much superior to one reported previously (4). EXPERIMENTAL  The lyophilized hypothalami were shipped to our laboratory, where they were pulverized on Dry Ice, defatted with acetone and petroleum ether (40-60"), and extracted with 2 N acetic acid at 8". Usually batches of 20,000 to 50,000 hypothalami were extracted five times with the use of 3 liters of 2 N acetic acid for each extract. All the det,ails of this extraction have been described (8). The extracts were heated to boiling and lyophilized.
Gel Filtration on Sephadex-Since t,he glacial acetic acid reextraction (4, 8) of lyophilized extracts did not lead to significant concentration of activity in the case of this particular batch, this step was omitted.
The lyophilized 2 N acetic acid extracts were suspended in 1 N acetic acid, centrifuged at 30,000 x g, and subjected to molecular sieving on a large column of Sephadex G-25 (fine) (15.5 x 180 cm) (8). The separation pattern was followed by the Folin-Lowry reaction (9) or by optical density readings at 278 nm. Phenol extraction and chromatography on CM-cellulose were described previously (8).
Countercurrent Distribution-Countercurrent distribution was carried out in an automatic glass apparatus (H. 0. Post Scientific Company, New York).
Two models were utilized-C-2 which has 100 cells with 50-ml capacity in each phase and A-4 which has 400 cells, with a 3-ml capacity in the lower phase and up to 5 ml in the upper phase. Certain modifications introduced in the automatic robot of model C-2 to allow for an adequate draining of the viscous upper phase were reported recently (10). Analytical grade pyridine was redistilled over sodium hydroxide pellets.
I-Butanol (J. T. Baker Chemical Company) was treated wit'h zinc and redistilled.
After distribution, the materials were recovered as described previously (8, 10). Reagent glacial acetic acid and ammonium hydroxide used in solvents and buffers were also redistilled.
The water in all t'he experiments was triple distilled in glass.
Homogeneity and Composition Tests-Thin layer chromatography and electrophoresis were carried out in plates coated with a 250~pm layer of cellulose MN 300 HR (Nacherey, Nagel, and Company, Duren, West Germany) or Avicel microcrystalline cellulose (Brinkmann Instruments, Inc., Westbury, New York). DeSaga-Brinkmann equipment was used for the separations. tion of release of LH in ovariectomized rats previously treated with estrogen and progesterone (13). The rats (three to five per group) were anesthetized with 150 mg of urethane per 100 g of body weight intraperitoneally 1 hour before injection of the samples. Each sample was dissolved in 0.5 ml of acidified saline (0.9% NaCl containing 0.01 M acetic acid) and injected into the jugular vein. Twenty minut.es after the injection, blood was collect,ed from the abdominal aorta and kept at 4" overnight.
The serum was then separated by centrifugation and assayed for LH by the double antibody radioimmunoassay for rat LH as described by Niswender el al. (14). Each sample was measured at least in triplicate.
LH concentration was expressed in terms of NIH-LH-S14, which was used to construct the standard curve for LH in the radioimmunoassay.
The increase of serum LH levels after injection of samples as compared to that after saline injection was used as the index of LH-RH activity. FSH-RH activity was measured by a specific assay based on stimulation of FSH release in vitro by pituitaries of male rats (15). FSH released into the incubation medium was measured by the method of Steelman and Pohley (16). The responses are given in terms of ovarian weight since the response is linear with respect to dosage over a certain range. Five to eight assay rats were used per group. NIH-FSH-S8 was used as the reference standard.
The significance of differences between groups was calculated by the new Duncan's multiple range test (17). In some experiments the FSH released was also measured by radioimmunoassay for rat FSH (18). The location of the LH-RH/ FSH-RH activity on the chromatograms or countercurrent distribution curves is based on assays carried out on a large number of fractions with the use of one single dose level of the liquid aliquot.
The recovery figures for LH-RH activity after each step were obtained from assays which employed two or more dose levels of the lyophilized LH-RH fraction as well as two or three doses of a preparation of highly purified LH-RI-I/ FSH-RH (4) as a standard.
The recoveries of FSH-RH activity were also computed by comparing the responses of active fractions versus those given by the standards, but are much less accurate, as in some cases only one dose of the unknown was utilized.
The doses are expressed in terms of dry weight. Pressor assays were performed as described by Dekanski (19). RESULTS

AND COMMENTS
When 240,000 lyophilized pig hypothalami weighing 5.74 kg were pulverized and defatted, the weight was reduced to 5.3 kg (Table I). This hypothalamic powder was extracted five times with 2 N acetic acid in batches equivalent to 30,000 to 45,000 hypothalami and lyophilized, yielding extracts of 2 kg. The residue from the extraction had no LH-RH activity in doses up to 500 pg. The extract was subjected to gel filtration on Sephadex G-25 in amounts of 50 t'o 100 g, which corresponded to 6,000 to 12,000 hypothalami.
The location of LH-RH activity with respect to vasopressin is shown in Fig. 1  Preparative countercurrent distribution of LH-RH concentrate in a system of 0.1% acetic acid-1-butanol-pyridine, 11:5:3, by the single withdrawal method.
Phenol extract (179.9 g) was extracted with the distribution solvent (1 liter of lower phase and 500 ml of upper phase) and 171.3 g of material which dissolved was loaded in tubes 0 to 19. One hundred cell train was filled with 50 ml of lower phase and 25 ml of upper phase. Two hundred fifty transfers were performed.
Folin-Lowry analyses were carried out on 10 d of lower phase (L) and 25 ~1 of upper phase (U). LH-RH activity was determined on l-p1 aliquots of upper phase, equivalent to approximately 0.8 rg dry weight. From Schally, Nair, and Carter (10). was in the same fractions as LH-RH.
Growth hormone-releasing activity was found in Fractions 757 to 856. Owing to the preparative nature of these separations, the LH-RH activity was spread over a large number of fractions.
However, Fractions 1357 to 1700 contained only about 8% of the total LH-RH activity applied and Fractions 457 to 856 were inactive.
Fractions 857 to 1156 were now active in doses of 50 pg per rat in LH-RH assays and at 100 pg per ml in FSH-RH assays. The recovery of LH-RH activity from Sephadex was about 100% within the error of the assay, but only 92% of the total LH-RH activity applied, corresponding to Fractions 857 to 1356, was used for further purification.
The yield of these LH-RH/ FSH-RH active fractions from 24 Sephadex columns was 731 g.

Extra&on
with Phenol-This material was extracted with 4 liters of redistilled phenol.
The LH-RH/FSH-RH active fractions were recovered from phenol by re-extraction into the aqueous phase after the addition of 35 liters of redistilled diethyl ether (8). The phenol extract (yield, 179.9 g) was active in the LH-RH assay in doses of 10 to 50 1.18, whereas the residue (496.8 g) was inactive in doses of 500 to 1000 pg (Table II).
FSH-RH activity in the same fractions was detectable in doses of 20 pg per ml. The recovery of LH-RH/FSH-RH in this step, as based on the assays for LH-RH activity, was 94%.
Countercurrent Distribution I-The LH-RH/FSH-RH activity in the phenol extract was further purified by countercurrent distribution in the system of 0.1 y. acetic acid-1-butanol-pyridine (11:5:3).
The details of this type of separation were reported previously (10). The single withdrawal method was used, and, after 250 transfers, LH-RH/FSH-RH activity was located in Fractions 130 to 230 (Fig. 2). In spite of very large amounts of material used, the mean K value for LH-RH/FSH-RH, determined to be 2.0 in analytical runs in this solvent system, did not, change in the present run. Lysine vasopressin was found in Fractions 60 to 99 (mean K = 0.76). The dry weight of combined LH-RH active fractions was 2.7 g. This material showed significant LH-RH activity in doses of 0.2 pg per rat and FSH-RH activity in amounts of 0.5 pg per ml. Other fractions were inactive in doses up to 20 pg. Since the recovery of biological activity was nearly quantitative (99%, see Table I), the purification factor of over 60 resulted in this run.
CM-cellulose Chromatography-The LH-RH/FSH-RH active material from countercurrent distribution I (2.7 g) was subjected to ion exchange chromatography on a CM-cellulose column. The location of LH-RH activity is shown in Fig. 3. After lyophilization of Fractions 191 to 229, 138 mg of material were obtained, which was active in LH-RH assays in doses of 5 ng and released FSH in vitro at levels of 10 ng per ml. Fractions 10 to 190 and 230 to 404 were inactive in doses 4 to 15 times larger than those utilized for LH-RH.
The purification factor was 19 to 20, and the combined purification factor from countercurrent distribution I and CM-cellulose was about 1200. The recovery of LH-RH activity in this step was again essentially quantitative (Table I).
Countercurrent Distribution II-The material from carboxymethylcellulose (138 mg) was repurified by countercurrent distribution in the A-4 apparatus.
The solvent system utilized was the same as that in distribution I: 0.1 y. acetic acid-butanolpyridine (11:5:3).
After the first 400 transfers were carried out, analyses (9) of the first 250 cells did not reveal any peptide materials.
Consequently, the countercurrent distribution was continued by recycling for an additional 400 transfers.
The pattern of distribution of peptide materials and the location of the LH-RH activity are shown in Fig. 4. The partition coefficient K = 2.06 was again in good agreement with previous work (4). Fractions 575 to 599 and 655 to 674 contained 3% of the total LH-RH activity and were not combined with the main LH-RH Fractions 600 to 654. After lyophilization of tubes 600 to 654, 55.4 mg of material were obtained, which released LH in viva in doses of 2 ng and FSH in vitro at levels of 4 ng per ml. The recovery of LH-RH activity in this step, calculated on the basis of one assay, was about 78%, but the actual recovery might have been higher.
The purification factor was only 2.5. This can be explained by the fact that the same solvent system was used in countercurrent distribution I. Countercurrent Distribution III-The final purification step consisted of countercurrent distribution for 900 transfers in the system of l-butanol-acetic acid-water (4: 1:5) in the A-4 apparatus. Instead of recycling, fresh upper phase solvent was continually introduced by the automatic filling device. The emerging fractions were collected by a fraction collector.

Issue of
The peptide profiles and the location of LH-RH/FSH-RH activity are shown in Fig. 5. Folin-Lowry (9) analyses of lOO+l aliquots of upper phase of Fractions 400 to 900 indicated that very little peptide material was present in these fractions.
The peptide peak with LH-RH/FSH-RH activity had a partition coefficient K = 0.22 and was well separated from other peaks. After lyophilization of contents of cells 215 to 269, 11.5 mg of material were obtained.
The purification factor in this final step was 5, and the recovery was about 98%. The over-all recovery of LH-RH activity from the 2 N acetic acid extract Lower phase was 3 ml and upper The number of transfers was 900. The solvents were mixed and equilibrated 20 hours before countercurrent distribution. Folin-Lowry analyses were done on loo-p1 aliquots of lower phase. Assays for location of LH-RH activity were carried out on 2 ~1 of lower phase per rat.
after six purification steps was 64%. The recovery of FSH-RH activity was about the same. In six steps which followed the acetic acid extraction, the LH-RH/FSH-RH activity was purified about 200,000 times. The over-all purification was about 500,000 times.
Biological Activity of LH-RHIFSH-RH-LH-RH assays in &JO showed that this material released LH in doses as small as 0.25 ng per rat (Table III).
Larger doses caused up to 20-fold elevation of plasma LH.
Significant stimulation of FSH and LH release in vivo was seen after administration of LH-RH/ FSH-RH into immature male rats or castrated male rats previously treated with testosterone (4, 7). The same material also released FSH and LH in vitro in doses as small as 0.5 ng per ml (Table IV).
Since the amino acid analyses of this material (see below) were in good agreement with those of a small batch of LH-RH/FSH-RH preparation, (12). obtained after 12 different purification steps (4), further homogeneity tests were deemed unnecessary. mic acid, proline, leucine, and tyrosine (Table V). The amino

Micromoles
Amino acid analysis after acid hydrolysis in the presence of acids accounted for 67.5% of the dry weight of LH-RH/FSH-RH thioglycollic acid (12) showed the presence of 2 moles of glycine preparation.
As in the case of thyrotropin releasing hormone, and 1 mole each of histidine, arginine, tryptophan, serine, gluta-other impurities such as bound acetate, ash, and carbohydrate from the columns did not introduce problems during convenz Carried out by Dr. H. Matsuo in connection with synthesis of tional degradation procedures or mass spectroscopic studies. LH-RH.
The failure to detect NHz-terminal amino acid by the dansyl