Sheep Pituitary Extract Research Articles

The combined use of size-exclusion and reversed-phase high-performance liquid chromatography for characterization of β-endorphin processing pathways

A method is described for the separation and analysis of multiple molecular forms of immunoreactive β-endorphin and its α-N-acetylated congenera by a combination of reversed-phase and size-exclusion high-performance liquid chromatography coupled with two specific radioimmunoassays. Both chromatographic procedures are fast ( < 50 min per analysis) providing good resolution and high recovery ( > 90% ). The solvents used in both systems are ultraviolet transparent ( < 214 nm), non-corrosive, low salt ( < 0.05 M) and after evaporation fully compatible with subsequent radioimmunoassay. We have evaluated these techniques using both synthetic and purified peptide standards and have applied these procedures to characterize immunoreactive β-endorphin and α-N-acetylendorphin in rat and sheep pituitary extracts, and the low levels found in sheep hypothalamus and rat ovary. These chromatographic procedures are not only applicable to the study of pro-opiomelanocortin-derived peptides, but also could be employed to examine the processing pathways of other biologically active polypeptides, in both central and peripheral tissue extracts.

Identification of a pituitary factor responsible for enhancement of plasminogen activator activity in breast tumor cells.

Sheep pituitary glands contain a protein that stimulates plasminogen activator (PA) activity 3- to 20-fold in serum-free cultures of T47D, MTW9/PL, and SC115 breast tumor cells. This protein was found to be similar to basic fibroblast growth factor (bFGF) in size, cationic nature, and affinity for heparin. Purified human placental bFGF, a homologue of human and bovine pituitary bFGF, was effective in stimulating mammary tumor cell PA at a concentration of 1 ng/ml. Antibodies to placental bFGF blocked the PA stimulatory activity of sheep pituitary extracts. Because of these properties, the active protein in sheep pituitary glands was identified as bFGF. This represents another function of bFGF, indicating that its spectrum of target cells is wider than previously thought. Because of previously established correlations between PA production in vitro and tumor growth in vivo, we suggest that bFGF may contribute to the growth of breast carcinomas in vivo. Other types of carcinomas may also be affected by bFGF.

Dissociation of the Hypocalcaemic Effect of Sheep Pituitary Extract from its Lipolytic Action

The relation between the calcium-lowering and lipid mobilizing effects of crude sheep pituitary extract given intravenously was investigated in rabbits. Pituitary extract produced hypocalcaemia and free fatty acid elevation in rabbits. The hypocalcaemic effect was not related to the lipolytic effect of the extract, since infusion of dextrose in water prevented the lipolytic effect without altering the hypocalcaemic effect of the pituitary extract.

Comparison of antisera to various gonadotropins as they effect the mouse vaginal cycle.

Antisera to 6 different gonado-tropins were given to young adult female mice and daily vaginal changes followed. Anti-sera to sheep luteinizing hormone virtually halted vaginal cycling. At comparable dose levels antisera to sheep FSH, HCG, and crude sheep pituitary extract failed to have this effect. It was concluded that there had been a cross-reaction between antibodies to the sheep luteinizing hormone and endogenous mouse pituitary hormones.

FERTILIZING LIFE OF FERRET SPERM IN THE FEMALE TRACT.

AbstractFor the determination of the fertilizing life of ferret sperm in the female tract, gonadotrophin was intraperitoneally injected to induce ovulation at various times after the deposition of epididymal sperm into the uterine horns. The animals were examined 48 or 60 hours after ovulation injection to assess the proportion of fertilized eggs, although a few of them were allowed to deliver young.Intraperitoneal injection of 50 hypophysectomized R.U. of sheep pituitary extracts or 90 I.U. of Human Chorionic Gonadotrophin invariably induced ovulation in ferrets that were in heat. Ovulation occurred about 24 to 36 hours after the administration of H.C.G. and an average of 14.5 eggs with a range of 8 to 24 eggs per animal was obtained.The average number of sperm in the cauda epididymides and the vasa deferentia of nine males was 219 millions with a range of 24 millions at the end of breeding season to 621 millions at the peak of breeding season.The percentage of total eggs that were fertilized was 73, 40, 24, 15, 3, and 0 when the ovulation injection was given at 0, 36, 72, 84, 96, and 108 hours after sperm deposition respectively. Since ovulation occurred 30 hours after ovulation injection the fertilizing life of ferret sperm in the female tract is therefore no more than 126 hours. The percentage of eggs fertilized was lower following the deposition of epididymal sperm than following mating (73% vs. 98%). There is evidence that the fertility of sperm in the female tract was higher if a large number of sperm was deposited, and that the penetration of sperm into the egg facilitated the denudation of the egg.The majority of the penetrated eggs (54 to 84%) were at the pronuclear stage 48 hours after insemination and ovulation injection (i.e., about 18 hours after ovulation), at 60 hours (30 hours after ovulation) the majority of them were either at pronuclear stage (26–100%) or at the 2‐cell stage (46–57%), and at 70 hours (40 hours after ovulation) most of them were at the 4–6 cell stage. The morphology of fertilization in the ferret and the time sequence after sperm penetration were described.

Adrenocorticotropin XXVII. On the presence of pig-tyre adrenocorticotropin in sheep pituitaries, and a simple method for the isolation of αs-adrenocorticotropin

A simple, rapid method for the preparation of highly purified ACTH by means of chromatography on CM-cellulose is described. Pig-type ACTH has been isolated from sheep pituitaries and appears to represent about 10% of the ACTH present. Evidence is adduced for the presence in sheep-pituitary extracts of a peptide having the sequence of ACTH but lacking the NH2-terminal hexapeptide and the COOH-terminal amino acid.

Immunological estimation of sheep growth hormone.

SUMMARY An electrophoretically homogeneous component obtained by starch gel electrophoresis of sheep growth hormone (GH) has been used to prepare antiserum in rabbits. By means of a haemagglutination—inhibition reaction, this antiserum was used to assay GH both in sheep pituitary extracts and in sheep sera. The values for the GH content of a number of pituitary extracts obtained by both immunological and biological assay methods were in good agreement. GH levels in sheep serum were found to range between 38 and 600 μg./100 ml.

Effect of sheep pituitary extract on hypercholesterolaemia in intact and hypophysectomized rats.

THE prolonged administration of cortisone or adrenocorticotrophic hormone (ACTH) has sometimes been found to cause a change in the concentration of cholesterol in serum in human beings and in experimental animals. While some workers1,4 report that they have found such an elevation, others have noted no effect7; still others have reported the opposite3.

Continuous zone electrophoresis of pituitary extracts

The application of continuous zone electrophoresis to the fractionation of sheep pituitary extract is described. Pituitary extract was fractionated into 12 fractions. Each contained 3, 4 or 5 components and each component was common to more than one fraction. It is suggested that the formation of protein complexes prevents resolution into mutually exclusive fractions. Preliminary evidence is given on the distribution of hormone activity.

Effect of Antigonadotrophic Serum on Testes of A-Strain Mice Treated with Estrogen

Summary1. Male A-strain mice treated simultaneously with estrogen and normal rabbit serum developed extensive interstitial cell hypertrophy and hyperplasia. Animals receiving an antigonadotrophic serum prepared by injecting a crude sheep pituitary extract into rabbits failed to show the same intensive interstitial cell changes. 2. These results have been interpreted as indicating that the effect of the excessive ICSH, presumed to be elicited from the pituitary gland under the influence of estrogen, has been nullified by the antigonado-trophic serum. The consequent suppression of the interstitial cell hypertrophy and hyperplasia, which normally occurs in this strain of mice when treated with estrogen, provides further evidence that the pituitary gland participates in growth and development of interstitial cell tumors. The possibility that the antiserum exerts an aspecific toxic effect is considered.

Progonadotrophic property of dilute antigonadotrophic serums.

SummaryIt has been shown that the majority of the antigonadotrophic serums of rats and rabbits will augment the effect of a crude sheep pituitary extract upon the uterine weight of immature female test rats when administered in sufficiently small doses. A possible explanation of this phenomenon is discussed.

A study of the origin and distribution of the antigonadotrophic substance in animals chronically treated with crude sheep pituitary extract.

Some few attempts have been made to determine the site of formation and the location of antihormones outside of the blood stream where they are readily demonstrable. Zondek and Sulman (1942) reported that urine and extract of liver, spleen, muscle, ovary, and testis of animals which had received repeated injections of prolan were lacking in antigonadotrophic activity. On the other hand de Fremery and Scheygrond (1941) were able to demonstrate antiprolan in the milk of a pregnant goat which had been protractedly treated with prolan. Gordon, Kleinberg, and Charipper (1939) finding that blockage of the reticuloendothelial system or splenectomy interfered with antihormone formation concluded that the reticuloendothelial system participated in the production of antigonadotrophic substances as would appear to be the case for other antibodies. It is now generally agreed that antihormones are antibody-like in nature (see Thompson, 1941)

STUDIES ON ANTIHORMONE SPECIFICITY WITH PARTICULAR REFERENCE TO GONADOTROPIC THERAPY IN THE FEMALE*

PROTRACTED administration of extracts containing gonad-stimulating hormones will frequently result in the formation of antihormones (1). With the appearance of these hormone inhibitors two choices present themselves: either to discontinue therapy and await the disappearance of the antihormones, which usually is a matter of 2 to 3 months, or to administer a gonadotropin from another source. Whether or not a response to another gonadotropic extract in the presence of antihormones can be anticipated has received little clinical consideration. Animal experiments, however, provide leading information and while these studies are too extensive to review here, a good summary in table form is presented by Zondek and Sulman (2). In general, in the rabbit, pregnant mare serum gonadotropin will form antihormones which are specific in their antagonistic action. Sheep pituitary extracts, on the other hand, elicit antigonadotropins which are nonspecific in that they will antagonize the gonadotropic activity of human, ox, horse, pig, rat and rabbit pituitaries, of human chorionic gonadotropin and of pregnant mare serum. Numerous reports are also listed by Zondek and Sulman (2) to show that human chorionic gonadotropin will form antihormones in the rabbit and that these sera are capable of inhibiting human pituitary activity. It is evident from the animal experiments that the source of the gonadotropic extract is a factor in the problem of antihormone specificity. One might, of course, consider the results as being due to extracts which for the most part were not prepared for clinical use. This does not appear to be a determining factor, however, as it has been shown that the clinically used combination of sheep anterior pituitary extract and human chorionic gonadotropin (Synapoidin) will form antigonadotropins in the rabbit and that the serum will inhibit the gonadotropic activity of pregnant mare serum, human chorionic gonadotropin, human pituitary and rat pituitary (3).

GONADOTROPIC MODIFYING ACTION OF SERA OF ANIMALS TREATED WITH HYPOPHYSEAL EXTRACTS

THE PRESENT communication concludes our study of the effects produced by the chronic treatment of animals with implants or extracts of pituitaries of the same species. In our last publication on this topic (Katzman, Wade and Doisy, 1939), we reported that the sera obtained from sheep during 262 days of treatment with sheep pituitary extract continued to exhibit progonadotropic activity when tested with the same pituitary extract in immature female rats. In no instance during this period did this serum manifest inhibitory activity. Data were also presented showing that the sera of pigs treated for as long as 364 days with pig pituitary extract failed to exhibit consistent augmentatory or inhibitory properties. At this time we wish to present the data which we have obtained from a continuation of the above studies. The animals Sh 2 (ewe), Sh 3 (ram), Sh 4 (ram) and Pg 2 (sow)

Luteinization of the Ovaries of Immature Hypophysectomized Parabiotic Rats with Gonadotrophic Hormone Preparations.

SummaryThe injection of LH, gonadotrophin prepared from the urine of pregnant women, and unfractionated sheep pituitary extracts into the nonhypophysectomized partner produced luteinization in the ovaries of the hypophysectomized partner of either hypophysectomized-gonadectomized or hypophysectomized-normal parabiotic rats.The results indicate that the failure of the ovaries of the hypophysectomized partner of hypophysectomized-gonadectomized pairs to develop corpora lutea is due to an insufficient secretion of LH following gonadectomy rather than to an unresponsive state of the ovaries of the hypophysectomized animal.

PROGONADOTROPIC AND ASPECIFIC EFFECTS OF THE SERUM OF A HORSE IMMUNIZED WITH EXTRACTS OF SHEEP PITUITARY GLANDS1

IN ORDER to obtain adequate amounts of antigonadotropic serum for use in physiologic studies, it was decided to use the horse as a source of this aterial. Soon after the beginning of the experiments to be reported here, Thompson (1937) reported that repeated injections of sheep pituitary extracts for long periods of time resulted in the production in the horse, first, of a gonadotropic-augmenting substance, then a gonadotropic-inhibiting substance. Collip (1937), Rowlands (1938, a, b), and Katsman, Wade, and Doisy (1937, 1939) have shown that injections of homologous pituitary material produce augmenting substances in recipient animals and that inhibitory substances do not appear even after long periods of time. These augmenting substances enhance the gonadotropic activity of concomitantly administered gonadotropins. The above results are of particular immunologic interest since Collip (1937) and Selye, Collip, and Thomson (1934b) had reported that injections of animals with homologous gonadotropins-would...

Purification of Antigonadotropic Sera by Enzymatic Digestion.

It has been shown by Coghill et al..1 that antitoxin in horse serum can be purified to a considerable extent without much loss of activity by digestion with the enzyme Taka-Diastase. Since the antitoxins as well as the antigonadotropic substances produced in the blood of animals over a long period with gonadotropic extracts are found in the globulin fraction of the serum, it was considered probable that the antigonadotropic serum could be purified by the same method. The results of our preliminary work support this view.Antigonadotropic serum of the horse and the goat were used in this study. The horse serum was obtained from a mare pony which had been injected 3 to 6 times a week for 3 years with a crude sheep pituitary extract (SAP). The goat serum was obtained from an adult female goat which had been injected 5 to 6 times a week for 2 years with a purified pregnancy urine preparation (PU).Both sera were digested with Taka-Diastase (obtained from Parke, Davis and Company, Detroit, Michigan) in a manner ...

GONADOTROPINS OF THE SWINE PITUITARY III. IMMUNOLOGICAL SPECIFICITY OF SWINE METAKENTRIN

THE ISOLATION OF a protein believed to be pure metakentrin (ICSH) was described in 1940 (1). In a preceding report (2) the latest method of preparation was described in detail and the results of experiments indicating that hormone activity and the pure protein are inseparable were discussed. Immunological methods offer an additional means of demonstrating the specificity (uniqueness) and purity of the isolated protein. Application of these methods as here described demonstrated that hog metakentrin can readily be distinguished immunologically not only from sheep pituitary extract, sheep metakentrin (ICSH) or beef pituitary extract but also from extracts of hog tissues such as muscle, neural and intermediate lobes of the pituitary and even anterior pituitary extract, purified thylakentrin (FSH). Preparation of source material. The hormone protein used as antigen was isolated from hog pituitary glands, according to the procedure described in the preceding paper (a).

PROGONADOTROPIC SERA OF ANIMALS TREATED WITH HYPOPHYSEAL EXTRACTS

IN 1936 WE (1, 2) reported that the serum obtained from rats which had been treated with chronic implantation of pituitaries of the same species, enhanced the gonadotropic action of such hypohyses when both were ad′ ministered subcutaneously in separate sites to immature female rats. The serum of normal rats did not produce this effect and the experimental serum alone did not induce gonadal stimulation. Augmentative activity was also induced in the serum of rats treated with an extract of rat pituitaries in place of the implants (3) Subsequent to our first report, Collip (4) and Rowlands (5) have reported that the serum of sheep treated with sheep pituitary extract exhibits an enhancing effect on the gonadotropic action of this extract in immature female rats. The latter investigator found, as we had found with the rat serum, that the serum of the treated sheep possessed

Production of Antihormones by Prolonged Administration of Pituitary Extract. Effect on Anterior Hypophysis

A cytological study has been made of the hypophyses of 2 dogs and 2 sheep which had received prolonged injections of extracts of sheep-pituitary glands, and of 2 dogs that had been injected with antihormone-serum. The striking physical changes in one of the dogs, namely, an atrophy of the thyroid, adrenals and gonads, a cessation of growth, and the development of obesity, have been reported elsewhere. The 2 dogs which were injected subcutaneously with the sheep-pituitary extract for 4 and 7 months respectively, developed anti-hormones which inactivated not only the injected pituitary extract, but also several of the hormones of their own hypophyses. The 2 dogs which were injected daily with 10 cc of canine antihormone-serum showed signs of moderate inactivation of the gonads, thyroids and adrenals. Twin ewes were injected with sheep-pituitary extract daily for 6 months. Their genitalia and mammary glands continually showed signs of activation, and at autopsy their gonads and uteri were found to be stimulated. No gonadotropic antihormone developed. A half-sister of these ewes served as a control. The hyophyses of the dogs injected with sheep-pituitary extract. The basophile cells show the most striking abnormalities consisting of: (1) a great increase in the size and number of the basophiles; (2) an excessive clumping and liquefaction of cytoplasmic granules; (3) the formation of many vacuoles of 3 different types: (a) clear round spaces containing a non-stainable substance or one removed by the technic employed; (b) scattered clear pale blue vacuoles which in some of the basophiles coalesced to become extensive vacuolar inclusions; (c) scattered deeply basophilic vacuoles which in some cells displaced the cytoplasm almost entirely.