Prolactin Preparations Research Articles

CONTAMINATION OF AN OVINE PROLACTIN PREPARATION WITH ANTIDIURETIC HORMONE

Department of Physiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72201, U.S.A. (Received 27 April 1976) The administration of various prolactin preparations to mammals has been shown to decrease urine flow (Lockett & Nail, 1965; Horrobin, Lloyd, Lipton, Burstyn, Durkin & Muiruri, 1971) and suppress diuresis induced by expansion of the extracellular fluid volume with saline (Lucci, Bengele & Solomon, 1975). It was assumed in these studies that the antidiuresis was due to prolactin. Antidiuretic hormone (ADH), however, may be present in certain ovine extracts from the pituitary which also could lead to a decrease in urine flow and suppression of diuresis (Rabkin, Swann, Shapiro & Isaacson, 1974). Consequently, we examined the possibility that ADH was present in an ovine prolactin preparation (NIH-P-S10) in quantities sufficient to cause antidiuresis. Experiments were performed on male, Sprague–Dawley rats anaesthetized with pentobarbitone. The rats were placed in the ventral position on

GROWTH HORMONE RADIOLIGAND ASSAY UNRESPONSIVE TO HUMAN PROLACTIN

A specific and sensitive radioligand assay for growth hormone with female rat liver plasma membranes is presented. Growth hormones of different species are able to displace labelled human growth hormone from membrane binding sites with parallel standard curves. Human prolactin V-L-S is not equal to 1 and the international human prolactin standard MRC 71/222 do not affect the assay. Human prolactin preparations with different growth hormone content displace human growth hormone tracer only to the same extent as they contain growth hormone when measured by radioimmunoassay.

Homologous radioimmunoassay for human prolactin

Radioimmunoassays for human prolactin in various biological fluids have been available since 1971. (1–7) Most of these assays have used heterologous systems with prolactin preparations from various sources, i.e. sheep, pig and primate with their corresponding antiserum. (1–5) Friesen et al. (6) find Sinha et al. (7) are the only authors who have described homologous assays using human prolactin as the reference standard and tracer as well and an anti-human prolactin antiserum. In the present study we have established a homologous radioimmunoassay using human pituitary prolactin. The separation method is based on the double antibody solid phase system. (8) Cross reactivity with human growth hormone (HGH), placental lactogen (HPL), the pituitary protein hormones and prolactins of various species were studied as were values found in normal subjects in basal conditions and after a TRH injection. The results of measuring basal levels and performing dynamic tests under various physiological and pathological circumstances will be reported subsequently.

Antidiuretic Action of Prolactin in the Rat with Diabetes Insipidus

Rats with hereditary hypothalamic diabetes insipidus, devoid of endogenous ADH, exhibited a prompt antidiuresis when injected subcutaneously or intraarterially with ovine prolactin. The antidiuresis was accompanied by a decrease in free water clearance and an increase in urine osmolality without a change in osmolal clearance or creatinine excretion. Measurement of PAH and insulin clearances indicated that prolactin had no effect on renal plasma flow or glomerular filtration rate. Prolactin injection caused a transient decrease in urinary sodium excretion, but proximal tubular sodium reabsorption, estimated by lissamine green transit time, was unaffected. The antidiuretic effect of prolactin could not be attributed to ADH contamination of the ovine prolactin preparation. Kidney cyclic AMP content was increased significantly 5 min after injection of prolactin. Thus, prolactin has an antidiuretic effect similar to that which occurs as a result of ADH action on the kidney and does not require either the release or the presence of ADH in order to cause the antidiuresis. Further, the impaired water excretion cannot be attributed to an increase in proximal tubular sodium reabsorption or to alteration of renal hemodynamics. It is suggested that prolactin has a direct ADH-like action on the kidney resulting in antidiuresis.

Purification and properties of an avian prolactin

Prolactin was isolated from chicken adenohypophyses by extraction of the frozen glands with alkaline ethanol and chromatography on Sephadex G100 and DEAE-cellulose. The resulting preparation was homogeneous in disc electrophoresis on polyacrylamide gel and had strong pigeon crop sac stimulating activity. The chicken prolactin had significant mammotrophic activity in pseudopregnant rabbits. Furthermore, a radioactively labeled preparation showed binding to mammary tissue in vivo. The amino acid composition of the purified avian prolactin closely resembles that of mammalian prolactin preparations and the isoelectric point is also similar in both cases.

Seasonal and sexual variations in the responsiveness of rabbit hearts to prolactin.

Rabbit hearts perfused by the Langendorff technique were studied. The addition of ovine prolactin (NIH-P-S-10) to the perfusate in a concentration of 50 ng/ml produced rapid increases in both the amplitude and rate of contraction in 33 adult male hearts studied in winter. Prepubertal male animals showed no response, and only 1 out of 12 adult females responded. Pretreatment for 10 days with 2.5 mg/day testosterone propionate led to minimal inotropic but not chronotropic responses in 2 out of 4 prepubertal males and 2 out of 4 adult females to prolactin. Clear responses to prolactin were seen in 5 adult males pretreated with reserpine. Propanolol consistently reversed both the inotropic and chronotropic actions of prolactin. The original experiments were performed in January and February. When tested in May, adult males failed to respond to prolactin and this situation, persisted until October when responsiveness again appeared. The same prolactin preparation and procedures were used throughout indicating that the changes must have been due to seasonal variations in the cardiac responsiveness to the hormone.

Human and monkey prolactin and growth hormone: separation of polymorphic forms by isoelectric focusing.

The feasibility of using isoelectric focusing for the separation of primate pituitary growth hormone from prolactin and for the characterization of polymorphic forms of these hormones was explored. In a pH 3--10 gradient, extracts of both human and cynomolgus monkey pituitaries were each resolved into 4 growth hormone components and at least 3 prolactin components, as shown by radioimmunoassay. In narrower gradients (of 2--3 pH units) greater resolution was achieved; the principal growth hormone components were well separated from the principal prolactin components but there was overlapping of some minor components. A partially purified human prolactin preparation was found to contain 4 prolactin components, one of which had a prolactin/growth hormone ratio of 760. Clinical grade human growth hormone was also resolved into at least 5 prolactin and 5 growth hormone components, many of which had higher pI values than those found in pituitary extract. Under the conditions used, both growth hormone and prolactin were found to be polymorphic with respect to isoelectric point. Some of the human prolactin components were found to contain less than 0.2% growth hormone by radioimmunoassay. Monkey growth hormone containing 0.01% prolactin was isolated. These findings demonstrate that isoelectric focusing is useful for the preparation of both growth hormone and prolactin which are essentially free of one another. Furthermore, the polymorphic forms were repeatedly found in preparations obtained by several methods and from 2 different species, suggesting that these forms are not artifacts.

Assay of Lactogenic Hormones Using Receptors Isolated from Rabbit Liver

Using 125-I-labelled ovine prolactin and receptors isolated from the livers of rabbits, a sensitive method has been developed suitable for the assay of ovine, bovine, porcine, human and rat prolactins. These hormones showed competitive displacement of 125-I-ovine prolactin which was in general agreement with their respective activities in the pigeon crop sac bioassay. Human and monkey growth hormones and human placental lactogen, which have marked prolactin-like actions on mammary tissue were also effective competitors. Non-primate growth hormones (ovine, bovine, equine and canine) which do not have prolactin-like activity gave little if any displacement as did human FSH, LH, TSH, ACTH and bovine insulin. Preparations of equine and canine prolactin of varying purity gave dose-response curves although their activity as competitors relative to ovine prolactin was poor and not related to their pigeon crop stimulating activity. This indicates species differences between prolactins in hormone-receptor interaction. Experiments with antiserum to human growth hormone have suggested an effective method of making the assay specific in species such as man in which prolactin is not the sole hormone with lactogenic activity.

The purification of human prolactin from frozen pituitary glands.

A method for the purification of human prolactin from frozen pituitary glands is outlined which permits as many as 1,000 glands to be processed at one time, with a final yield of 40–50 μg prolactin per gland. The prolactin obtained is identical in its chemical characteristics and immunological potency to the most highly purified human prolactin preparations currently available.

Radioreceptor Assay for Prolactin and Other Lactogenic Hormones

A radioreceptor assay with a sensitivity of 5 nanograms per milliliter has been developed for mammalian and avian pituitary prolactin, placental lactogenic hormones, and humnan growth hormone, using a membrane receptor preparation isolated from rabbit mammary glands. Prolactin preparations inhibited the binding of [(125)I]prolactin to receptors in direct proportion to the biological potency of these preparations. Thus, the radioreceptor assay provides a convenient and simnple assay for hormones which have lactogenic activity.

Pigeon crop sac stimulation due to a factor produced by plerocercoids of the tapeworm, Spirometra mansonoides.

The activities of a factor produced by plerocercoid (spargana) larvae of the tapeworm, Spirometra mansonoides have been compared to several mammalian hormones. Harlow et al. (1) found that plerocercoids as well as an acetone extract prepared from the plerocercoids exhibited activity similar to that of insulin. Mueller and Reed (2) found that implantation of plerocercoids into propylthiouracil-treated, growth-arrested rats produced a growth response comparable to that of administered thyroxin. Steelman et al. (3) compared the plerocercoid growth factor to growth hormone and Garland et al. (4) found that spirometrid plerocercoids induced sulfation factor (somatomedin) in hypophysectomized (hypox) rats. However, there is no mention in the literature of possible similarities between the plerocercoid factor and prolactin. Bern and Nicoll (5) compiled a list of the actions attributed to prolactin in the vertebrates. These actions include: stimulation of larval growth and reduction of lipid deposition in reptiles, and stimulation of growth and lipogenesis in birds and mammals. Later work (6) has shown that prolactin hypersecretion can produce nearly normal growth in human subjects whose level of growth hormone is below normal. It is obvious that prolactin enhances growth in several groups of vertebrates. One of the most striking effects of prolactin is seen in the Order Columbiformes. Prolactin preparations from many vertebrate species have the ability to stimulate the crop sac of pigeons and doves to produce crop “milk” (5). Crop sac stimulation has subsequently been used as a definitive bioassay for prolactin. In addition, somatotropin has lactogenic activity (7, 8) and exhibits other actions similar to those attributed to prolactin (5). The objectives of the experiments described in this paper were to determine: (a) if plerocercoid growth factor (PGF) will stimulate the production of pigeon crop “milk” and (b) if the projected “prolactinlike” activity of PGF on the pigeon crop sac might be used as an assay system for PGF activity.

A summary of the biological activities of the various lots of pituitary hormones produced under the programs of the National Institute of Arthritis, Metabolism and Digestive Diseases.

BEGINNING in late 1956 with the distribution of commercial preparations of bovine growth hormone and ovine prolactin, workers in endocrine research the world over have been given many lots of pituitary hormone preparations, which now include ovine and bovine growth hormone, prolactin, luteinizing and thyroid—stimulating hormones, and ovine and porcine follicle—stimulating hormone. This program, originally supported by several of the National Institutes of Health, has been supported since 1959 by a single grant from the National Institute of Arthritis, Metabolism and Digestive Diseases.

Radioimmunoassay of rat prolactin comparing prolactin obtained from anterior pituitary organ culture with that distributed by the national institute of arthritis and metabolic diseases

Rat prolactin was purified by preparative disc gel electrophoresis from anterior pituitary organ culture medium. Rabbit antiserum to rat prolactin was absorbed with serum from hypphysectomized rats to increase its specificity. A double antibody radioimmunoassay was developed using the above hormone and its antiserum, and also using the rat prolaction and antiserum distributed by National Institute of Arthritis and Metabolic Diseases. Standard curves over the range of 0.1 to 20 ng/ml were established with various combinations of rat prolactin and its antisera from the two different sources. When the two prolactin preparations were used as reference standards in an assay utilizing the same labeled prolactin and antiserum, the standard curves were paralled to each other. However, prolactin obtained from National Institute of Arthritis and Metabolic Diseases was bound to the antiserum to a greater extent that was the prolactin obtained from culture medium, indicating a difference in specific immunoreactivity between the two preparations. Thus, for an unknown sample the standard curve using prolactin from organ cultures gave a value 2 to 3 timmes higher than the standard curve using the National Institute of Arthritis and Metabolic Diseases prolactin. Rat growth hormone, follicle stimulating hormone and luteinizing hormone up to 100 ng/ml did not interfere with the assay, indicating the specificity of the system. Measured with a radioimmunoassay system using the National Institute of Arthritis and Metabolic Disease prolactin as the standard, and the antiserum to prolactin in the medium, the following prolactin levels (ng/ml) were obtained: hypophysectomized, 9 ± 1; diestrus, 29 ± 7; proestrus, 70 ± 25; fro 6 h were probably due to the lack of ovarian estrogens. Low prolactin levels in non-suckled lactating rat have also been reported by other workers 6,16. The dramatic 13-to 14-fold increase following suckling for 30 min was in good agreement with the results of Amenomori et al. 6 and Amenomori and Meites 16. The rapid effect of the suckling stimulus on elevating serum prolactin levels has also been demonstrated in the cow and goat 17. An increase in serum prolactin content of pituitary-grafted ovariectomized rats was related to the number of grafts and confirmed similar observation by Chen et al. 18 in hypophysectomized-ovariectomized animals. The known effect of estrogen in stimulating prolaction secretion 5 was also observed in the present study.

THE EFFECT OF PROLACTIN AND GROWTH HORMONE PREPARATIONS ON THE CASEIN PRODUCTION IN MOUSE MAMMARY GLAND TISSUE EXPLANTS

The enhanced casein production in mammary gland explants from pregnant mice, measured by 32P incorporation, has been described as a suitable assay for prolactin. The specificity of this system was tested by immunoassay of casein after exposure of the explants to various preparations of prolactin and growth hormone.

The response of the cutaneous glands to steroids

The subject of this paper is the way that the activity of skin glands, both sebaceous and apocrine, is controlled by steroid hormones, and how, with respect to the sebaceous glands of the rat at least, this control is mediated in situ by pituitary hormones. In the immature or castrated rat, as in many other species including man, the sebaceous glands are stimulated by androgens and inhibited by estrogens. After hypophysectomy, however, the effect of testosterone is insignificant, though the effects of androstenedione and of 5α-dihydrotestosterone remain substantial. The response to testosterone has been restored by preparations of growth hormone, prolactin or thyrotropic hormone. Steroids are converted in the skin, and it is suggested that a pituitary hormone may mediate the response of the sebaceous glands by influencing this conversion or by affecting the balance between free and conjugated testosterone. The three pairs of specialized apocrine glands in the rabbit all resemble the sebaceous glands in their responses to testosterone and estradiol. These glands, like many other cutaneous glands in different mammals, produce pheromones which are concerned with establishing dominance in territory (chin and anal glands) or act as sex attractants (inguinal glands). The production of pheromones, some of which may themselves be steroids, could be biologically significant for steroid transformations in skin.

Application of disc electrophoresis for production of antiserum to rat prolactin

For the production of the antiserum to prolactin, especially of the mouse or the rat, a large amount of normal or tumorous anterior pituitaries is necessary for prolactin preparation as antigen. Studies on analytical disc electrophoresis have found that the rat anterior pituitary (RAP) contains much larger amount of prolactin and growth hormone than other hormones.The present paper deals with a trial for the production of the antiserum to rat prolactin (RPL) by administering RPL segment of polyacrylamide gel after electrophoresis instead of extracted RPL preparation.

Some immunological properties of rabbit antiserum to mouse growth hormone.

As a fundamental experiment for the study on the role of growth hormone in mouse mammary gland, some immunological properties of rabbit antiserum to mouse growth hormone (MGH) were studied.Antiserum was obtained by immunizing New Zealand White male rabbits with MGH preparation extracted from mouse anterior pituitaries. The precipitin titer of the antiserum by precipitin ring test against MGH preparation was 1: 16. The antiserum did not react with mouse serum, and extracts of brain, liver, kidney, spleen and heart. The immunoelectrophoretic pattern of polyacrylamide gel after electrophoresis of anterior pituitary with the antiserum or the reaction of antiserum with MGH and mouse prolactin (MPL) preparations on the Ouchterlony agar plate showed two precipitin arcs or lines; one appeared to be due to MGH and another due to MPL. The absorption of the antiserum with MPL preparation resulted in the development of a single precipitin line between the absorbed antiserum and MGH preparation on the Ouchterlony agar plate. Further, immunoelectrophoretic pattern of polyacrylamide gel after electrophoresis of mouse pituitary homogenate with the absorbed antiserum showed one precipitin arc at the corresponding site of MGH band. Inhibition by the antiserum of the biological activity of MGH was proven in rat tibia test and in body and mammary growth of a mouse.All the results have demonstrated that the antiserum absorbed with MPL preparation is specific only to MGH.

Radioimmunoassay of rat prolactin: Comparison of rat prolactin preparations isolated from the ‘granular fraction’ of pituitary tumour transplants and from normal pituitary glands

The performance in various procedures for radioimmunoassay of a rat prolactin preparation isolated from the ‘granular fraction’ of transplants of prolactin-producing pituitary tumours is compared to that of a prolactin preparation isolated from pooled frozen normal pituitary glands. Antisera titration curves and competitive inhibition curves indicate that the two preparations resemble each other closely. Analysis of covariance of competitive inhibition curves indicates that the small differences observed between the two preparations in recent assays can be ascribed to experimental error. Not withstanding a marked difference in biological activity between the two preparations as measured by the local pigeon crop assay, the reported experiments indicate that radioimmunoassays based on either of the two preparations will give approximately the same values when the prolactin estimates in biological material are expressed in unit weight aequivalents of the preparation employed. Expression in unit weight aequivalents should therefore be preferred to an extrapolation from these to i.u. of biological prolactin activity.

The influence of pituitary hormones on the response of the sebaceous glands of the male rat to testosterone.

SUMMARY Testosterone implants releasing about 0·2 mg./day had no effect on sebum production in hypophysectomized-castrated rats, even though treatment by a similar dose of testosterone has been shown to effect a marked increase in sebaceous activity in rats with intact pituitaries. A preparation of porcine growth hormone, and a preparation of prolactin (luteotrophin) which had no growth hormone activity, were each capable of fully restoring the response of the sebaceous gland in hypophysectomized male rats to testosterone, though the pituitary hormones had no effect by themselves. The stimulation of sebaceous activity was measured both by the increase in sebum production and by the increased incidence of cell division in the sebaceous glands. The responses of the preputial glands and seminal vesicles to testosterone were independent of the presence of the pituitary, and were not significantly enhanced by the hypophysial hormones.

Quantitative analysis of prolactin by disc electrophoresis and its relation to biological activity.

SummaryThe quantitative analysis of prolactin was investigated by disc electrophoresis. A linear relationship was found between the amount of standard prolactin preparation used and the optical density of the band obtained, indicating that this method can be employed for the quantitative determination of prolactin. One-2 mg of fresh pituitary from mature female rats was sufficient for clear detection of prolactin. A high correlation was obtained between the optical density and the biological activity of the eluate of the band. Chronic administration of large doses of prolactin to rats decreased the optical density of the prolactin band to 59% of that in the control pituitary. It was also demonstrated that prolactin released in vitro from the anterior pituitary of rats could be detected by disc electrophoresis.