Mouse Prolactin Research Articles

Interaction of mouse prolactin with mouse hepatic receptors

Lactogenic receptors are usually studied in heterologous systems where prolactin is derived from one species and receptors prepared from another. In such systems the foreign prolactin could be seen as a growth hormone by the host tissue. We have therefore developed a homologous radioreceptor assay using secreted mouse prolactin (smPRL) and mouse hepatic receptors. In this system, monovalent anions augment the smPRL-receptor interaction in the order F − > Cl − > Br − > I −. Divalent cations (Mg 2+, Ca 2+, Sr 2+), phosphate and acetate also increase smPRL binding. Temperature and pH optima are at 8°C and pH 8.3, respectively. Under optimum conditions, the percent total, specific and nonspecific binding are 55%, 45% and 10%, respectively. At infinite receptor concentration the maximum specific bindability of labeled smPRL is 50%. The effects of ions on binding of smPRL to the receptor show that hydrophobic forces participate in smPRL-receptor coupling. The biphasic dissociation kinetics show initial and final rate constants of 1.56 × 10 −4/s and 7.62 × 10 −6/s, respectively. The lactogenic receptor does not bind mouse growth hormone; however, it binds both mouse placental lactogen (mPL) and smPRL with equilibrium association constants of 3.90 × 10 8 M −1 and 2.25 × 10 8 M −1, respectively, suggesting that smPRL and mPL share biological roles by acting through the same receptor.

Nucleotide sequence of a growth-related mRNA encoding a member of the prolactin-growth hormone family.

As part of the proliferative response to serum, mouse 3T3 cells produce a set of growth-related mRNAs identified by hybridization to cloned cDNAs. One of these mRNAs, which is about 1 kilobase long, appears within a few hours after stimulation of resting cells with serum or platelet-derived growth factor and reaches a high level during the transition from the G1 to the S phase of growth. This mRNA is translated in vitro into a protein of approximately 25 kilodaltons. The corresponding cloned cDNA of 791 base pairs has been sequenced; it contains a single open reading frame that encodes a protein of 224 amino acids with extensive sequence homology to mammalian prolactins. The initial 29-amino acid segment of the encoded protein resembles the signal sequences of prehormones. That the growth-related protein is not mouse prolactin is indicated by comparison of its predicted amino acid composition with that of mouse prolactin and by the distinct fragment patterns seen when restricted mouse DNA is probed with the cloned cDNA or rat prolactin cDNA. Therefore, the growth-related protein appears to be a new member of the prolactin-growth hormone family. Because of its relationship to prolactin and growth hormone and its association with cell proliferation, the protein has been called "proliferin."

Complete amino acid sequence of mouse prolactin

Complete amino acid sequence of mouse prolactin

Electrophoretic separation of prolactin secreted from mouse pituitary glands in vitro

Prolactin secreted from the mouse pituitary gland in organ culture was characterized after disc or SDS-polyacrylamide gel electrophoresis. Some pituitary glands were cultured with 3H-labelled leucine for 24 h to obtain radioactive prolactin. In disc electrophoresis, immunoreactive, receptor-bindable and 3H-incorporated prolactins formed a single band with the same relative mobility (0.5). Prolactin migrating at r f = 0.5 was extracted and re-analysed by SDS electrophoresis. A single stained and radioactive band was observed at the same position with an apparent molecular weight of 23 000 regardless of denaturing in the presence of absence of dithiothreitol. No other band was detected. These results indicate that mouse prolactin synthesized and secreted in organ culture is homogeneous and that mouse prolactin is a single-chain molecule.

Development and characterization of a homologous radioimmunoassay for deer mouse ( [formula omitted][formula omitted][formula omitted]) prolactin

A highly specific and sensitive homologous radioimmunoassay has been developed for the secreted form of prolactin from the deer mouse Peromyscus maniculatus bairdii . Peromyscus serum and pituitary homogenates displayed parallel dilution response curves, and no cross reaction was seen with either mouse prolactin, mouse growth hormone or rat prolactin. The assay was sensitive to 25 picograms per tube and the intra- and inter-assay coefficients of variation were 5 and 3.6%, respectively. In addition, we have demonstrated that Peromyscus prolactin does not show parallel displacement in a homologous radioimmunoassay utilized for measuring prolactin in the common laboratory mouse.

Isolation, purification, and characterization of deer mouse ( Peromyscus maniculatus bairdii) prolactin

Prolactin (PRL) secreted by Peromyscus maniculatus bairdii anterior pituitaries was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. Peromyscus PRL (pmPRL) eluted from Sephadex G-100 with an elution-tovoid volume ratio of 1.9 and at a salt concentration of 100 m m NaCl on DEAE-cellulose. Electrophoretic homogeneity of the hormone was demonstrated in several gel systems. On 7 1 2 % alkaline polyacrylamide gels, pmPRL migrated with an R f of 0.65. The molecular weight of pmPRL was estimated at 24,000 to 26,000 by sodium dodecyl sulfate electrophoresis and molecular exclusion chromatography. The amino acid composition of pmPRL was similar to other mammalian PRLs including two tryptophan residues and three disulfide bonds. Leucine was found to be the NH 2-terminal residue. Circular dichroism (CD) spectra indicated an α-helix content of 55 ± 5%, a value typical for prolactin molecules. However, in the region of side-chain absorption, the CD spectrum displayed a weak, asymmetric, negative band near 299 nm, with the CD returning to slightly positive values at 292 nm. This CD pattern is not typical of other secreted or stored prolactins. In the pigeon crop-sac assay, pmPRL showed a prolactin-like activity of 21 IU/mg.

Conformational studies of secreted mouse pituitary prolactin.

A secreted form of mouse pituitary prolactin has been shown to contain only a single tryptophan residue. All previously reported prolactins contain two tryptophans. Circular dichroism spectra indicate that secreted mouse prolactin is conformationally similar to stored forms of prolactin previously isolated from several other species, including its alpha-helix content (65%). However, like secreted rat prolactin, secreted mouse prolactin shows no tryptophan signal in its circular dichroism spectrum. All stored forms of prolactin studied to date display distinct tryptophan signals. This suggests the possibility that secretion of prolactin may be accompanied by modification of the protein's tertiary structure.

The development and characterization of a homologous radioimmunoassay for mouse placental lactogen.

A highly specific and sensitive homologous radioimmunoassay (RIA) has been developed for mouse placental lactogen(mPL). Mouse pregnancy serum and placental extracts showed parallel dilution-response curves, and no significant cross-reaction was seen with either mouse growth hormone or mouse prolactin. The sensitivity of the assay ranged from 0.5 to 1.0 ng/ml, and the intra- and inter-assay coefficients of variation were 5 and 11%, respectively. By RIA, mPL levels were detectable on day 9 of pregnancy (1 ng/ml) and increased until day 14 (100-150 ng/ml) in both C3H and BALB/c strains. On days 14-18 of pregnancy, mPL levels were maintained (95-125 ng/ml) in C3H mice, while they continued to increase (greater than 250 ng/ml) in BALB/c mice. In both strains, a midpregnancy peak in prolactin-like activity was detected by a radioreceptor assay but not by the mPL RIA.

Isolation, purification, and characterization of mouse placental lactogen.

Mouse placental lactogen was purified 1840-fold from BALB/c placentae from days 14-18 of gestation with an overall yield of 29%. The purification procedure included alkaline homogenization and extraction, ammonium sulfate precipitation, hydrophobic interaction chromatography on Phenyl-Sepharose, ion-exchange chromatography on CM- and DEAE-cellulose, and gel exclusion chromatography on Sephadex G-100. On 10% alkaline polyacrylamide gels, mouse placental lactogen had an Rf of 0.19. Electrophoresis in gels containing NaDodSO4 showed a single band with a mobility corresponding to a Mr of 23,000 +/- 1000. The isoelectric point, determined by isoelectric focusing in 8 M urea/5% 2-mercaptoethanol, was 7.1. When tested in the pigeon crop sac assay, 10 micrograms of mouse placental lactogen produced stimulation comparable with that evoked by 10 micrograms of ovine prolactin. In the rabbit mammary gland radioreceptor assay, mouse placental lactogen was 150% more potent than ovine prolactin in displacing 125I-labeled ovine prolactin from rabbit mammary gland membranes. Iodinated purified mouse placental lactogen could be displaced from rabbit mammary gland membranes by mouse placental lactogen, mouse prolactin, and ovine prolactin. Ovine prolactin was 45% as avid as mouse placental lactogen is displacing 125I-labeled mouse placental lactogen from rabbit mammary gland membranes. Mouse placental lactogen did not crossreact with antisera to mouse prolactin or mouse growth hormone in a radioimmunoassay.

The amino acid composition of secreted mouse prolactin, growth hormone, and hamster prolactin: The presence of one tryptophan in mouse prolactin

The amino acid compositions and the electrophoretic properties of secreted mouse prolactin (mPRL), mouse growth hormone (mGH), and hamster prolactin (haPRL) were determined. The amino acid compositions of secreted mPRL and haPRL were similar to the compositions of other rodent prolactins, except that secreted mPRL contained only one tryptophan residue rather than the usual two. The composition of secreted mGH was similar to that of rat growth hormone. On 10% alkaline polyacrylamide gels, mPRL, mGH, and haPRL migrated with R f 's of 0.54, 0.21, and 0.69, respectively. The molecular weights of mPRL, mGH, and haPRL, determined by SDS-gel electrophoresis, were 23,000, 21,000, and 22,000, respectively.

Homologous radioimmunoassay for secreted mouse prolactin

A highly specific, homologous radioimmunoassay has been developed for the secreted form of mouse prolactin using hormone isolated and purified from the media from long-term pituitary cultures. Mouse pituitary homogenates and mouse serum give parallel dilution response curves in the assay, and no cross-reaction is seen with either mouse growth hormone or mouse placental lactogen. The assay is sensitive to 40 pg per tube, and the reproducibility and precision of the assay are within acceptable limits. Antiserum generated to secreted mouse prolactin cross-reacts with both the secreted and stored forms of the hormone; however, the secreted form shows greater immunopotency. Secreted prolactin also shows greater immunopotency when compared with stored prolactin using an antiserum generated to the stored form of the hormone.

The lactogenic response of mouse mammary explants to mose prolactin and growth hormone.

The lactogenic response of mouse mammary explants to mouse prolactin (mPRL) and mouse growth hormone (mGH) was studied. Mouse mammary explants were cultured, in a defined medium, in the presence of varying concentrations of either mPRL or mGH and the rate of 3H amino acid incorporation into casein was measured. The lactogenic activity of mPRL was compared with that of ovine prolactin (oPRL) and found to be directly comparable. MGH, when compared to oPRL, was found to be less active in stimulating casein synthesis.

Metabolism of prolactin in mice with a high incidence of mammary tumours: evidence for greater conversion into a non-immunoassayable form.

Monomeric mouse prolactin containing small amounts of 125I-labelled prolactin was administered to adult female mice of a high (C3H/St) and low (C57BL/St) mammary tumour strain. Their endogenous prolactin had been suppressed with 2-bromo-alpha-ergocryptine. The chromatographic profile, on Sephadex G-100, of prolactin in the serum of mice injected with mouse prolactin was compared by direct measurement (radioactivity count) and by radioimmunoassay (RIA) at several intervals after injection. With both methods, the injected hormone was found in the serum in predominantly two molecular sizes, the so-called 'big' and 'little' forms. Although 'little' prolactin in both strains constituted a constant 80% of the total hormone at most intervals by direct measurement, it comprised a comparatively smaller proportion by RIA. In addition, the RIA-determined 'little' prolactin, after reaching maximum levels at 15 min, progressively decreased with time, the decrease being greater in the C3H/St than in the C57BL/St strain. Similar experiments with mouse growth hormone revealed no such discrepancies between the radioactivity counts and the RIA measurements. A fraction of both 'big' and 'little' forms in the C3H/St strain failed to precipitate completely after the material had been incubated with an antiserum to mouse prolactin. These results demonstrate that the prolactin injected into mice is metabolized in serum into two non-immunoreactive forms, one that elutes with the same elution volume on Sephadex G-100 column as the monomer and the other that elutes as the 'big' form. Furthermore, the loss of immunoreactivity of monomeric mouse prolactin is greater in the high-tumour C3H/St strain than in the low-tumour C57BL/St strain. Endogenous immunoreactive prolactin, on the other hand, was found mainly in the 'big' form in the serum of female mice of the C3H/St strain under basal conditions, whereas it was present only in the 'little' form in comparable mice of the C57BL/St strain, even though pituitary extracts of both strains contained mainly the 'little' form. These results support the concept that monomeric prolactin in the systemic circulation of the tumour-prone C3H/St strain is largely in a non-immunoreactive form.

Isolation and partial characterization of secreted mouse pituitary prolactin

Prolactin secreted by mouse anterior pituitaries was purified by gel filtration on Sephadex G-100. Electrophoretic homogeneity of the purified hormone was demonstrated in several gel systems, and electrophoresis in the presence of sodium dodecyl sulfate indicated an apparent molecular weight of 21 000 ± 2000. Mouse and ovine prolactin displayed parallel dose vs. response curves in radioreceptor binding studies, indicating that these two hormones compete for identical receptor sites on rabbit mammary membranes. Comparative peptide mapping studies carried out on tryptic digests of mouse and ovine prolactin suggested only partial homology between the two hormones. Internally labeled monomeric mouse prolactin was observed to undergo aggregation following storage at −20°C for 2 months.

Plasma prolactin and progesterone during the estrous cycle in the mouse.

SummaryThe concentrations of prolactin and progesterone in plasma of Line C mice during different stages of the estrous cycle were measured by radioimmunoassay. During the afternoon of proestrus, prolactin rose to a peak concentration at 1900 hr. The pattern of progesterone consisted of two major surges, beginning during the late afternoon of proestrus and the morning of metestrus.The author is indebted to Mr. Jerome J. Cymerman for assistance with the vaginal smears and to Dr. Y. N. Sinha for providing the materials for the radioimmunoassay of mouse prolactin.

Effect of antiserum to rat prolactin on milk yield and food intake in the rat.

Department of Applied Pharmacology, School of Pharmacy, Hebrew University, Jerusalem, Israel (Received 5th December 1974) Anti-ovine prolactin injected into the duct of a rabbit mammary gland inhibits the local lactogenic response of prolactin in the same duct (Saji & Crighton, 1968), and inhibits the proliferative effect of concurrently-injected prolactin in the pigeon crop-sac assay (Hayashida, 1962). Anti-ovine prolactin also decreases the weights of the prostate and seminal vesicle of male rabbits (Asano, Kanzaki, Sekiguchi & Tasaka, 1971). Anti-mouse prolactin has been reported to suppress the growth of mammary tumours in mice (Sinha, Lewis & Vanderlaan, 1974) and to inhibit the biological activity of mouse prolactin as measured by the pigeon crop-sac test (Kosugiyama, Mori & Nagasawa, 1970). Anti-rat prolactin injected into lactating rats lowered the progesterone secretion rate (Yoshinaga, 1973), while anti-human placental lactogen injected to pregnant rats during Days 1 to 5 of gestation reduced the weight of the

Mouse prolactin obtained by pituitary organ culture in a serum-free medium.

Mouse prolactin was purified by organ culture of pituitaries and electrophoresis of the medium. Mouse pituitaries were organ-cultured in 9-cm Petri dishes containing Waymouth's medium (MB 752/1) supplemented with penicillin (50 units/ml), streptomycin (100mug/ml), and bovine insulin (0.12 I.U./ml) for 8 days. Prolactin-rich culture medium was half-saturated with ammonium sulfate and centrifuged. The pellet was subjected to analytical disc electrophoresis (10% acrylamide). Gels were sectioned into 2-mm segments. Prolactin was eluted in 0.04M phosphate buffer (pH 7.2), dialyzed and lyophylized. Two hundred and forty ml medium in which 360 pituitaries were cultured yielded 29.3 mg lyophylized mouse prolactin. Although the preparation contained 2 other bands on acrylamide gel disc electrophoresis, a single precipitin line was seen in immunodiffusion and immunoelectrophoresis, showing the identity of their antigenicity. From these results, two other proteins in the preparation were suggested to be deamidated prolactin.

Radioimrnunoassay of mouse prolactin prolactin levels in isograft-bearing orchidectomized mice

Two groups of 24 (C57BL X CBA)F 1 mice were orchidectomized at the age of 6–8 weeks and received a hypophyseal isograft under the kidney capsule. One group received progesterone pellets implanted subcutaneously. Each group was divided into four sub-groups, which received oestrone in the drinking water at four dose levels: respectively 1 mg/l, 250 g/l. 12.5 g/l and 1.25 g/l. Plasma samples were taken from each mouse by puncture of the retrobulbar venous sinus on days 7, 21, 190, 270, 360 and 450 (day of operation = day 0), To obtain statistically reliable estimates of tumour data 290 mice of the same hybrid combination were divided into two similarly treated groups. Each group was divided only in three sub-groups, which received respectively the three higher oestrone dose levels. The assay results in the group of mice receiving no implants of progesterone pellets were suggestive of a dose-response relationship between oestrone and plasma prolactin values extending over the whole range of the oestrone dose levels employed. The higher levels of prolactin observed in the 1 mg/l sub-group compared to those in the 250 g/l sub-group, however, could be ascribed to the contribution by the in situ glands, which enlarged tumourously only in all the mice in this sole sub-group. A real oestrone doserelated secretory response of the isografts was observed in the three sub-groups respectively on 250 g/l, 12.5 g/l and 1.25 g/l. This oestrone-dependent response of the isograft was also reflected in the estimates of the degree of enlargement of the isografts in the respective sub-groups. In contrast only suggestive evidence was obtained for a dose-response relationship between either oestrone or plasma prolactin levels induced by this steroid on the one hand and mammary tumour incidence or the average age attained by the tumour-bearing mice in the various sub-groups on the other hand. The results indicated that continuously increased rather than “pharmacologically” high prolactin levels maintained in the circulation were mandatory for the induction of mammary tumours. Progesterone was found to have a clear-cut synergistic effect on the carcinogenic action of continuously increased plasma prolactin levels. More mammary tumours developed earlier in the presence of progesterone. The results in the mice on 1 mg/l oestrone in their drinking water indicated a “protective” action by higher dose levels of oeslrone against the induction of mammary tumours. The induction of pituitary tumours—to a size incompatible with survival by compression of the brain before the induction of mammary tumours—could not satisfactorily explain this for all cases, since in progesterone supplemented mice the development of tumorous enlargement of the pituitary by 1 mg/l oestrone in drinking water was completely prevented und yet the mammary tumour incidence declined.

Studies of prolactin secretion in mice by a homologous radioimmunoassay.

We have developed a sensitive homologous radioimmunoassay for mouse prolactin (mPRL) capable of measuring concentrations of PRL as low as 1 ng/ml of mouse serum and studied PRL secretion in mice under several physiological conditions. Newborn mice of C3H/St strain had low concentration of PRL in sera at 2–5 days of age which increased with age. Adult females of C3H/St strain contained significantly more PRL in their pituitary glands (7.9 μg/mg) than comparable females of C57BL/St strain (5.6 μg/mg); in contrast, however, they had lower PRL levels in sera (26.6 ng/ml) than the C57BL/ St females (84.5 ng/ml). The females of both strains possessed greater concentration of PRL in their pituitary glands, but in sera only C57BL/St females showed higher levels than males. Castration of C3H mice reduced pituitary PRL in both sexes but decreased serum PRL in males only. Stilbestrol in the diet of castrated mice, however, enhanced pituitary and serum PRL levels in either sex. Adult C3H mice had depressed serum conc...

Luteolytic action of prolactin during estrous cycle of the mouse.

In the rat prolactin has been shown to exent a luteotropic action an newly formed corpora lutea and a lutmlytic effect on older corpora lutea (1). During each estrous cycle of the rat, luteolysis of corpora lutea from previous cycles occurs (2, 3). Recent work from our laboratory has shown that the proestrous surge of prolactin during the estrous cycle is responsible for luteolysis of the old corpora lutea (4). Billeter and Fluckiger (5) also reported a luteolytic action by prolactin when injected daily in cycling rats with an ergot drug (CB 154) for 21 days.In the mouse prolactin produces a luteotropic action on the corpora lutm (2), but a luteolytic action for prolactin has not been described. A brief report by Kwa and Verhofstad (6) states that during the estrous cycle of the mouse, blood prolactin rises beginning late in proestrus and reaches a peak early in estrus. It was of interest therefore to determine in the mouse whether inhibition of prolactin secretion by the drug ergocornine (EC) during proe...