Different molecular forms of circulating prolactin (PRL) are known to occur in several species. As no such information was available in dogs, we assessed the molecular profile of circulating PRL in bitches. Pooled sera from covertly (CTRL) and overtly pseudopregnant (PSPT) diestrous bitches with high or low (> 10 or < 10 ng x mL(-1), respectively) serum PRL (measured by ELISA) were analyzed by Sephadex G-100 and Concanavalin A-Sepharose column chromatography. Four serum PRL fractions were identified and termed big-big, big (> 67 kDa), native (23 kDa) and fragmented (< 20) kDa) PRL. The percentages of these fractions were roughly similar in CTRL and PSPT animals, irrespective of their serum PRL levels (higher in PSPT than in CTRL bitches). A large proportion of glycosylated PRL (between 69 and 100%) was also detected in these sera. We conclude that in dogs, circulating PRL occurs in multiple molecular forms, whose relative abundance is comparable in covertly and overtly pseudopregnant bitches.

Isolation of glycosylated 26 kDa rat prolactin and subsequent proper carbohydrate characterization has so far not been reported. In the present work the hormone isoform was isolated to 95% homogeneity by preparative electrophoretic separation on Mini Prep Cell of rat pituitary homogenate. The isoform was then investigated by 2-mercaptoethanol gradient electrophoresis, Cleveland's sequential SDS-PAGE, digestion with endoproteinase Asp-N and N-glycanase. The glycosidic part of the isoform was examined in O-profiling and its monosaccharide composition obtained by FACE and HPAE-PAD analysis. The outcome of the experimental data is: 1) in contrast to unglycosylated 23 kDa rat prolactin, intra-chain S-S bridging is not affected in 26kDa rat prolactin, neither by transiting through a thiol gradient nor in sequential nonreducing/reducing SDS-PAGE; 2) the conformational availability of Asp residues involved in the endoproteinase Asp-N attack is the same in 23- and 26 kDa rat prolactin; the glycan moiety apparently does not cause steric hindrance at this level; 3) no glycosidic N-linkage could be detected, only O-linkage(s); 4) 26 kDa rat prolactin is no glycosyl-phosphaditylinositol-anchored protein; 5) in O-profiling an oligosaccharide chain of Mr +/- 1.4 kDa was recorded; 6) the monosaccharide composition obtained in FACE is peculiar in the sense that next to Fuc, Man, GalNac, GlcNac and NeuAc also Rib was determined; 7) HPAE-PAD analysis identified NeuAc subtypes; 8) in vitro, glycosylation of rat prolactin modulates immune recognition through steric hindrance of the access to the epitope sites.

Milk is primarily regarded as a food furnishing essential nutrients for infant growth and development, but milk can also serve as a vehicle for mother to neonate transfer of molecules that regulate development. A wide array of biologically active compounds such as hormones, cytokines and enzymes are present in milk, especially early milk. The premise that prolactin (PRL) in milk is an important and possibly essential developmental factor for the newborn is explored. Both PRL and structurally modified isoforms are abundant in early milk and gradually diminish with the progression of lactation. Milk PRL is absorbed and biologically active in the neonate. Assays of PRL variants, experimental paradigms to test them as developmental regulators and the body of evidence supporting the hypothesis that milk PRL regulates differentiation and maturation of neonatal neuroendocrine, reproductive, and immune systems is presented.

It has previously been suggested that the mammary cell could produce prolactin (PRL). This hypothesis was investigated by incubation with [35S]methionine-cysteine followed by SDS-PAGE, immunoblotting and autoradiography of immunoprecipitated PRL, and by electron microscopic analysis after incubation without or with cycloheximide. Immunoreactive 14-, 23-, 25-, 32- and 36-kDa PRL forms were radioactive. By two-dimensional electrophoresis analysis, immunoreactive and radioactive spots, of about 25 kDa and high molecular weight, were also detected. After incubation of mammary epithelial cells with cycloheximide, immunogold electron microscopy showed a drastic decrease of labelling in organelles involved in synthesis and secretion, compared to those incubated in control medium. These results make it possible to conclude that lactating mammary tissue is able to synthesize PRL.

To study the localization of PRL in mammary epithelial cells (MEC) and to characterize PRL forms in serum, milk, and mammary tissue, two groups of lactating rats, a control group and a bromocriptine-treated group, were compared. In serum and milk from control rats, two forms of PRL (25 and 23 kDa) were detected by immunoblotting. In bromocriptine-treated rats, only the 25-kDa form was present. In mammary tissues from control rats, 25-, 23-, and 14-kDa forms of the hormone were detectable, whereas only 25- and 14-kDa forms were present in bromocriptine-treated rats. Immunofluorescence and immunogold electron microscopy revealed that in MEC from control rats, PRL was located in the organelles involved in endocytosis and was also present in rough endoplasmic reticulum, Golgi apparatus, secretory vesicles, and lumen of the acini. In bromocriptine-treated rats, a decrease in the labeling was evidenced in endosomes and multivesicular bodies. On the contrary, labeling associated with the rough endoplasmic reticulum and secretory vesicles was increased. Thus, even when the amount of circulating PRL detected by RIA was strongly decreased, PRL was always detectable in MEC and in the lumen of the acini, suggesting an active participation of MEC in the transport of PRL to milk.

Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities, G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about 1/30th the mitogenic activity of bovine PRL; G-ePRL was approximately 1/10th as active as ePRL. Glycosylation of G-ePRL at Asn31 was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29-37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man3, GlcNAc2, GalNAc1, Fuc0.6, Gal0.2, NeuAc0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn31 carbohydrate unit is a fucosylated complex mono- and /or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.

Rat pituitary homogenates were submitted to differential and density gradient centrifugation. Subcellular fractions as well as the purified secretory granules were examined in electron microscopy, radioimmunological techniques, protease digestion, alkaline treatment and immunoblotting. The global outcome of these experiments was that: 1) the glycosylated rPRL was foremost recorded in the crude secretory granular fraction, also in the microsomal fraction and the cytosol, but virtually not in the plasma membrane fraction; 2) in purified secretory granules glycosylated rPRL appeared as an array of near Mr, such as was formerly obtained by enzymatic deglycosylation; 3) protease digestion and ice-cold alkaline treatment of the secretory granules showed that 23,000 rPRL appears in three different physicochemical states in these organelles: unsequestered within a closed system, membrane-bounded and bound state; 4) likewise treatment of microsomal vesicles showed that 23,000 and glycosylated rPRL are sequestered in these bodies, but apparently 23,000 rPRL appears as both integral membrane-bound and released from the lumen, whereas glycosylated rPRL is chiefly retained as an integral membrane protein. 5) dopamine alters the pattern of glycosylation as well in Mr as in relative percentages of the molecular variants. The systematical occurrence of the array of near Mr glycosylated rPRL is biosynthesized as a pool of proteins with a different degree of glycosylation. On the basis of our data, we speculate that selection of definite molecular variants from this pool could play an important role in the biological function of 23,000 rPRL and that oligosaccharides could perhaps target the glycosylated forms of rPRL to specific sites of action.

Abstract Prolactin cells derived from the anterior pituitaries of female rats were cultured in the presence of tunicamycin, swainsonine, castanospermine, beta-hydroxynorvaline and monensin in order to study their effect on the post-translational processing of the M(r) 17,000, 23,000 and 26,000 prolactin molecular forms. Sodium-dodecyl-sulphate polyacrylamide electrophoresis and subsequent immunoblotting revealed that: 1) tunicamycin, swainsonine and castanospermine, compounds that are essentially known as inhibitors of the N-glycosylation processus, had no effect on M(r) 17,000, 23,000 and 26,000 rat prolactin; 2) betahydroxynorvaline, which has been assumed to inhibit processing of pre-prolactin to mature 23,000 prolactin, did not increase the synthesis of 26,000 rat prolactin. In case of inhibition of the processing of a pre-prolactin to mature prolactin, one would expect an increase of the pre-prolactin; consequently, we could not establish the 26,000 rat prolactin, we revealed in immunoblotting, as a pre-prolactin; 3) monensin affected the post-translational processing of 17,000 and 26,000 rat prolactin, but left the 23,000 mature form intact. This is an important finding for the following reasons: monensin blocks the transport of secretory and membrane proteins, and this blockade prevents the cleavage of these molecules; indeed, production of 17,000 rat prolactin, a form of cleaved prolactin, was inhibited. Monensin also affects glycosylation and 26,000 rat prolactin has been identified as a presumably O-iinked glycosylated variant. The fact that its synthesis is inhibited by monensin treatment, but not by inhibitors of the N-linked process, particularly tunicamycin, and that 26,000 rat prolactin is susceptible to mild alkali and decomposition via beta-elimination are decisive arguments in favour of the O-linked glycosidic linkage.

Prolactin and GH cells from rat pituitary glands were separated into three main fractions on discontinuous Percoll gradient layers. SDS-PAGE and subsequent immunoblotting of these fractions revealed that:(1) multiple rat prolactin (rPRL) molecular variants were present in total culture, Percoll layer 1 and 2; four variants were clear-cut: Mr approximately 23,000, Mr doublet approximately 25,000-26,000, Mr approximately 40,000 and Mr approximately 42,000;(2) cell cytosol from Percoll gradient layer 1 was particularly enriched in prolactin;(3) cells from gradient layer 1 secreted into the culture medium only prolactin in detectable amounts;(4) three distinct molecular forms of rat growth hormone (rGH) were recorded in layer 3: Mr approximately 36,000, 24,000 and 20,000; the 20,000 variant was paramount; and (5) cells from layer 3 secreted both rPRL and rGH into the culture medium. Reduction experiments showed that, on the one hand, 42,000 and 40,000 rPRL variants and, on the other hand, 36,000 rGH variants are disulphide-bridged dimers. An important finding was the presence of glycosylated rPRL and rGH: indeed Concanavalin A-Sepharose 4B affinity chromatography indicated that 26,000 rPRL and 24,000 rGH display a very strong affinity for lectin. Competitive inhibition tests showed that this affinity is specific and not due to hydrophobic binding. When rPRL was submitted to deglycosylation in conditions specific for O-linked glycoproteins, the 26,000 rPRL variant disappeared. The biological role of glycosylated rPRL is as yet unknown.

Rat pituitary homogenates were submitted to differential and density gradient centrifugation. Subcellular fractions as well as the purified secretory granules were examined in electron microscopy, radioimmunological techniques, protease digestion, alkaline treatment and immunoblotting. The global outcome of these experiments was that: 1) the glycosylated rPRL was foremost recorded in the crude secretory granular fraction, also in the microsomal fraction and the cytosol, but virtually not in the plasma membrane fraction; 2) in purified secretory granules glycosylated rPRL appeared as an array of near Mr, such as was formerly obtained by enzymatic deglycosylation; 3) protease digestion and ice-cold alkaline treatment of the secretory granules showed that 23,000 rPRL appears in three different physicochemical states in these organelles: unsequestered within a closed system, membrane-bounded and bound state; 4) likewise treatment of microsomal vesicles showed that 23,000 and glycosylated rPRL are sequestered in these bodies, but apparently 23,000 rPRL appears as both integral membrane-bound and released from the lumen, whereas glycosylated rPRL is chiefly retained as an integral membrane protein. 5) dopamine alters the pattern of glycosylation as well in Mr as in relative percentages of the molecular variants. The systematical occurrence of the array of near Mr glycosylated rPRL is biosynthesized as a pool of proteins with a different degree of glycosylation. On the basis of our data, we speculate that selection of definite molecular variants from this pool could play an important role in the biological function of 23,000 rPRL and that oligosaccharides could perhaps target the glycosylated forms of rPRL to specific sites of action.

Recent findings indicate that interleukin-1 (IL-1) and interleukin-6 (IL-6), cytokines secreted by immunologically activated monocytes and macrophages modulate neuroendocrine function. The site of production (centrally, peripherally), the site of action and the physiological significance of IL-1 and IL-6 as "classical hormones" are questioned.

Early detection of subjects with a propensity to obesity might be of great help for setting up preventive intervention studies. In this study we tested whether the development of obesity in Wistar rats, given ad libitum cafeteria foods, could be predicted by a low prolactin (PRL) response to 5-hydroxytryptophan (5HTP), as an index of low hypothalamic serotoninergic tonus. Basal and 5HTP-stimulated (50 mg/kg body weight i.p.) PRL were measured by RIA in 15 young male Wistar rats, whose pelleted diet was afterwards supplemented with cafeteria foods. In the tested animals an increase of PRL between 4 and 56 times the basal value was observed 60 min after the 5HTP injection. After 2 months of feeding, marked inter-individual differences in weight gain between the cafeteria fed animals were observed. After 10 months of feeding, median body fat percentage, assessed by dual X-ray absorptiometry, of the overfed rats was significantly higher than that of control animals: median (range): 41.2%(28.9 - 51.5%) vs 25.1 (18.0 - 32.2%)(p < 0.0001). The PRL response at the start of the experiment was neither correlated with the monthly weight increases, nor with the fat mass percentage at the end of the experiment, suggesting that a pre-existing low hypothalamic serotoninergic tonus is probably not involved in the overeating and ultimate overweight of cafeteria diet fed animals.

Isolation of glycosylated 26 kDa rat prolactin and subsequent proper carbohydrate characterization has so far not been reported. In the present work the hormone isoform was isolated to 95% homogeneity by preparative electrophoretic separation on Mini Prep Cell of rat pituitary homogenate. The isoform was then investigated by 2-mercaptoethanol gradient electrophoresis, Cleveland's sequential SDS-PAGE, digestion with endoproteinase Asp-N and N-glycanase. The glycosidic part of the isoform was examined in O-profiling and its monosaccharide composition obtained by FACE and HPAE-PAD analysis. The outcome of the experimental data is: 1) in contrast to unglycosylated 23 kDa rat prolactin, intra-chain S-S bridging is not affected in 26kDa rat prolactin, neither by transiting through a thiol gradient nor in sequential nonreducing/reducing SDS-PAGE; 2) the conformational availability of Asp residues involved in the endoproteinase Asp-N attack is the same in 23- and 26 kDa rat prolactin; the glycan moiety apparently does not cause steric hindrance at this level; 3) no glycosidic N-linkage could be detected, only O-linkage(s); 4) 26 kDa rat prolactin is no glycosyl-phosphaditylinositol-anchored protein; 5) in O-profiling an oligosaccharide chain of Mr +/- 1.4 kDa was recorded; 6) the monosaccharide composition obtained in FACE is peculiar in the sense that next to Fuc, Man, GalNac, GlcNac and NeuAc also Rib was determined; 7) HPAE-PAD analysis identified NeuAc subtypes; 8) in vitro, glycosylation of rat prolactin modulates immune recognition through steric hindrance of the access to the epitope sites.

To gain an insight in the routing, processing and export of rat prolactin, rat pituitary cells were cultured in serum-free medium in the presence of cycloheximide, carbonyl cyanide m-chlorophenylhydrazone, Brefeldin A and monensin. The potential influence of these perturbants, whose well documented effects are the altering of protein synthesis and transport, was studied on rat prolactin molecular size isoforms appearing in cellular extracts and in culture medium. The outcome of the culture experiments as recorded in vertical SDS-PAGE, thiol gradient electrophoresis and sequential SDS-PAGE followed by prolactin specific immunoblotting and densitometry, was as follows:(1) at the cellular level we were able to characterize a novel 36 kDa protein as a disulphide-bridged oligomeric precursor prolactin, which is presumably rapidly transformed in the cis/medial Golgi; to designate monomeric rat prolactin as an early Golgi protein and t o advance evidence that the main processing of the glycosylated rat prolactin is a cis/medial Golgi event;(2) in release none of the perturbants disturbed the relative distribution of monomeric and glycosylated rat prolactin, the main molecular size isoforms currently secreted by untreated pituitary cells, or induced the appearance of transformed molecular size isoforms;(3) the secretion mode indicates that rat prolactin is released via the regulated pathway in the presence of the perturbants used.

The modulation of both the molecular size heterogeneity and the relative distribution of rat prolactin variants, synthesized and secreted in vitro by rat pituitary cells in the course of postnatal ontogeny and in gestation, lactation and weaning was investigated by SDS-PAGE, immunoblotting, radioimmunological techniques and O-sialoendopeptidase digestion. The outcome of the experiments is as follows: 1) from day 1 of postnatal life 20-, 23-, 26-, 40-44 kDa and oligomeric rat prolactin isoforms were stored and secreted; 2) perinatal life is characterized by a high degree of variability of prolactin size isoforms and their respective repartition in storage and release; in addition to the major variants, transient ones of M, 25-, 28-, 33- and 36 kDa were secreted and/or stored; 3) O-sialoglycoprotease digestion of pituitary cell lysate gave good evidence for 25 kDa prolactin being a glycoform; 4) at 1 month of age 16 kDa rat prolactin appeared and persisted over the whole postnatal span (1 day-->1 year) but only in stored form; 5) the physiology of gestation was essentially characterized by the M(r)-modulation of the glycoform (26 kDa-->26.3 kDa) and the virtual absence of stored 26 kDa rat prolactin at week 1 of pregnancy; 6) in lactation and weaning uncommon multiple banding was observed in secreted oligomeric prolactin; 7) in pregnancy, lactation and weaning the differential distribution of released and stored prolactin isoforms displayed a considerable intra- and intervariability; 8) in the vast array of size isoforms observed in all our experiments monomeric 23 kDa prolactin was always the dominating variant. In conclusion, the molecular size heterogeneity and the differential distribution of secreted and stored rat pituitary prolactin is considerably influenced by age and physiological stimuli. The nature of polymeric prolactin and of the transient variants is presently unclear, and the exact physiological role of molecular heterogeneity modulation is unknown, both in humans and rat, but the patterns of change we observed in definite stages of life, suggest that this phenomenon is important in the maturation of the hypothalamus-pituitary axis and in the metabolic and hormonal changes accompanying gestation.

Hyperphagia in rats fed a cafeteria diet might be related to the palatability of the diet or to diet-induced changes in central neurotransmitters regulating the feeding behavior. In this study the central serotonergic tonus in adult male Wistar rats was evaluated in vivo after 6 weeks of feeding a cafeteria diet by the prolactin response to the administration of 5-hydroxytryptophan (5HTP), the immediate serotonin precursor. Blood was taken just before, 30, 60 and 90 min after the ip injection of 50 mg/kg 5HTP for the determination of prolactin concentrations were comparable between cafeteria fed rats and control rats, fed normal laboratory chow (12.7 +/- 5.4 vs 7.7 +/- 4.5 ng/ml). The 5HTP-stimulated prolactin secretion in the cafeteria diet fed rats, determined by the peak value (95.8 +/- 17.2 vs 119.1 +/- 27.0 ng/ml) as well as by the integrated area under the curve (5478 +/- 774 vs 5916 +/- 2275 ng/ml. 90 min) was not significantly lower than in the control rats. In conclusion, our results did not show a significantly decreased 5HTP-induced prolactin release in cafeteria-fed rats, suggesting that a low hypothalamic serotonergic tonus is probably not involved in the overeating of this dietary-induced obesity model.

A single-step procedure was devised to separate PRL cells from the rat anterior pituitary gland. After dissociation, cells were centrifuged on a Percoll gradient. Three layers were recovered. The composition of the different layers was evaluated using immunocytochemistry (with antisera to the six pituitary hormones), and in situ hybridization [with DNA complementary to PRL or to GH messenger RNA (mRNA)]. Both methods yielded identical values. PRL cells were recovered in the lower density layer (layer 1) with a good yield (that is 81% of the total PRL cells of the initial cell suspension) and in addition, markedly enriched (indeed 85% of the cells in layer 1 stained for PRL). A second layer (layer 2: intermediate density) contained most of the remaining PRL cells which were, however, heavily contaminated mainly by GH cells and cells that did not stain for any of the known pituitary hormones. A third layer (layer 3: higher density) was enriched in GH cells to 93%(representing, however, only 10% of the initial pituitary GH cells). In addition, PRL and GH were measured by RIA in culture medium and in cell lysates. Hormone biosynthesis was monitored by polyacrylamide gel electrophoresis and autoradiography after culture in the presence of [35S]methionine. These experiments confirmed that layer 1 was enriched in cells containing, and producing, PRL and depleted from GH cells. Cells in layer 2 contained and produced more GH than PRL. PRL cells from layer 1 responded to dopamine and to vasoactive intestinal polypeptide in the same way as PRL cells in the unseparated pituitary cell population. In contrast PRL cells in layer 2 had a lower basal secretion rate but a higher response to vasoactive intestinal polypeptide. Unless this represents a paracrine effect of non-PRL cells, PRL cells in layer 2 exhibit different properties and may therefore form a distinct subpopulation of PRL cells.