Nonglycosylated Prolactin Research Articles

Molecular form identification of anterior pituitary gland-secreted prolactin in chicken

Endocrine changes during bird reproduction are well documented. Prolactin (PRL) exhibits a strong relationship between incubation and broody behavior. The molecular forms of PRL in the anterior pituitary gland during the reproductive cycle have already been previously identified but not those in the secreted form. To identify the molecular forms of secreted PRL during the reproductive cycle, we thus monitored the physiological status and incubation behavior of 10 Silkie hens by a video recording system over 1–2 years. Nine out of ten mature hens exhibited incubation behavior multiple times during the experiment. Ten hens demonstrated two interesting features. In a typical clutch, hens spent 10–15 min in the nest to lay an egg. Once they spent over 1 h in the nest, the nest occupancy increased incrementally. This shift in the nest occupancy occurred 7–10 days before the incubation onset and was highly repeatable. Based on the behavior of the hens, we cultured the anterior pituitary gland during four stages (premature non-laying, laying, trans, and incubation) with physiological PRL-releasing factor, vasoactive intestinal peptide (VIP). Based on our two-dimensional protein analysis, glycosylated PRL (G-PRL) displayed several isoforms with varying isoelectric points (pI), whereas we could detect one primary signal for non-glycosylated PRL (NG-PRL). However, 3–4 NG-PRL isoforms were detected in the anterior pituitary gland. These results suggested that secreted PRL, especially from the trans and incubation stages, contains various isoforms and it is post-translationally glycosylated and phosphorylated.

Regulation of Prolactin Release at the End Stage of Chicken Embryogenesis.

Difference of onset of increase of PRL content in the anterior pituitary gland and plasma PRL concentration during the late stage of chicken embryogenesis is well known. To investigate the disagreement, changes in PRL content and PRL mRNA levels, and the effects of vasoactive intestinal polypeptides (VIP) on PRL release and PRL mRNA expression were examined using western blot analysis and real-time PCR quantification. Changes in SPRL content were strongly correlated with PRL mRNA levels. The increase in PRL content on day 17 of incubation may be caused by the increase in PRL mRNA levels on day 16 of incubation. Additionally, the effects of VIP on PRL release from the embryonic anterior pituitary gland were not observed until day 18 of embryogenesis. These results suggest that increased levels of PRL mRNA and PRL content in the anterior pituitary gland are closely correlated. However, the increased expression of PRL mRNA observed on day 17 and the initiation of PRL release from the anterior pituitary gland on day 19 were differentially regulated. According to the results of western blot analysis, the proportion of glycosylated PRL (G-PRL) and non-glycosylated PRL (NG-PRL) in the anterior pituitary gland at the end stage of development differed from the proportion of PRL released from the anterior pituitary gland. According to the results of two-dimensional western blot analysis, no isoforms with different isoelectric points were detected in the culture medium on days 19 and 20. These data suggest that the peptide chains of G-PRL and NG-PRL were not modified. In conclusion, the differentiation of PRL-producing cells and the maturation of the hypothalamus and anterior pituitary gland were completed at the end stage of incubation, and that different factors regulated the initiation of PRL mRNA expression before day 18 of incubation.

Pièges diagnostiques des hyperprolactinémies chez la femme

Prolactin is a major hormone, involved in gonadotroph axis regulation. Hyperprolactinemia induces gonadotropin deficiency and therefore hypogonadotropic hypogonadism. It should be suspected in front of menstrual cycle abnormalities, infertility and/or galactorrhea. If drugs and/or PRL adenoma represent the vast majority of causes of hyperprolactinemia, other etiologies and misleading diagnosis of hyperprolactinemia should be searched for. After eliminating a pregnancy, in women of childbearing age, the first step is to interpret the result of hyperprolactinemia, according to the assay technique used. Indeed, the major active form of prolactin is the 23kDA non-glycosylated prolactin. However, some assays interfere with macroprolactinemia, an inactive form of prolactin, including glycosylated prolactin bound to an IgG immunoglobulin. Its presence in the serum is misleading as it may induce increased levels of prolactin, usually below 100ng/mL. The diagnosis of macroprolactinemia has major issues as pituitary MRI does not need to be performed. Furthermore, neither treatment nor follow-up of patients with macroprolactinemia are necessary. It should be suspected in the presence of normal menstrual cycles. Drugs inducing hyperprolactinemia usually raise prolactin levels below 100ng/mL. If prolactin level is higher than 250ng/mL, the main diagnosis is pituitary macro-adenoma. If prolactin ranges between 100 and 250ng/mL, it is usually related to a micro-adenoma or a necrotic macro-adenoma. A mixed PRL/GH should always be suspected. If prolactin level is below 150ng/mL, in the presence of a large hypothalamic-pituitary tumor, the major diagnosis is hyperprolactinemia due to pituitary disconnection. Ectopic secretions of prolactin remain very rare. A new etiology of hyperprolactinemia is loss of function mutation of prolactin receptor.

Cosecretion of Glycosylated Prolactin and Non-Glycosylated Prolactin from Childhood to the End of Puberty

Background: Glycosylated prolactin (G-PRL) is considered as the major post-translational modification of prolactin (PRL) showing reduced lactotropic and mitogenic activities compared to non-glycosylated prolactin (NG-PRL). Aim: To evaluate the evolution of G-PRL in normoprolactinemic children and adolescents and to analyze possible variations in glycosylated/total prolactin (T-PRL) ratios. Methods: T-PRL, G-PRL and NG-PRL were evaluated in 111 healthy female and male children and adolescents (4.1–18 years), classified as group 1 (Tanner I), group 2 (Tanner II–III) and group 3 (Tanner IV–V). G-PRL and NG-PRL were identified by chromatography on concanavalin-A-Sepharose. Results: G-PRL/T-PRL (median-range): females, group 1: 0.59 (0.17–0.77), group 2: 0.56 (0.31–0.78), group 3: 0.60 (0.38–0.79); males, group 1: 0.64 (0.39–0.80), group 2: 0.61 (0.24–0.79), group 3: 0.62 (0.35–0.90); the p value is not significant among the different groups in both genders. G-PRL/T-PRL ratios do not change when comparing low (first quartile) versus high (third quartile) T-PRL levels in the different groups. Conclusion: Our study would appear to support cosecretion of G-PRL and NG-PRL from childhood to the end of puberty. Such cosecretion would not be dependent on sex steroid levels. It is important to point out that puberty does not change the proportions of G-PRL and NG-PRL.

Reversed-phase high-performance liquid chromatography method for the determination of prolactin in bacterial extracts and in its purified form

Reversed-phase high-performance liquid chromatography methodology for the determination of human prolactin (hPRL) in bacterial periplasmic space or in purified preparations has been developed. The technique, based on the high hydrophobicity of the hPRL molecule, allows its separation from the bulk of bacterial proteins. The precision for periplasmic shock fluid analysis was characterized by relative standard variations of 3–7% for intra-day and of 3–25% for inter-day determinations. Accuracy, evaluated by recovery tests, was of the order of 90%, a calibration curve being constructed with the use of a lyophilized osmotic shock fluid extract, which provided a stable, readily prepared internal reference. Sensitivity was of the order of 0.5 μg of hPRL. The methodology developed also provided a tool for comparing the hydrophobicity of glycosylated and non-glycosylated prolactin molecules obtained from several different species and of different preparations of native or biosynthetic human prolactin.

Biologic activity and plasma clearance of prolactin-IgG complex in patients with systemic lupus erythematosus.

To characterize the clinical findings in hyperprolactinemic systemic lupus erythematosus (SLE) patients with or without macroprolactinemia (big, big prolactin [PRL]) due to anti-PRL autoantibodies (PRL-IgG complex), and to assess the bioactivity and structure of big, big PRL. Twenty-seven SLE patients with hyperprolactinemia (HPRL) were studied. Patients with (n = 8) or without (n = 19) big, big PRL were identified by gel filtration chromatography and affinity chromatography for IgG. PRL concentrations in serum and fractions by gel filtration chromatography and affinity chromatography were characterized by immunoradiometric assay (IRMA), Nb2 bioassay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, and clearance studies. SLE patients without big, big PRL had significantly higher levels of disease activity, cause-proven HPRL, and menstrual disturbances compared with patients with big, big PRL (P < or = 0.05). The big, big PRL fractions by Western blotting revealed a single 23-kd nonglycosylated PRL. The Nb2:IRMA ratio of the samples with big, big PRL was significantly higher than that of the samples without big, big PRL (P < or = 0.02). However, bioactivity of big, big PRL in the Nb2 cells was very similar to that of 23-kd nonglycosylated PRL. Clearance studies in rats demonstrated that the PRL-IgG complex was eliminated more slowly than monomeric PRL (little PRL). We demonstrated that the PRL-IgG complex was formed by 23-kd nonglycosylated PRL that was noncovalently bound to IgG and showed that the complex was fully active in vitro. This result suggests that the absence of symptoms of HPRL or lower levels of lupus activity in these patients is not explained by lower bioactivity of the complex. Instead, because of the large molecular size of the complex, the PRL does not easily cross the capillary walls. Delayed clearance of the PRL-IgG complex may account for increased serum levels of PRL in SLE patients with anti-PRL autoantibodies.

In VitroRelease of Isoforms of Prolactin from Pituitary Glands of Turkey Hens at Different Physiological Stages

To study thein vitrorelease of PRL isoforms, anterior pituitary glands from medium white turkeys at various physiological stages were stimulated by cVIP in a perifusion system. Pituitaries were cut into hemi-pituitaries after collection and placed into separate perifusion chambers. Medium (M199) was continuously perifused through the system and pituitaries were stimulated with cVIP (10−7M). Total PRL content was monitored by RIA and, the ratio of immunoreactive PRL isoforms in the perifusate was estimated by Western blotting. After exposure to X-ray film for autoradiography, the relative intensity of the bands was analyzed by densitometry. All the perifused pituitaries responded to cVIP stimulation by increasing the release of PRL. Two immunoreactive bands with relative molecular weights of 24 and 27 kDa were detected by Western blotting. The immunoreactive band corresponding to the glycosylated isoforms of PRL (27 kDa) was predominant in samples from egg-laying and incubating hens and the band corresponding to the nonglycosylated isoform (24 kDa) was predominant in samples from out-of-lay and molting stages. No changes in the ratio of isoform released were detected during cVIP stimulation. Our data clearly show that glycosylated and nonglycosylated PRL isoforms are released by the pituitary glandin vitroin the same relative proportion that was previously observed in pituitary extracts and thus are likely to reflect the secreted forms of PRL in the blood during various physiological stages. In addition, the PRL-releasing activity of VIP does not affect the ratio of isoforms secreted by the pituitary glandin vitro.

Synthesis and release of glycosylated prolactin in transfected cells with human prolactin complementary deoxyribonucleic acid.

To analyze how the synthesis and release of glycosylated PRL (G-PRL) is regulated, we transfected human PRL complementary deoxyribonucleic acid (cDNA) into three different cell lines consisting of GH3 cells that originated in rat pituitary tissue, Chinese hamster ovary cells, and COS-1 cells generated from monkey renal tissue. 35S-labeled PRLs produced by the cells were immunoprecipitated with anti-human PRL antiserum, and the ratios of G-PRL to total PRL were compared. PRLs of 23 kDa and 25 kDa were detected in the cell lysate and medium. The 25-kDa PRL was confirmed to be a glycosylated form by endoglycosidase treatments. The ratios of G-PRL/total PRL were 0.17-0.33, which were similar in lysates and media and among different cell lines. Pulse-chase experiments revealed that the autonomaous secretion rates of G-PRL and non-glycosylated PRL were almost identical. These results indicate that synthesis and secretion kinetics of human PRL may not be affected by its glycosylation in the cells transfected with PRL cDNA.

Regulated secretion of prolactin by the mouse insulinoma cell line beta TC-3.

Our aim is to use cultured cells capable of regulated protein secretion for the production of recombinant proteins that require particular types of post-translational modifications. Here we have generated a stable transfected beta TC-3 cell line, beta TC-IPR9, that secretes high levels of recombinant prolactin. Transfected cells synthesize both the 27 kDa glycosylated and a 23 kDa nonglycosylated prolactin; the 23 kDa nonglycosylated species was secreted preferentially when cells were placed in secretion medium containing isobutylmethylxanthine (IBMX) and high concentrations of glucose, K+, and Ca2+. When the cells were cultured in medium containing low concentrations of glucose, K+, and Ca2+, most of the prolactin and insulin were not secreted; much of the prolactin was proteolytically converted to a 16 kDa form. Within the first 30 minutes after transferring the cells to medium containing secretagogues there was a 20-fold increase in the rate of secretion of prolactin; all of the 16 kDa species was secreted. The recombinant cells could be cycled several times between medium in which prolactin was biosynthesized and medium in which it was secreted. Preferential secretion of proteolytically processed prolactin in a medium without contaminating proteins offers an example of the advantage of this technology for production of other recombinant proteins.

Glycosylated and non-glycosylated prolactin forms are increased after opioid administration as part of surgical anaesthesia.

Previous studies have shown that non-glycosylated prolactin (NG-PRL) increased more markedly than glycosylated hormone (G-PRL) after TRH or metoclopramide stimulation. The aim of the present study was to determine whether such results could be extended to opioid-induced PRL stimulation. Open and prospective study. Using a newly developed IRMA specific for NG-PRL, we determined G-PRL and NG-PRL immunoreactivities after administration of 0.8-1.2 mg of the opioid drug phenoperidine as part of an anaesthesia. Ten male patients anaesthetized for surgical treatment of a prolapsed lumbar intervertebral disc. Samples were obtained hourly pre and post-operatively, and every 15 minutes during operation for determination of plasma PRL, NG-PRL and G-PRL. Plasma cortisol, ACTH and GH levels were measured in an attempt to differentiate the respective roles of stress and opiate agonists in the variations of PRL levels during surgery. A dramatic increase in PRL levels was observed in all patients from an average of 300 +/- 90 to 1200 +/- 330 mU/l (mean + SEM) 30 minutes after drug administration. The proportion of G-PRL immunoreactivity was not significantly different when basal (25.2%) and stimulated (27%) values were compared (P > 0.05), and when mean increments of NG-PRL and G-PRL were compared (345 and 348%, respectively). The opioid drug induced a significant decrease in cortisol levels after injection and during operation (from 585 +/- 63 to 99 +/- 51 nmol/l) with a concomitant decrease in ACTH levels. GH levels were not significantly altered during anaesthesia but were significantly greater (P < 0.05) after than before surgery (5.0 +/- 1.3 vs 0.98 +/- 0.54 mU/l, respectively). We conclude from the present and from previous data that opioid induced anaesthesia is accompanied by an increase in both glycosylated and non-glycosylated PRL and that different PRL secretagogues may induce distinct responses in terms of PRL molecular forms.

Glycosylation of human prolactin regulates hormone bioactivity and metabolic clearance

To analyze the role of individual glycosylation pattern on PRL biopotency, monomeric prolactin (PRL), secreted by human prolactinoma cells in culture, was isolated by gel filtration and separated by affinity chromatography on Concanavalin A-Sepharose or Lentil-Agarose. These lectins allowed the isolation of PRL glycoforms containing either biantennary, mannose-rich or fucosylated complex carbohydrate structures, respectively. Endoglycosidase treatment and carbohydrate content of PRL was found to be consistent with N-linked oligosaccharides of mannose-rich structure and complex units terminated in sialic acid. Mannose-rich PRL and PRL with biantennary oligosaccharides promoted cell growth of rat lymphoma cells to a diminished extent compared to non-glycosylated PRL (NG-PRL), indicating that the two major types of carbohydrate structure are able to decrease the intrinsic bioactivity of PRL. Metabolic clearance of the various forms of PRL in rats was also found to be highly dependent upon hormone glycosylation. The various glycosylated forms (G-PRLs) proved to be totally eliminated from the circulation within 60 min, faster than NG-PRL 10% of which was still present at that time. Mannose-rich or biantennary G-PRLs were differently cleared in a biphasial fashion with a similar rapid phase of about 2 min followed by distinct slow phases of 12 and 27 min, respectively. The presence of fucose did not alter this distribution. In contrast, NG-PRL was eliminated with a half-time of approximately 5 min, followed by a very slow disappearance over several h. It thus appeared that glycosylation increased the metabolic clearance rate of PRL from 0.13 +/- 0.07 ml/min for NG-PRL to 0.47 +/- 0.12 ml/min for PRL with biantennary carbohydrate chains and 0.8 +/- 0.2 ml/min for the hormone with mannose-rich oligosaccharides. The distribution of PRL to target and elimination organs was also found to be different according to the carbohydrate structure present in the hormone. NG-PRL and mannose-rich G-PRL showed higher incorporation in liver than biantennary G-PRL which was preferentially eliminated by the kidney. Altogether, the current data show that addition of oligosaccharides to PRL as well as carbohydrate structure contribute to modulate both the duration of the hormone in the blood and its distribution to different organs. It is proposed that glycosylation may selectively down-regulate PRL action at individual target tissues.

Endocrinology: Serum pattern of different molecular forms of prolactin during normal human pregnancy

In this study, the molecular heterogeneity of prolactin was analysed in serum from normal women throughout pregnancy. Lectin affinity chromatography and denaturing polyacrylamide gel electrophoresis under reducing and non-reducing conditions, followed by Western blotting and immunostaining were used to resolve and identify the molecular variants of prolactin. During the first trimester, large molecular forms (64 and 53 kDa) and those corresponding to glycosylated and non-glycosylated prolactin (25 and 23 kDa, respectively) were present either under reducing or non-reducing conditions. The 64 and 23 kDa were the predominant species at this stage of gestation. As pregnancy progressed, the 64 kDa variant, which did not bind to concanavalin A, decreased until disappeared at the third trimester of gestation. The unbound/bound ratio of serum prolactin to concanavalin A increased only at the third trimester; however, the relative proportions of concanavalin A-bound prolactin did not show statistically significant changes along the gestational period. The results demonstrated the occurrence of changes in the heterogeneity of prolactin during gestation and further confirmed previous observations that various forms of non-glycosylated prolactin are indeed the predominant species in serum from normal women throughout pregnancy.

Immunoradiometric analysis of circulating human glycosylated and nonglycosylated prolactin forms: spontaneous and stimulated secretions

Immunoradiometric analysis of circulating human glycosylated and nonglycosylated prolactin forms: spontaneous and stimulated secretions

Immunoradiometric analysis of circulating human glycosylated and nonglycosylated prolactin forms: spontaneous and stimulated secretions.

The monoclonal antibodies (MAbs) obtained in mice immunized with human PRL coupled to an anti-PRL MAb were screened for their ability to distinguish the glycosylated (G-) and nonglycosylated (NG-) forms of PRL. The 431-29 MAb exhibited high affinity binding for NG-PRL but little or no cross-reactivity to G-PRL. Using this antibody in conjunction with other MAbs which equally recognized both forms, we developed 2 immunoradiometric assays which were used to determine the amount of G- and NG-PRL in plasma. In 85 normal subjects, NG-PRL baseline levels averaged 6.6 +/- 3 micrograms/L, and represented 76 +/- 8% of the total PRL immunoreactivity. In 74 pregnant women, this proportion was significantly higher during the last 2 trimesters (84 +/- 4% and 85 +/- 6%), as compared to the first trimester (76 +/- 7%). In 6 healthy volunteers studied over 24 h, 79% of the NG-PRL peaks detected using the cluster algorithm occurred concomitantly to a G-PRL peak. The mean NG-PRL/PRL ratio was significantly higher during NG-PRL pulses (81 +/- 9%) than during valleys (71 +/- 12%). Similarly, this ratio was significantly increased during TRH or metoclopramide stimulated PRL secretion (to 88 +/- 7% and 86 +/- 6%, respectively). We conclude that 1) NG-PRL is the predominant immunoassayable form of PRL in plasma; 2) both G- and NG-PRL are cosecreted but NG-PRL is the main PRL form released during spontaneous or pharmacologically induced PRL secretion.

On the nature of serum prolactin in two patients with macroprolactinemia

To further characterize the high molecular weight (MW) forms of prolactin (PRL) present in patients with macroprolactinemia. Case reports with laboratory investigations. Academic medical centers. Two patients with macroprolactinemia. Measurements of PRL concentrations before and after chromatographic separations. The majority of serum PRL had an estimated MW of at least 669 kd in the first patient and approximately 171 kd in the second. During a pregnancy, a new form of PRL (MW 291 kd) appeared in the first patient's serum and persisted for at least 3 years. Immunoprecipitation and polyacrylamide gel electrophoresis under reducing and denaturing conditions revealed that the largest form of PRL (MW 669 kd) was composed mostly of 25 kd glycosylated PRL; intermediate forms (171 kd and 291 kd) were composed of roughly equal portions of 25 kd glycosylated PRL and 23 kd nonglycosylated PRL, whereas "little" PRL in these patients was composed primarily of 23 kd nonglycosylated PRL. Injection of the first patient's serum into rats demonstrated that the human PRL (hPRL) immunoreactivity was cleared from the serum more slowly than the PRL from sera containing predominantly little hPRL; after stimulation with thyrotropin-releasing hormone in the second patient, serum PRL concentrations decayed more slowly than observed in normal subjects. Large forms of serum PRL are at least partially glycosylated. These large forms are heterogeneous, both within and among patients. Delayed clearance may account for increased serum PRL concentrations in patients with macroprolactinemia.

6 Endometrial prolactin and implantation

The role of prolactin in human implantation and human endometrial function is still unclear. Synthesis of prolactin from human endometrium and decidua was first demonstrated in 1977. Prolactin mRNA isolation from term decidua subsequently confirmed expression of the prolactin gene in human endometrium. More recently, a glycosylated form of prolactin has been isolated from both the human pituitary and human endometrium. It appears that predominately glycosylated prolactin is secreted in the late luteal phase of the menstrual cycle, with increasingly greater amounts of non-glycosylated prolactin secreted as pregnancy advances. The biological significance and regulation of prolactin glycosylation is uncertain, although glycosylated ovine prolactin has only 20-33% of the lactogenic activity of ovine prolactin. Pituitary lactotropes, of ectodermal origin, produce pituitary prolactin and are regulated by dopamine, oestradiol and thyrotropin releasing hormone. Decidual cells, of mesodermal origin, are not influenced by these pituitary secretogogues. Progesterone and calcium both appear to stimulate pituitary and decidual prolactin secretion, while arachidonic acid seems to inhibit decidual prolactin release. More recently, insulin like growth factor-1, a polypeptide growth factor implicated in the oestrogen promoted growth reproductive tissues, has been found in high concentration in the pig uterus and has been shown to stimulate the synthesis and secretion of prolactin from human decidua. Prolactin receptors have not been reported on endothelium of blood vessels and no clear evidence exists that endometrial prolactin may modulate the secretion of endothelin, endothelium derived relaxing factor or other potentially important substances controlling menstruation. Prolactin does appear necessary for the normal production of macrophage activating factors, including interferon, and may have a local immunomodulatory role upon human implantation for this reason. Human alpha 2-interferon has sequence homology to ovine trophoblast protein-1 and is found in high concentration in human pregnancy and fetal tissues. There is some evidence that interferon may reduce human endometrial prolactin secretion, but whether endometrial or decidual prolactin is critical to human implantation awaits further study.

Ontogeny of Glycosylated and Nonglycosylated Forms of Prolactin and Growth Hormone in Porcine Pituitary during Fetal Life

Although prolactin (PRL) and growth hormone (GH) were long considered to be nonglycoprotein hormones of the pituitary, glycosylated forms of these hormones have nevertheless been discovered in recent years. We determined the ontogeny of glycosylated and nonglycosylated PRL and GH during the fetal life of the pig, an animal in whose pituitary the glycosylated variant of PRL has been found in high (40%) concentrations. Swine fetuses of both sexes from lean and obese animals of Duroc x Yorkshire crosses were examined at 60, 75, 90, and 105 days of age. No appreciable differences related to sex or phenotype were noted in any of the parameters measured; therefore, data for all animals within an age group were combined. Such averages revealed considerable amounts of GH in the fetal pituitary as early as 60 days of age, whereas PRL, although detectable by radioimmunoassay and immunoblotting at all ages tested, was not present in significant amounts until 105 days of age. From its first appearance, however, almost 70% of the PRL synthesized in the fetal pituitary was of the glycosylated type. In contrast to PRL, both the glycosylated and nonglycosylated forms of GH showed a steady rate of increase throughout the observation period. The immunoblotting analyses also revealed in the fetal pituitary several intensely staining 8- to 12-kDa PRL-immunoreactive peptides of unknown identity. The occurrence of significantly greater concentrations of glycosylated PRL than of non-glycosylated PRL in the fetal pituitary during late gestation offers new possibilities for the role of this hormone in the development of swine fetus.

Comparisons of glycosylated and nonglycosylated amniotic fluid prolactins in normal second- and third-trimester pregnancies

Prolactins in glycosylated and nonglycosylated form were isolated from 218 second- and third-trimester amniotic fluid samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose paper, and probed with anti-human prolactin serum and protein A labeled with iodine 125. The glycosylated and nonglycosylated prolactin bands were exposed by autoradiography. The bands were analyzed by densitometry, and the ratio of glycosylated to total (glycosylated + nonglycosylated) prolactin was calculated (glycosylated/total ratio) and compared with matched prolactin radioimmunoassay measurements. Both radioimmunoassay-measured and nonglycosylated prolactin decreased from the second to the third trimester whereas glycosylated prolactin concentrations remained unchanged. Concomitantly, glycosylated/total prolactin ratios increased significantly (p less than 0.001). The correlation of radioimmunoassay-measured prolactin was closer to nonglycosylated prolactin (r2 = 0.32, p = 0.00001) than to glycosylated prolactin (r2 = 0.02, p = 0.06). We suggest that the proportion of amniotic fluid glycosylated prolactin increases with length of gestation and that glycosylation of prolactin is inversely related to total prolactin secretion. Furthermore, prolactin's immunoreactivity correlated more closely to the nonglycosylated-prolactin variant, suggesting that radioimmunoassay of prolactin poorly recognizes the glycosylated prolactin form.

The Proportion of Glycosylated Prolactin in Serum Is Decreased in Hyperprolactinemic States

The proportion of serum PRL that is glycosylated has been determined, and the effects of physiological and pathological hyperprolactinaemia on this proportion have been examined. Glycosylated and nonglycosylated PRL were immunoprecipitated from serum and subjected to polyacrylamide gel electrophoresis under reducing and dissociating conditions. Separated proteins were then transferred to nitrocellulose paper by electroblotting and detected immunologically with anti-PRL antiserum and 125I-labeled protein-A, followed by autoradiography. The proportion of total monomeric PRL present in the glycosylated form was then estimated by densitometric scanning of autoradiograms. In normal individuals glycosylated PRL was predominant, accounting for about 72% of the circulating monomeric PRL. This proportion was markedly decreased (ranging from undetectable to about 60%) in the serum of women who were pregnant or were lactating postpartum and also in patients with hyperprolactinemia caused by a pituitary tumor. The results suggest that under basal conditions the majority of PRL secreted from the pituitary is glycosylated, but with physiological or pathological hyperprolactinemia the capacity for glycosylation is exceeded.

Concentrations of glycosylated prolactin in the pituitary gland and plasma of lean and obese barrows.

We measured glycosylated PRL (G-PRL) in the pituitary gland and plasma of lean and obese barrows (castrated male pigs) by immunoblotting. We found that G-PRL constituted 40% or more of total monomeric PRL in the pituitary gland of these animals. Furthermore, pituitary G-PRL concentrations in obese barrows averaged 58% higher than in those of lean controls. The non-glycosylated PRL concentrations were also higher in the pituitaries of obese barrows in comparison to lean controls, but the magnitude of the difference was smaller (42%). Plasma concentrations of the two forms, or total immunoreactive PRL measurable by radioimmunoassay, did not differ significantly between the two groups. The pituitary data suggest that the state of PRL secretion is altered in obese barrows, and that the alteration is more pronounced for G-PRL than for the non-glycosylated counterpart.