Influence of sulpiride treatment on the level of prolactin and immunoglobulins in the peripheral blood of mares during the postpartum period.

The aim of this study was to determine the effect of increased levels of prolactin (PRL) on the concentration of immunoglobulins in the blood, colostrum and milk of mares. The study was conducted on 12 mares of the Polish Pony breed (6 in the control and 6 in the experimental group). To induce hyperprolactinaemia in mares of the experimental group, 750 mg sulpiride was administered orally once a day. The initial PRL concentration was 52.22 ± 11.21 ng/ml in the control group and 49.39 ± 10.12 ng/ml in the experimental group. In the subsequent days, the concentration of PRL dynamically changed. Statistical analysis showed highly significant differences (P < 0.01) between the groups. The concentration of immunoglobulins in the blood plasma was at the same level during the experimental period (32.97-29.08 mg/ml in the experimental group and 28.60-18.11 mg/ml in the control group). Statistical analysis showed highly significant differences between the groups in blood plasma immunoglobulin level (P < 0.01). The highest immunoglobulin concentration was obtained within 12 h after parturition in the control and the experimental group (23.49 ± 2.12 mg/ml and 26.94 ±1.72 mg/ml, respectively). The lowest values were obtained on day 12 after parturition in the experimental group (10.15 mg/ml ± 1.47 mg/ml) and on day 7 after parturition in the control group (14.30 mg/ml ± 2.48 mg/ml). In conclusion, this study did not provide evidence that the lactogenic hormone prolactin is involved in the transfer of immunoglobulins into the colostrum in horses.

Black porgy (Acanthopagrus schlegeli) prolactin cDNA sequence: mRNA expression and blood physiological responses during freshwater acclimation.

To investigate the consequences of freshwater (FW) transfer, we studied the prolactin (PRL) cDNA sequence and its mRNA expression, and physiological responses in the black porgy (Acanthopagrus schlegeli). We cloned and characterized cDNA encoding its PRL from the pituitary gland. Black porgy PRL cDNA consists of 1492 bp and encodes a protein of 212 amino acids including 24 signal peptides. Reverse transcription-PCR showed the PRL mRNA expression in the pituitary gland. Expression of pituitary gland PRL mRNA was significantly higher during FW acclimation. Furthermore, we studied the stress responses and osmoregulatory abilities of black porgy in changing salinities. Plasma cortisol, glucose, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) significantly increased in the fish immediately after transfer to FW. We also identified significant changes in the fish in terms of plasma ions (Na(+), Cl(-), Ca(2+)) and osmolality during the acclimation period. These results suggests that PRL plays an important role in hormonal regulation in osmoregulatory organs, thereby improving the hyperosmoregulatory ability of black porgy in freshwater.

The prolactin of European sea bass (Dicentrarchus labrax L.): cloning of cDNA and efficient expression in Escherichia coli.

The cDNA encoding sea bass (Dicentrarchus labrax) prolactin (sbPRL) was obtained by reverse transcription-polymerase chain reaction (RT/PCR) from pituitary RNA with degenerate primers designed on the basis of the cDNAs of the two PRLs (tPRL188 and tPRL177) from the tilapia, Oreochromis niloticus. The sbPRL cDNA encodes a preprotein of 212 amino acids composed of a putative signal peptide of 24 residues and a mature protein of 188 amino acids that is the homologue of tiPRL188. The cDNA coding for the mature protein was cloned into the pAX4a+ expression vector and expressed efficiently in Escherichia coli as a beta-galactosidase-fusion protein. To split the fusion protein, a sequence encoding the hexapeptide, (Asn-Gly)3, that contains three Asn-Gly hydroxylamine-cleavable bonds, had been previously introduced by PCR upstream of the sbPRL cDNA. N-terminal sequencing confirmed that the cleaved product corresponded to sbPRL. An antiserum raised against the recombinant hormone detected by immunoblotting a single band in sea bass pituitaries and two bands in tilapia pituitaries, suggesting the occurrence of a single PRL form in sea bass.

Prolactin heterogeneity: a limitation on the evaluation of results from prolactin assays due to differences in immunoassays and the different bioactivities of prolactin forms.

Prolactin exists in biological fluids in several molecular forms. This raises two questions: (1) whether the assay of prolactin by immunotechniques is valid and reliable and (2) whether the different forms have different physiological roles, which might be exploited to improve diagnostic accuracy and data interpretation by the use of appropriate methods. To investigate these questions, prolactin from human amniotic fluid was separated, by concanavalin A-Sepharose affinity chromatography, into bound, retarded and unbound fractions (bound prolactin fraction, retarded prolactin fraction, unbound prolactin fraction), which were characterized by electrophoresis, immunoblotting and glycan detection blot. Virtually no contamination was found in the bound prolactin fraction, and the unbound prolactin fraction and retarded prolactin fraction were 74-83% pure according to densitometry of the electrophoretic and immunoblot patterns. High variability was found among the individual patterns. Glycan detection in the blotted fractions revealed that the bound prolactin fraction bands corresponding to M(r)25,000-29,000 were weakly glycolysated, whereas the bands of M(r)60,000-64,000 were significantly glycan-positive. Immunoreactive bands of unbound prolactin fraction and retarded prolactin fraction also stained positively for glycans. Using two commercial prolactin kits, the bound prolactin fraction forms were virtually undetectable. To demonstrate that the prolactin forms may depend on the hypothalamic state, two behaviourly different breeds of cattle were used as an animal model for studying hypothalamic activities. The number of immunoreactive bands, representing the prolactin forms, and the change of the forms in response to thyroliberin differed strikingly among the groups. The bioactivity of the forms was examined in bovine granulosa, oviductal, endometrial and spleen cells, and in murine splenocytes, the latter being activated by concanavalin A or allogeneically to create in vitro conditions that may have relevance for situations in vivo. The rate of incorporation of [3H]thymidine in murine splenocytes was dose-dependently enhanced only by bound prolactin fraction. The increase was abolished by purified anti-prolactin antiserum. However, the standard prolactin from the kits inhibited the proliferation even in low dose (1.25 microgram/l) and the inhibition was abolished in part by bound prolactin fraction. Thymidine incorporation into the bovine cells was significantly increased by low concentrations (2 micrograms/l) of unbound prolactin fraction and retarded prolactin fraction. Oviduct epithelial cells and splenocytes were stimulated by unbound prolactin fraction but not by retarded prolactin fraction in a dose of 16 micrograms/l. Thymidine incorporation into granulosa cells was inhibited by retarded prolactin fraction (16 micrograms/l) but not by unbound prolactin fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Impaired response of free alpha-subunits after luteinizing hormone-releasing hormone and thyrotropin-releasing hormone stimulations in beta-thalassemia major.

In order to clarify whether the damage in gonadotropin secretion due to iron overload in patients with beta-thalassemia is of pituitary or hypothalamic origin, 14 euthyroid patients (8 females and 6 males, age 15-24 years) affected by beta-thalassemia major with hypogonadotropic hypogonadism were studied. Luteinizing-hormone (LH), follicle-stimulating hormone (FSH) and free alpha-subunit (FAS) were measured during LH-releasing hormone (LH-RH) stimulation test, and thyroid-stimulating hormone (TSH), prolactin (PRL) and FAS during thyrotropin-releasing hormone (TRH) stimulation test. During LH-RH stimulation, the mean basal LH, FSH and FAS levels were similar to those found in normal prepubertal children, but the peak values were lower than those found in such children. Also during TRH stimulation, the mean peak values of FAS were lower than those of normal prepubertal children, but the TSH response was normal. The lack of response of gonadotropins and FAS to LH-RH cannot exclude hypothalamic failure; however, the normal response of TSH to TRH, in spite of the poor response of FAS, indicates that the origin of hypogonadotropic hypogonadism is the pituitary damage concerning not only the gonadotroph but also the thyrotroph cells.

Immunocytochemistry and immunoblotting of avian prolactins using polyclonal and monoclonal antibodies toward a synthetic fragment of chicken prolactin.

A major obstacle in the production of specific antibodies toward chicken prolactin (PRL) has been overcome by mimicking a putative epitope of the molecule using the synthetic decapeptide Lys-chPRL 59-67. This peptide represents the highest hydrophilicity peak of the amino acid sequence of chPRL that was recently derived from the nucleotide sequence. Polyclonal mouse antisera against the fragment specifically recognized the lactotropes in the cephalic lobe of the chicken pars distalis as illustrated by immunocytochemical double staining experiments. Monoclonal antibody production yielded antibodies that specifically labeled purified turkey PRL upon SDS-PAGE separation and immunoblotting. Turkey and chicken PRL showed a very similar polymorphism with respect to their apparent molecular weights, including the occurrence of a glycosylated variant of chicken PRL. The monoclonal antibodies were finally used to demonstrate the presence of PRL-like immunoreactivity both in the pituitary gland and in the brain of the quail. In the brain, immunoreactive neurons were in the nucleus accumbens and in the lateral parts of the ventro-medial hypothalamus, partly similar to those described in the rat.