Prolactin variants in ram adenohypophyses vary with season.

Secretion of PRL in sheep is affected by photoperiod being highest during the spring and summer, lowest in fall and winter. The objectives of this study were to determine if 1) the production of variant forms of PRL, and 2) immuno- and bioactivities of PRL (iPRL and bPRL) differ during times of the year selected to represent periods of low, transitional and high PRL secretion. Twelve mature rams were maintained on pasture and killed in October, December, and April (n = 4/month). Individual pituitary glands were dispersed, cells obtained, and fixed for immunocytochemical flow cytometry, extracted with 0.01 N NaHCO3 or cultured in serum-free, defined media. The Mr of PRL extracted from cells immediately following dispersion ranged from 14-140K, with significantly more bands greater than 40K being detected from rams sacrificed in December than from those killed in October and April (P less than 0.01). No bands of PRL greater than 25K were observed when samples were reduced with beta-mercaptoethanol prior to electrophoresis, indicating that the high Mr forms were disulfide-linked aggregates. Culture media from October and April contained variants of PRL that ranged from 22-40K but those greater than 25K were generally not observed from cells harvested during December. Extracts of cells after 24 h in culture contained fewer high Mr species during December than had been present in initial extracts from that month. In contrast, during April more high Mr intracellular forms were present after culture than had been detected prior to culture during that month. The percentage of lactotrophs averaged 50.0 +/- 2.5, 47.4 +/- 5.7, and 59.4 +/- 5.5 for October, December, and April, respectively. Initial lactotroph content (pg/lactotroph) of iPRL was higher (P = 0.06) in April (46.0 +/- 17.0) when compared to October and December (8.0 +/- 2.0 and 20.0 +/- 10.0, respectively). In contrast, the bPRL content of initial extracts was higher (P = 0.05) in December (267.0 +/- 68.0) than in October (101.0 +/- 35.0), but not than in April (190.0 +/- 70.0). Although iPRL and bPRL concentrations in culture media were similar for the 3 months, the intracellular iPRL (P less than 0.001) and bPRL (P less than 0.0001) content after culture was greatest during April. In summary, in addition to the well-documented seasonal changes in blood concentrations of PRL, different molecular forms of PRL were found within the pituitary at different times of the year and seasonal variations in iPRL and bPRL did not occur in parallel.

Bioactive and immunoreactive variants of prolactin in milk and serum of lactating rats and their pups.

Prolactin (PRL)-like bioactivity (in Nb2 lymphoma assay) and immunoreactivity (in radioimmunoassay (RIA)) in rat milk, maternal and neonatal sera and in neonatal rat pituitary cultures were investigated. The PRL-like bioactivity in the water-soluble fraction of rat milk was high and exceeded its immunoreactivity 5.8-, 4.0- and 2.1-fold, on days 2, 12 and 22 of lactation respectively. The elevated bioactivity to immunoreactivity (B/I) ratio of PRL in milk was not due to the presence of interleukin-2 (IL-2) in milk, since the proliferation of the CTLL-2 murine T cells, which are not sensitive to PRL, was promoted by IL-2 but not by milk. Serum levels of immunoreactive PRL were low in sera of non-weaned rat pups on days 2, 12 and 22 postpartum. Similar to milk, the B/I ratio of PRL in sera of rat pups was high and decreased with time postpartum. Pituitary glands of pups obtained on days 2, 12 and 22 secreted progressively increasing amounts of PRL in vitro; the B/I ratio ranged between 1.2 and 2.1 without a significant change. The relative concentrations of size variants in milk were not proportional to those in serum of lactating rats on day 2 postpartum as assessed by Sepharcryl S-100 HR gel permeation chromatography and Nb2 bioassay or RIA. Size variants of biologically active PRL were abundant in early milk and gradually diminished as lactation progressed: a partially resolved peak representing monomeric to dimeric PRL variants (relative molecular weights ranging between 18 k and 42 k) became progressively narrower between days 2 and 22. Biologically active and immunoreactive PRLs displayed disparate elution profiles. The elution profile of PRL in sera of neonatal rats on day 2 post-partum was different from that of maternal serum or milk. The major immunological (and possibly biological) PRL-like activity eluted as two adjacent peaks at 2.2 k and 1.5 k, raising the possibility that fragments of milk-borne PRL were absorbed from the gut after partial proteolytic degradation. In contrast with PRL, GH (which is present in rat milk only in minute concentrations) did not show heterogeneity in sera of 2-day-old rat pups in gel permeation chromatography. The present results demonstrate that the concentrations of PRL-like activity in rat milk and newborn rat serum have been grossly underestimated because levels have been measured by RIA. The high B/I ratio of PRL in milk and neonatal sera is due to the presence of PRL-related compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin-releasing hormone mediates growth hormone release induced by milk and nursing in neonatal rats.

Previous evidence from this laboratory suggested that growth hormone (GH) release induced by milk in vitro and by nursing in vivo from neonatal rat pituitary glands is mediated by an alternative GH-releasing factor(s) (GRF) distinct from GH-releasing hormone (GHRH(1-43) ). In the present experiments we tested whether thyrotropin-releasing hormone (TRH) could fulfil the criteria of this alternative GRF in neonatal rats. The water-soluble fraction of rat milk (infranatant, prepared by ultracentrifugation) and its methanol/acetic acid extract (milk-borne peptides) stimulated GH release from perifused pituitary glands obtained from 2-day-old rats. Dialysis of the infranatant (mol wt cut-off: 2,000) against 500 volumes of culture medium at 4°C eliminated its GH-releasing activity in the perifusion system, while the infranatant retained its full GRF-like activity when incubated at 4°C without dialysis. The milk-borne GRF eluted as a single peak and coeluted with TRH in a combined gel permeation chromatography (Sephadex G-10) and perifusion set-up. Prolactin secretion was also stimulated simultaneously with the release of GH induced either by milk or by TRH. In a stepwise C(18) reversed-phase chromatography, milk-borne GRF was highly hydrophilic and coeluted with synthetic TRH. The in vitro GH-releasing bioactivities of synthetic TRH and a milk extract purified in C(18) reversed-phase chromatography were abolished by proline-specific endopeptidase. Thus, TRH and milk-borne GRF displayed similar molecular weights, hydropathic characteristics and proteolytic enzyme resistance. In vivo, nursing (which has been reported as a potent stimulus of GH secretion even in the absence of milk-intake) increased serum GH levels in 2-day-old pups. A supramaximal dose of TRH (10 ng/g intraperitoneally) stimulated GH release in 2-day-old pups separated from their mothers for 6 h to a similar extent as nursing. Nursing-induced levels of serum GH were not further elevated by TRH. This failure of TRH to further increase serum GH levels was not due to a maximal GH output by the neonatal pituitary gland, since the GH release induced by the serotonin precursor 5-hydroxy-L-tryptophan was augmented either by TRH or by nursing. These data provide evidence that the milk-borne GRF-like activity in vitro is indistinguishable from TRH, and suggest that TRH (probably of hypothalamic origin) might be the mediator of the nursing-induced release of GH in vivo as a physiological GRF in neonatal rats.

Brain structures mediating the suckling stimulus-induced release of prolactin.

Suckling-induced prolactin release is a widely studied neuroendocrine reflex, comprising a neural afferent and a humoral efferent component. The information on the brain structures involved in this reflex is fairly limited. The present studies focused on this question. The following hypothalamic interventions were made in lactating rats and the dams were tested for the suckling-induced prolactin response: (i) unilateral or (ii) bilateral frontal cuts at the level of the anterior and posterior hypothalamus; (iii) administration of 5,7-dihydroxytryptamine or (iv) 6-hydroxydopamine into the hypothalamic paraventricular nucleus (PVN) to destroy serotonergic and catecholaminergic innervation of the cell group, respectively; (v) lesion of the medial subdivision of the PVN; and (vi) horizontal knife cuts below the PVN. Bilateral posterior and bilateral or unilateral anterior frontal cuts caused blockade of the suckling-induced release of prolactin. Likewise, most dams receiving 5,7-dihydroxytryptamine in the PVN did not respond to the suckling stimulus. Immunocytochemistry revealed that, in those rats which did not show a rise in plasma prolactin, there were almost no serotonergic fibres and terminals in the PVN, while in dams which exhibited a response, numerous serotonergic elements were evident. 6-Hydroxydopamine treatment did not cause significant alteration in the prolactin response. Lesion of the medial, largely parvocellular subdivision of the PVN, or horizontal knife cuts below this cell group, blocked the hormone response. The findings demonstrate for the first time that: (i) interruption of the connections between the brain stem and the hypothalamus interferes with the prolactin response to the suckling stimulus; (ii) serotonergic fibres terminating in the hypothalamic PVN are involved in the mediation of the suckling stimulus; and (iii) within the PVN, neurones in the medial, largely parvocellular subdivision of the cell group take part in the transfer of the neural signal, eventually inducing prolactin release.

Salsolinol is a putative endogenous neuro-intermediate lobe prolactin-releasing factor.

The isolation and identification of a prolactin-releasing factor (PRF) from the neuro-intermediate lobe of the pituitary gland has been pursued for over a decade. Using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD) and gas chromatography/mass spectrometry (GC/MS) (R)-salsolinol (SAL) (a dopamine-related stereo-specific tetrahydroisoquinoline) was found to be present in neuro-intermediate lobe as well as median eminence extracts of male, intact-, and ovariectomized female rats. Moreover, analysis of SAL concentrations in neuro-intermediate lobe revealed parallel increases with plasma prolactin in lactating rats exposed to a brief (10 min) suckling stimulus following 4-h separation. SAL appears to be a selective and potent stimulator of prolactin secretion in vivo and it was without effect on the secretion of other pituitary hormones. We have also found that SAL can elevate prolactin release, although to a lesser extent, in pituitary cell cultures as well as in hypophysectomized rats bearing anterior lobe transplants under the kidney capsule. Lack of interference of SAL with [3H]-spiperone binding to AP homogenates indicates that SAL does not act at the dopamine D2 receptor. Moreover, [3H]-SAL binds specifically to homogenate of AL as well as neuro-intermediate lobe obtained from lactating rats. Taken together, our data clearly suggest that SAL is synthesized in situ and this compound can play a role in the regulation of pituitary prolactin secretion.

Effect of posterior pituitary denervation (PPD) on prolactin (PRL) and alpha-melanocyte-stimulating hormone (alpha-MSH) secretion of lactating rats.

Previous data have clearly suggested that the posterior pituitary (PP), consisting of neural lobe (NL) and intermediate lobe (IL), has a role in the control of anterior pituitary PRL secretion. However, basic aspects of this regulatory mechanism like (1), the role of an intact hypothalamic innervation of the PP as well as (2) the site of production of previously found PRL releasing substance(s) have not yet been characterized. Denervation of the PP (PPD) is an effective method for having a selective lesion of the innervation of PP, indeed, PPD results in a disappearance of neurosecretory materials from NL and tyrosine hydroxylase (TH) immunoreactivity from IL, leaving blood supply of all three lobes intact. Blood samples were taken from freely moving sham an PP-denervated lactating rats before and after 4-h separation from their pups and during the suckling stimulus. PPD blocks separation-induced depletion but only attenuates suckling induced release of PRL. Furthermore, it doubles plasma level of alpha-MSH during the entire sampling period, which has been used as a marker for in vivo secretory activity of IL cells. Lack of the separation-induced depression in plasma PRL of PPD animals can be partially restored by normalizing the diabetes insipidus with treatment of a vasopressin analogue, 1-desamino-8-D-arginine-vasopressin (dDAVP). In contrast, dDAVP, neither alone nor in combination with oxytocin (OXY), can change PPD-induced elevation of plasma alpha-MSH as well as attenuation of PRL response induced by suckling. It is concluded that: (1) contribution of the THDA system parallel to the confirmed role in the regulation of alpha-MSH seems to be crucial for the depletion of plasma PRL induced by separation but not for the elevation due to suckling stimulus, (2) intact hypothalamic innervations of both NL and IL, regulating water intake and the secretion of alpha-MSH, respectively, are necessary for normal secretory responses of AL during lactation, (3) as well as for the presence of PRF activity in PP, (4) which does not solely responsible for suckling-induced PRL release. Therefore, an interplay between several substances produced by NIL of the pituitary gland must have been responsible for the intact regulation of PRL secretion during lactation.

Effect of adrenalectomy and dexamethasone treatment on prolactin secretion of lactating rats.

The contribution of corticosteroids to the control of prolactin secretion in lactating rats was investigated. The prolactin response to domperidone (20 microg/kg b.w., i.v.), a dopamine receptor antagonist and to domperidone plus formalin stress was tested in adrenalectomized and/or dexamethasone-treated continuously nursing rats. Animals were adrenalectomized on the 3rd day of lactation and tested on the 7th day of lactation. Dexamethasone was injected s.c. 24 h before testing (400 microg/kg b.w.) and on the day of testing (200 microg/kg b.w.). Domperidone caused a significant rise in plasma prolactin levels. The prolactin response to domperidone was twice as high in solely adrenalectomized dams and in solely dexamethasone-treated rats compared to controls. In adrenalectomized animals treated with dexamethasone, the prolactin response to domperidone was like in controls. Formalin injection to either adrenalectomized plus domperidone-treated animals or to animals injected with dexamethasone plus domperidone, resulted in a statistically significant depletion of plasma prolactin. In controls and in adrenalectomized animals receiving dexamethasone and domperidone, the prolactin response to formalin was very similar, i.e., plasma prolactin levels did not change after the administration of formalin. The present findings suggest that in lactating rats, corticosteroids are involved in the prolactin response to domperidone and to formalin stress.

Effect of neonatal treatment with monosodium glutamate on dopaminergic and L-DOPA-ergic neurons of the medial basal hypothalamus and on prolactin and MSH secretion of rats.

The effect of neonatal treatment with monosodium L-glutamate (MSG) on the dopaminergic systems of the medial basal hypothalamus has been investigated using tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) immunocytochemistry. Changes in plasma levels of prolactin (PRL) and alpha-melanocyte-stimulating hormone (MSH) have also been determined in intact and in MSG-treated rats after inhibition of TH by alpha-methyl-p-tyrosine (alpha-MpT) or without inhibition of enzyme activity. Monosodium glutamate resulted in a 40% reduction in the number of TH immunopositive tuberoinfundibular neurons, but no change in the number of AADC-positive tuberoinfundibular nerve cells, indicating that this reduction has occurred mainly in TH-positive but AADC-negative elements, i.e., in L-DOPA-ergic neurons. In contrast, MSG did not cause changes in the number of TH and AADC immunoreactive neurons of the periventriculohypophysial and tuberohypophysial dopaminergic systems, and it did not influence basal plasma PRL levels. alpha-methyl-p-tyrosine has increased plasma PRL concentrations in both control and MSG-treated rats of both sexes, but significantly higher responses were detected in females. None of the treatments had any effect on plasma MSH level. These findings suggest that MSG affects primarily L-DOPA-ergic neurons located in the ventrolateral part of the arcuate nucleus, but not dopaminergic neurons situated in the dorsomedial part of the arcuate nucleus; neither PRL nor MSH secretion is altered by MSG; a significant sex difference exists in the pituitary PRL response to inhibition of TH, and this response is not affected by MSG.

Effects of anandamide (endogen cannabinoid) on anterior pituitary hormone secretion in adult ovariectomized rats.

It has been shown that the main psychoactive component of marihuana, delta 9-tetrahydrocannabinol (THC) has mainly inhibitory effects on pituitary luteinizing hormone (LH), prolactin (PRL) and growth hormone (GH) and has no or little effect on follicle stimulating hormone (FSH) secretion. Recently the purification and availability of the putative endogenous ligand for the cannabinoid receptor, anandamide (arachidonyl ethanol-amide, anandamide) (ANA) provided us the opportunity to compare the effects of THC and ANA on the female neuroendocrine system in ovariectomized (OVX) rats. OVX was performed three weeks prior to the experimental period to avoid cyclic differences. OVX rats were injected i.p. with either THC or ANA (0.02 mg/kg.b.w./day, respectively) or vehicle for two weeks. The results show that both ANA and THC decrease serum LH level although THC with a higher degree. No significant differences were observed in serum FSH level. Both drugs decreased serum PRL. Serum GH was increased after THC administration and significantly decreased after ANA. The results indicate that ANA and THC alter pituitary hormone secretion, mainly by inhibitory action. The site of action requires further investigations.

Rat milk stimulates pituitary growth hormone secretion of neonatal pituitaries in vitro.

Aqueous extracts of rat milk stimulated growth hormone (GH) secretion from superfused pituitaries of two-day old rats. The GH stimulatory effect of milk increased with the time elapsed postpartum; growth hormone releasing hormone and thyrotropin releasing hormone seem to be the major milk borne GH releasing factors. These results indicate that milk intake may play a role in maintaining the high plasma GH levels observed in the neonatal period.

The effects of cannabinoids on the regulation of reproduction.

It has been shown that the main psychoactive component of marihuana, delta9-tetrahydrocannabinol (THC) has mainly inhibitory effects on the regulation of reproduction. Recently, the purification and availability of the endogenous ligand of the cannabinoid receptor, arachidonyl ethanolamide, anandamide, (ANA) and its specific long lasting antagonist, the SR 141716 (SR) provided us the opportunity to compare the effects of THC and ANA on the neuroendocrine regulation of reproduction. ANA decreases serum luteinizing hormone (LH) and prolactin (PRL) levels in rats of both sexes. It has no action on serum follicle stimulating hormone (FSH) level. When ANA was administered to pregnant rats it resulted in an increase of the duration of pregnancy and in the frequency of stillbirths. The postnatal development of hypothalamo-pituitary axis in offspring was temporarily inhibited. In conclusion, we found that exogenous and endogenous cannabinoids have only slightly different effects on the reproductive parameters. These effects may occur via the central cannabinoid receptor. It is possible that the sites of action are at both hypothalamic and pituitary levels. The results further support the view that ANA may be a central neurotransmitter or neuromodulator.

In vitro control of prolactin (PRL) and growth hormone secretion of neonatal rat pituitary glands: effects of ovine PRL, salmon calcitonin, endothelin-3, angiotensin II, bromocryptine and somatostatin.

Bromocryptine potently decreased prolactin (PRL) secretion of pituitary glands of 2-day-old rats in vitro (up to 85% inhibition; ED50 between 0.1 and 1.0 nM) without altering the bioactivity to immunoreactivity (B/I) ratio. Bromocryptine tended to suppress growth hormone (GH) secretion although the effect did not reach statistical significance. Angiotensin-II (A-II; 1-1000 nM) stimulated PRL secretion in a dose-dependent manner without affecting secretion of GH. The B/I ratio of PRL secreted in response to A-II was increased. Somatostatin (SRIF) had no effect on PRL secretion but inhibited GH secretion in a dose-dependent manner; significant inhibition (50%) was observed at 100 nM. A 6-h exposure to ovine PRL (oPRL) in concentrations equipotent with 1.2-120 ng/ml rat PRL (rPRL) in the Nb2 bioassay had no effect on immunoreactive rPRL secretion. Salmon calcitonin (sCT) and endothelin-3 (ET-3; 0.1-100 nM) failed to inhibit secretion of PRL or GH. PRL secretion was slightly stimulated by sCT with no apparent dose-response relationship. The present findings suggest that neonatal pituitary glands do not display autoregulation of PRL secretion, and sCT and ET-3 (either endogenous or milk-derived) may not function as PRL inhibiting factors in 2-day-old pups. Thus, the receptors of PRL, sCT and ET-3 on lactotropes, or their functional coupling with inhibition of basal PRL secretion, occur at a later stage of development. The specificity of the PRL releasing factor (PRF) activity of A-II at this age is unique for established PRFs and might reflect a physiological function of PRL in osmoregulation. The increased B/I ratio of PRL secreted in response to A-II may be due to the release of specific PRL variants, and might be a sign of functional heterogeneity among lactotropes. The differential sensitivity of PRL and GH to the applied secretagogues suggests that the intracellular regulation of PRL and GH are compartmentalized in the mammosomatotrope cell.

Neuroendocrine control of immunoreactive growth hormone and bioactive prolactin secretion in neonatal rats: ontogeny and interactions between the serotonergic, cholinergic and alpha 2-adrenergic systems.

The effects of the alpha 2-agonist clonidine (CLO), the serotonin (5-HT) precursor 5-hydroxy-L-tryptophan (5-HTP), the 5-HT2/histamine (H1) antagonist cyproheptadine (CYPRO), the muscarinic cholinergic antagonist atropine (ATR), and an affinity-purified polyclonal anti-rat growth hormone-releasing hormone (rGHRH) immunoglobulin on serum concentrations of growth hormone (GH) and prolactin (PRL) were tested in 2- and 10-day-old litter-mate rat pups. Serum levels of GH and PRL were detected in RIA and Nb2 lymphoma bioassay, respectively. The effects of two different drugs either alone or in combination with each other were evaluated by two-factor analysis of variance. The data indicated that secretion of GH and PRL was regulated by alpha 2-adrenergic, serotonergic and cholinergic mechanisms; the pathways regulating the two hormones, however, were distinct. 5-HTP stimulated GH secretion as early as day 2 postpartum via cholinergic mechanisms not involving GHRH; this pathway was also present in 10-day-old pups. An additional serotonergic pathway was functional in 10-day-old pups which mediated CLO-induced release of GH, and did not include cholinergic transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin-synthesizing and prolactin-releasing activity of fetal and early postnatal rat pituitaries: in vivo and in vitro studies using RIA, reverse hemolytic plaque assay and immunocytochemistry.

In vivo and in vitro prolactin (PRL)-synthesizing and PRL-releasing activity of fetal (days 12-22) and early postnatal (days 1-10 after birth) rat pituitaries were studied by means of radioimmunoassay (RIA), reverse hemolytic plaque assay and immunocytochemistry. Using RIA, PRL could first be detected, both in the pituitary and in the serum, on day 17 of fetal development. From this day on, pituitary PRL gradually increased, the rise was particularly marked during the postnatal period and became depressed for the first 10 days of postnatal life. On fetal day 18, 12-15% of monodispersed pituitary cells displayed PRL immunopositivity, but only 3-5% of PRL-positive cells were plaque-forming, i.e. released PRL. By the end of gestation 19-25% and on postnatal day 10 42-45% of all pituitary cells were PRL cells and 31-35 and 15-17% of PRL-positive cells, respectively released PRL. Both pre- and postnatal PRL cells in monolayers were insensitive to TRH treatment. Pituitary primordia immunocytochemically and radioimmunologically negative for PRL (13- to 14-day-old fetal) when placed in serum-free organ culture were able to synthesize and release PRL. Fetal pituitary exhibited a highly regular increasing pattern of daily PRL release during a 7-day-culture period. Data obtained both in vivo and in vitro did not exhibit any sex differences. The present findings are consistent with all those observations suggesting an early emergence of fetal rat pituitary lactotrophs. The in vitro results support the concept that Rathke's pouch cells have substantial degree of independence from extrapituitary regulatory actions in the expression and further progression of specific functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat milk inhibits the mitogenic response of Nb2 lymphoma cells to prolactin.

Prolactin-like bioactivity in the rat milk was observed using the lactogen specific Nb2 lymphoma assay. The water soluble fraction (infranatant) of pooled milk samples obtained on days 2, 12 or 22 postpartum stimulated Nb2 cell growth in the range of 0.08-2.5 microliters/well. Higher concentrations of day-12 and day-22 (but not day-2) milk infranatant, however, decreased Nb2 proliferation in a dose-dependent fashion. [3H]-thymidine incorporation used as an indicator of cell growth was decreased by 21%, 49% and 83% at the doses of 5, 10, 20 microliters/well concentrations of day-22 milk infranatant, respectively. Milk infranatant did not reduce cell viability as assessed by Erythrosin B exclusion test. Addition of exogenous rat PRL (NIH B-6) at concentrations of ED50-ED90 did not restore the Nb2 proliferation rate decreased by milk infranatant. Saturating doses of PRL (ED100-ED400) resulted in maximal cell growth, but failed to counteract the inhibitory effect of milk infranatant. The relative molecular weight of the putative Nb2 cell inhibitor of rat milk is between 10 kDa and 30 kDa as determined by ultrafiltration and dialysis. The inhibitory activity of milk infranatant is stable at physiological pH, but is destroyed upon acidification. Thirty min of incubation at 37 degrees C enhanced but 30 min of incubation at 100 degrees C only slightly decreased the calculated total inhibitory effect of milk infranatant. These initial results indicate the presence of a water-soluble antimitogenic factor in rat milk (rMAF) which inhibits the Nb2 lymphoma cell response to prolactin in a non-competitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular heterogeneity of prolactin in lactating rats and their pups: biological and immunological activities in pituitary gland, serum and milk.

Prolactin (PRL)-like bioactivities (in Nb2 lymphoma assay), immunoreactivities (in RIA) and B/I ratios in rat milk, maternal and neonatal pituitary glands and sera were investigated. The PRL-like bioactivity in the water-soluble fraction of rat milk (infranatant prepared by ultracentrifugation) exceeded its immunoreactivity 3-7-fold. The elevated B/I ratio was in part due to the presence of a glycosalated PRL (G-rPRL)-like material, since 5-70% of the PRL-like bioactivity was recovered from the glycosylated fraction of rat milk infranatant prepared by concanavalin-A affinity chromatography. We were unable to detect PRL-like immunoreactivity in the glycosylated fraction of rat milk, and calculated that the maximal cross-reactivity of G-rPRL in the RIA is less than 3.8%. In day 12 milk, over 80% of the G-rPRL-like bioactivity eluted from a Sephadex G-100 column as a high apparent molecular weight (Mr) substance (approximately 50 kD), while the rest eluted as a monomeric G-rPRL (24-25 kD). The PRL-like bioactivity in the nonglycosylated fraction eluted in three peaks (Mr: 50, 24 and 16 kD), while two immunoreactive peaks occurred (Mr: 24 and 8 kD). The concentration of rPRL-like immunoreactivity in rat milk increased during the first days of lactation, remained high in midlactation, and declined by the end of lactation. The PRL-like bioactivity in the nonglycosylated fraction of rat milk displayed a similar timecourse. G-rPRL-like bioactivity in rat milk, however, changes inversely, i.e. decreased between days 2 and 18 postpartum then increased by day 22. The concentration of high Mr PRL-like bioactivity in rat milk was greatly reduced by day 22 from day 2 postpartum. No PRL-like bioactivity or immunoreactivity was recovered from the IgG fraction (prepared by protein A affinity chromatography) of rat milk. The B/I ratio in day 2 maternal pituitary glands was close to 1. In neonatal pituitaries and in maternal sera, however, the B/I ratio was slightly elevated (2-3). The B/I ratio in day 2 neonatal serum was between 6 and 22, while the B/I ratio of PRL secreted by day 2 neonatal pituitary glands in vitro was 1. The present results demonstrate that the concentrations of PRL in rat milk and neonatal serum have been grossly underestimated because levels were detected by RIA. The high B/I ratio reflects the presence of PRL variants. Milk appears to be the most likely source of PRL variants in the circulation of the neonate.(ABSTRACT TRUNCATED AT 400 WORDS)

Separation and suckling-induced changes in serum growth hormone levels of lactating rats and their pups.

The effects of separation and suckling on serum growth hormone (GH) levels of lactating rats and their pups were studied on days 1-3 of lactation. The litter size was adjusted to 8 pups/dam without respect to sex. The separation of pups from their mother for 5 h resulted in significant decrease in GH level in the pups. After 30 or 60 min of suckling the level of GH in serum of the pups reached the values of the control group, i.e. non-separated pups. If the pups were attached to pre-suckled mothers, thus consuming less milk, their serum GH levels increased in a significantly lesser extent. It is concluded that some factor(s) are present in milk which stimulate(s) GH secretion of the pups. Due to episodic secretion, high standard error occurred in every group of mothers studied. Mean GH level of freely behaving lactating rats were rather constant. On the other hand, the 5 h separation period and 30 min of suckling seemed to result in decrease and increase in serum GH values, respectively. By the end of a 60 min suckling period, GH declined to low levels again, hence it is likely that the separation and suckling stimuli synchronized GH-episodes. When the litter was replaced with hungry foster pups at 30 min of suckling, GH-decay by 60 min appeared to be less consequent.

Dual role of glucocorticoids in suckling-induced prolactin secretion.

The exact contribution of corticosteroids to the control of prolactin secretion in lactating rats is poorly understood. Therefore, the present studies were focused on the effect of adrenalectomy and dexamethasone treatment on the suckling-induced prolactin release. Animals were adrenalectomized on the 3rd day of lactation and tested on the 7th day of lactation. In adrenalectomized animals, the suckling stimulus failed to induce the characteristic increase in plasma prolactin levels. Dexamethasone pretreatment (400 microg/kg b.w. s.c. 24, 48, 72 h before testing) of adrenalectomized rats restored this prolactin response. The same treatment with dexamethasone given to control animals attenuated the suckling stimulus induced prolactin response. The present findings indicate that corticosteroids are essential for a basic prolactin response of lactating rats.

Evidence of prolactin immunoreactivity in the bone marrow of untreated multiple myeloma patients.

The role of prolactin (PRL) in the physiological regulation of the immune system and in hematopoiesis is well known. There is also evidence of the significance of PRL in several pathological conditions such as autoimmune diseases and some malignancies, e.g. colon and breast carcinomas and also B cell malignancies. Multiple myeloma is known as a B cell malignancy. It is the result of malignant transformation of a single clone of neoplastic plasma cells that synthesize abnormal amounts of monoclonal immunoglobulins or immunoglobulin fragments. In our present studies, the possible expression of PRL in bone marrow cells obtained from diagnosed multiple myeloma (17 cases) or nonmyeloma (5 cases) patients was examined by the method of immunocytochemistry. Samples obtained from those multiple myeloma patients (13 cases) who had not received chemotherapy for 6 months prior to these studies showed a positive immunocytochemical reaction for PRL. Bone marrow smears of patients diagnosed with multiple myeloma who had received chemotherapy within 6 months of the study and also the smears of patients without diagnosed multiple myeloma failed to show a positive immune reaction for PRL. In the case of a patient who was examined prior to and also after a period of 3 months of chemotherapy, the PRL-immunopositive bone marrow cells had disappeared due to the treatment. According to the light microscopic analysis of the cell morphology, PRL-immunopositive cells in the bone marrow were mainly, but not exclusively, plasma cells. There was no correlation between the positive PRL staining of cells and the type of monoclonal immunoglobulin or the ratio of plasma cells detected in the bone marrow. Taken together, our results indicate a possible role of PRL in multiple myeloma. Further experiments are necessary to identify the prognostic value of PRL in multiple myeloma.

Mammary epithelial cells of lactating rats express prolactin messenger ribonucleic acid.

The presence of prolactin (PRL) mRNA in the mammary gland, placenta, and pituitary gland of lactating and pregnant rats was investigated by polymerase chain reaction (PCR). Polyadenylated RNA was prepared from total RNA samples by oligo(dT)-cellulose chromatography, and complementary cDNAs were synthesized. A standardized amount of cDNA from each sample was used as the template in a Taq PCR under high-stringency conditions. PCR amplified a signal with the predicted size of approximately 375 bp in mammary and pituitary glands of lactating and pregnant rats, and in placentae of pregnant rats. This band specifically hybridized with a probe overlapping the entire sequence of the mature rat (r) PRL mRNA in Southern blot analysis. When the rPRL-specific primers were used, PCR revealed no signal in the liver or in lactating mammary gland explants cultured in vitro for 48 h, while the same cDNA preparations gave strong signals for beta-actin. The viability of the mammary gland explants was also suggested by their ability to secrete immunoreactive casein in vitro. PRL mRNA was localized in the epithelium of alveoli and ducts of the lactating mammary gland by in situ hybridization. These data provide evidence that the PRL gene is expressed in the mammary gland of pregnant and lactating rats, and suggest that the mammary gland might contribute to PRL in milk by de novo synthesis. Thus, while the placenta is an exogenous source of PRL-like activities for the fetus in utero, the mammary gland might take over this function after birth.