Evidence for an inhibitory influence of rat placental lactogen on prolactin release in vitro

Incubation of placental tissue from Day 11 pregnant rats for increasing periods of time resulted in proportionately more rat placental lactogen (rPL) release. The amount of placental tissue incubated correlated directly with the amount of rPL released into the medium. When placentas were coincubated with anterior pituitaries from ovariectomized rats, prolactin release was significantly inhibited. When media from incubations which had contained varying numbers of Day 11 placentas for 24 h were added to vials containing anterior pituitaries, prolactin release was inhibited, proportionate to the amount of rPL in the media. Media from incubations of Day 9 placentas, which contained very little rPL, had no effect on prolactin release. When medium containing anterior pituitary tissue was incubated for 24 h, pituitaries removed, and the medium incubated with placental tissue for an additional 24 h, there was no difference in prolactin levels compared to incubation medium not containing placental tissue. Addition of a trypsin inhibitor to the medium containing placental tissue did not augment the amount of prolactin remaining after a 24-h incubation. Thus it would appear that the placenta does not release a substance into the medium that destroys prolactin. This suggests that secretions from the placenta, presumably rPL, can exert a negative feedback on prolactin secretion at the level of the anterior pituitary.     

Inhibition of nocturnal prolactin surges in the pregnant rat by incubation medium containing placental lactogen

Rats hysterectomized on Day 7 or 8 of pregnancy continued to have nocturnal prolactin surges 1 day later. Conditioned medium obtained from incubation of Day 11 placentas infused via the jugular vein completely blocked this nocturnal surge, indicating a negative feedback of placental secretions on prolactin. Infusion of an ultrafiltrate of the conditioned medium which only contained molecules with Mr above 10,000 also blocked the prolactin surge. Next, it was determined whether this feedback of placental secretions on prolactin may work by way of hypothalamic dopamine. Levels of dopamine in hypophysial stalk blood from pregnant rats on Day 12, a time when secretion of placental lactogen is high, were not different from those in rats in which placental lactogen was absent. It is concluded that termination of prolactin surges at midpregnancy may be due to feedback of placental secretions, possibly placental lactogen, on the hypothalamus and/or pituitary. However, these experiments do not support the hypothesis that this inhibition is mediated by alteration in hypothalamic dopamine secretion.     

Rat placental luteotropin: initial secretion and luteolytic quality

The secretion of placental lactogen begins early in pregnancy. Previous studies indicate that rat placental lactogen (rPL) is secreted from Day 8 of pregnancy and that it is luteolytic as well as luteotrophic. This study establishes the onset of both the luteotrophic and the luteolytic effects of placental lactogen in pregnant rats subject to timed hypophysectomy. Pregnancy was preserved in all groups with the administration of dydrogesterone (9 beta, 10 alpha-pregna4,6-diene-3, 20 dione), a progesterone analog, and diethylstilbestrol, an estrogen analog. Plasma progesterone and 20 alpha-hydroxypregn-4-ene-3-one (20-OHP) were measured in serial serum samples by RIA. The data indicate that rPL is secreted as early in pregnancy as the seventh day. Rats hypophysectomized on Day 6 of pregnancy or later had ovaries that contained corpora lutea that secreted increasing quantities of progesterone during pregnancy. On Day 16 serum progesterone values were lowest in animals operated on Days 4 and 5 compared to animals operated on Days 6 or 8. The 20-OHP serum values from animals operated on Days 4 and 5 declined steadily from Day 8 to Day 16. These findings indicate progestational incompetency, which was confirmed morphologically. Thus, rPL secretion begins by Day 7 and it is both luteotrophic and luteolytic.     

Gallium-67 distribution in pregnant mammals.

Studies on the distribution of 67Ga in pregnant rabbits showed a marked concentration of the isotope in uterine tissue and flushings of 5-day pregnant rabbits 24 hours after isotope injection. The isotope no longer localized throughout the uterine tissues and flushings on injection of 7- to 8-day pregnant rabbits but was specifically associated with the blastocysts and sites of implantation. As the gestation time progressed 67Ga increasingly concentrated in placental and mammary tissue while the concentration in serum and thymus tissue decreased. The marked concentration in placental tissue was readily discernible on total-body scans of pregnant rabbits. Column chromatography of uterine washings and placental extracts from pregnant rabbits and thymus extracts from estrous rabbits showed most of the isotope associated with moieties greater than or equal to 200,000 MW whereas the isotope in "milk" and extracts of mammary tissue was bound to components of 25,000-35,000 MW. Extracts of thymus from pregnant rats showed a marked, selective loss of 67Ga binding in two peaks of approximately 50,000 and 120,000 MW compared to the chromatographic profile of extracts from normal rat thymus.     

Investigation of alkaline phosphatase activity in the serum of pregnant rats

An investigation was undertaken to determine if the placental alkaline phosphatase of the rat enters the maternal circulation and to study some of its characteristics. Unlike human, rat placental alkaline phosphatase was found to be heat labile and the alkaline phosphatase activity in the serum of both pregnant and non-pregnant rats was also found to be heat labile. Also unlike the human, the alkaline phosphatase activity in rat serum does not increase as pregnancy progresses to term. In an endeavour to establish if the rat placental enzyme is present in the serum of the pregnant rat, the characteristics of the enzyme in both placental extracts and serum of non-pregnant and 1-, 2- and 3-week pregnant rats were studied using the techniques of heat stability at 56°, gel filtration through Sephadex columns, disc gel electrophoresis, and L-phenylalanine inhibition. The presence of rat placental alkaline phosphatase in maternal serum could not be positively demonstrated by any of these procedures, suggesting that rat placental alkaline phosphatase does not enter the maternal serum.     

Physiological involvement of placental endothelin-1 and prostaglandin F2alpha in uteroplacental circulatory disturbance in pregnant rats exposed to heat stress

Several studies suggest that heat stress affects placental functions including uteroplacental circulation, subsequently leading to pregnancy failure and birth weight reduction. To clarify the involvement of endothelin and placental prostaglandin (PG) systems in the uteroplacental circulation during heat stress, we examined the effects of i.v. administration of the endothelin receptor antagonist bosentan and the cyclooxygenase inhibitor indomethacin on uteroplacental blood flow and on placental PGE2 and PGF2alpha levels and their 13,14-dyhydro-15-keto-metabolites (PGEM and PGFM, respectively) in heat-exposed or non-heat-exposed pregnant rats. The administration of bosentan or indomethacin did not change uteroplacental blood flow in non-heat-exposed pregnant rats. In contrast, heat reduced uteroplacental blood flow in pregnant rats, but the reduction was reversed by the administration of bosentan or indomethacin before heat exposure. Heat did not change placental PGE2 or PGEM levels, but in pregnant rats it increased placental PGF2alpha and PGFM levels, which were reversed by bosentan or indomethacin. Our results suggest that the activation of placental endothelin receptor and PGF2alpha systems are involved in the uteroplacental circulatory disturbances produced by heat. PGF2alpha systems activated by heat may be involved in the vasoconstricting effects of endothelin-A and -B receptors during heat exposure.     

Interrelation of prolactin and calcitonin

Intraperitoneal prolactin injection (3.5 U/200 g bw, daily, for 5 days) caused a marked rise in blood calcitonin concentration in female Wistar rats. It is common knowledge that exogenous calcitonin administration results in an obvious drop of blood prolactin level. Hence, the interrelation between anterior pituitary lactotropic function and blood calcitonin level may be regarded as a negative feedback, since prolactin activates calcitonin production and secretion, with the latter, in its turn, inhibiting adenohypophysial lactotropic function.     

Demonstration of potent, gonadotropin-like biological activity in the serum of rats during midpregnancy

The gonadotropin-like activity (GnLa) of serum from pregnant rats was measured using the rat interstitial cell testosterone (RICT) bioassay. Serum GnLA was elevated on day 9 of pregnancy, peaked at 7.2 μg rat LH-RPl equivalents/ml on day 11 and declined to undetectable levels by day 15. Serum LH, measured by homologous RIA, was consistently low (<20 ng/ml) during pregnancy, except near term.Rat placental lactogen (rPL), which was measured in the same serum samples by rat radioreceptor assay (RRA), reached maximal concentrations on days 12 and 13 of pregnancy.These data suggest the presence in pregnancy serum of a potent-gonadotropin-like hormone, different from pituitary LH, whose origin is unknown. Furthermore, there are discrepancies between the times of appearance of this GnLA and rPL.     

Suckling-induced prolactin release potentiates mifepristone-induced lactogenesis in pregnant rats

Suckling, starting at 19:00 h on Day 18 of pregnancy, induced a significant increase in serum prolactin concentration at 20:00 h on Day 19 of pregnancy, but no increase in mammary gland casein or lactose content. Mifepristone (2 mg/kg) injection at 08:00 h on Day 19 of pregnancy induced significant increases in casein, but not in lactose, 24 h after administration. Mifepristone alone did not induce prolactin secretion, indicating that lactogenesis was induced by placental lactogen in the absence of progesterone action. When mifepristone was injected into suckling rats, serum prolactin concentrations were higher than in the untreated suckling rats. Casein in these rats increased significantly 12 h after mifepristone administration and lactose at 24 h after. If the suckling mifepristone-treated rats were given two injections of bromocriptine (1.5 mg/kg) at 12:00 h on Days 18 and 19 of pregnancy, serum prolactin concentrations were not increased by suckling, but casein and lactose concentrations in the mammary gland showed values similar to those obtained in the mifepristone-treated non-suckling rats. Mifepristone can therefore potentiate suckling-induced prolactin release in pregnant rats, demonstrating a direct central inhibitory action of progesterone on prolactin secretion. This suckling-induced prolactin secretion, unable to induce casein or lactose synthesis in the presence of progesterone, enhanced significantly synthesis of these milk components in the absence of progesterone action (rats treated with mifepristone). Fatty acid synthase, which is stimulated by the suckling stimulus in lactating rats, was not modified by mifepristone or suckling in pregnant rats.     

Catecholamine mechanisms in the feedback effects of estradiol benzoate on the release of LH and prolactin

The role of hypothalamic catecholamines and luteinizing hormone releasing hormone (LHRH) in the negative feedback effect of estradiol benzoate (EB) on luteinizing hormone (LH) release was studied in chronic ovariectomized rats. Administration of 10 micrograms EB decreased plasma LH levels and increased LHRH content in the medial basal hypothalamus (MBH) 1 day after injection. Inhibition of dopamine and norepinephrine synthesis with alpha-methyl-p-tyrosine (alpha-MT) reduced the LHRH content in the MBH in both oil- and EB-treated animals and partially reversed the decrease in plasma LH levels. Inhibition of norepinephrine synthesis with fusaric acid decreased LHRH content in both oil- and EB-treated rats but had no effect on plasma LH levels. The results suggest that at least a portion of the inhibitory effect of EB on LH release is due to the stimulation of an inhibitory dopaminergic mechanism which reduces LHRH release from the MBH. This feedback mechanism is apparently not susceptible to dopaminergic receptor blockade since administration of pimozide had no effect on LH levels. The stimulatory feedback effect of EB on prolactin release was studied in the same animals. alpha-MT and EB produced additive effects on plasma prolactin levels whereas fusaric acid blocked the EB-induced increase in plasma prolactin levels. Pimozide appeared to potentiate the effect of EB on prolactin release. The results reconfirm the possible role of noradrenergic neurons in the release of prolactin induced by EB and also suggest that EB stimulates a dopaminergic mechanism which is inhibitory to prolactin release but is normally masked by increased noradrenergic activity.     

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature,pubertal, pregnant,lactating and ovariectomised rats

Summary We have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a ³²P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.     

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature, pubertal, pregnant, lactating and ovariectomised rats

We have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a 32P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.     

Acute embolization of the uteroplacental circulation: uterine blood flow and placental CO diffusing capacity

In an effort to determine to what extent the fetal sequalae following repeated embolization result from decreased area of placental exchange or from decreased uterine blood flow, we injected microspheres into the uterine circulation of the pregnant ewe. We measured total UBF continuously and sampled fetal blood gases in 6 chronically instrumented ewes following repeated injections of 1 to 2 million 25 mu microspheres into the common internal iliac artery at 30 min intervals. Embolization resulted in an immediate 25 to 30% drop in uterine flow, with partial recovery to about 85% of its control value within 30 min after injection. A linear relation existed between uterine blood flow and fetal O2 tension. A slightly accelerated decrease in O2 content with a more rapid increase in CO2 tension and [H+] was seen when uterine flow decreased below 150 ml X min-1 X kg fetal wt-1. Following repeated injections fetal descending aortic O2 tension and content decreased 34 and 82% respectively, while PCO2 and [H+] increased 28 and 84% respectively. Placental diffusing capacity for CO increased 117% after repeated embolization. Most of this increase could be accounted for by the fetal hypoxia and acidosis, although some of it may have resulted from distension or recruitment of vessels in the placental exchange area, or a more uniform distribution of placental blood flows. These studies suggest that the acute changes in fetal blood gas values following embolization result from a reduction in blood flow rather than from a reduced placental exchange area.     

Effects of acute and chronic trazodone administration on serum prolactin levels in adult female rats

Trazodone was tested for its ability to elevate serum prolactin levels in mature female rats. When the drug was administered acutely to female rats at doses up to 80 mg/kg ip, it induced a clear rise in serum prolactin levels, with a minimum effective dose of 20 mg/kg; blood trazodone levels at these doses were between 1.6–2.4 μg/ml. However, trazodone could not be considered to be a potent stimulator of prolactin secretion, since the injection of haloperidol at 2 mg/kg elevated serum prolactin to values twice those seen in animals receiving the 80 mg/kg dose of trazodone. When trazodone was administered chronically in the diet for two or four weeks, at an average daily dose of 80 mg/kg, serum trazodone levels were found to be 100–200 ng/ml when measured at each stage of the estrous cycle. Serum prolactin levels in trazodone-treated animals, however, did $\text{not}$ differ from those in control rats. Moreover, drug-treated animals showed normal proestrus surges in serum prolactin. The results of these studies thus indicate that acutely, at very high doses, trazodone probably can stimulate prolactin secretion modestly in female rats. However, when consumed chronically at 80 mg/kg/day, the drug has no effects on serum prolactin levels. Therefore, if trazodone stimulates prolactin secretion by altering neurotransmission across dopamine and/or serotonin synapses in brain, it is probably not potent in these actions, at least as concerns those dopamine and serotonin neurons that influence the secretion of prolactin.     

Initial permeability of the 19-day foetus to nickel

The administration of 63NiCl2 to 19-day pregnant rats produced a limited transfer of nickel to the foetus. An experimental model based on the incubation of the conceptuses still connected to the maternal organism resulted in an appreciable net transfer of nickel through the amniotic membranes. The placenta retained a significant amount of radioactive nickel as did the lining of the uterine wall. The circulation of nickel through the foetus following the direction maternal blood----placenta----foetus----amniotic membranes----endometrium----maternal venous blood, is postulated. This transfer could provide an additional protection to placental barrier against metal toxicity.     

Bacterial endotoxin induced hypothermia in pregnant rats: Role of tumor necrosis factor-α

Intraperitoneal (ip) administration of the lowest dose of Escherichia coli lipopolysaccharide (LPS) that elicits a maximal febrile response in non-pregnant rats when studied in a neutral ambient temperature (EC₁₀₀—160 μg/kg) produces a transient “regulated” hypothermia in near-term pregnant rats. The current experiments have been carried out to determine the role of tumor necrosis factor-α (TNF-α) in mediating this hypothermic response. Chronically instrumented non-pregnant and pregnant rats were housed and studied in a neutral ambient temperature and allocated to one of two experimental series depending upon whether they received ip recombinant rat TNF-α (rrTNF-α) in doses ranging from 0.1 to 1000 μg/kg or they received an antibody to tumor necrosis receptor I (TNF R1 Ab) – which neutralizes its cell surface mediated activity – before receiving an EC₁₀₀ dose of E. coli LPS. Intraperitoneal rrTNF-α elicited fevers in non-pregnant but not in near-term pregnant rats. In near-term pregnant rats, transient hypothermias predominated following ip rrTNF-α and occurred at doses ranging from 10 to 1000 μg / kg. As well, ip administration of TNF RI Ab eliminated the transient hypothermia following ip administration of an EC₁₀₀ dose of E. coli LPS in near-term pregnant rats. These data taken together provide evidence that TNF-α plays an important role in mediating the transient regulated hypothermia that occurs in near-term pregnant rats following ip administration of an EC₁₀₀ dose of E. coli LPS.     

A model of preeclampsia in rats: the reduced uterine perfusion pressure (RUPP) model

Preeclampsia is defined as new-onset hypertension with proteinuria after 20 wk gestation and is hypothesized to be due to shallow trophoblast invasion in the spiral arteries thus resulting in progressive placental ischemia as the fetus grows. Many animal models have been developed that mimic changes in maternal circulation or immune function associated with preeclampsia. The model of reduced uterine perfusion pressure in pregnant rats closely mimics the hypertension, immune system abnormalities, systemic and renal vasoconstriction, and oxidative stress in the mother, and intrauterine growth restriction found in the offspring. The model has been successfully used in many species; however, rat and primate are the most consistent in comparison of characteristics with human preeclampsia. The model suffers, however, from lack of the ability to study the mechanisms responsible for abnormal placentation that ultimately leads to placental ischemia. Despite this limitation, the model is excellent for studying the consequences of reduced uterine blood flow as it mimics many of the salient features of preeclampsia during the last weeks of gestation in humans. This review discusses these features.     

The effects of the cholecystokinin antagonist, proglumide, on prolactin secretion in the rat

Since cholecystokinin produced important effects on prolactin secretion following its intraventricular injection in ovariectomized rats, we have evaluated the effects of the cholecystokinin antagonist, proglumide, to assess the physiologic significance of CCK in the control of prolactin release. Conscious rats of either sex were used following implantation of third ventricular and/or intravenous cannulae for the administration of proglumide. Blood samples were drawn from conscious animals at various times after injection of the compound. Intraventricular injection of 1 or 10 micrograms of proglumide produced a dramatic decline in plasma prolactin levels in either castrate or intact male rats. Similar results were found following the intravenous injection of 10 or 100 micrograms of the drug. These results contrasted sharply with the findings in ovariectomized females in which the intraventricular injection of the same two doses of proglumide used in males produced a dose-related elevation of prolactin which was opposite to the delayed lowering of prolactin following the intravenous injection of the same doses of the compound used in males. These results indicate that proglumide can lower prolactin in male rats and suggests a physiologically significant role of CCK in the control of prolactin secretion in the male. There appears to be a sex difference in the response since the results contrasted sharply in ovariectomized female rats. The results in the females are puzzling and it is apparent that further studies are needed to determine whether or not CCK has a physiologically significant role to play in prolactin secretion in the female. Since previous results have shown that CCK has no effect on the release of prolactin by the pituitary directly these interactions are presumably taking place in the hypothalamus.     

Early embryonic development in the rat following in utero exposure to alcohol and caffeine

The influence of both alcohol and caffeine on early embryonic development was investigated in pregnant rats. Compared to the corresponding controls, a high incidence of resorptions and abnormal embryos was induced following treatment of the animals with alcohol (0.015 ml/g body weight, 12.5% v/v, i.p.) on gestational days 6 through 12 and with caffeine (25 mg/kg body weight, i.v.) on gestational day 10. In addition, embryonic growth was severely affected. Reduction of placental blood circulation and impairment of cellular proliferation may account for the observed deleterious effects on the embryo.     

Liquid chromatography–tandem mass spectrometry analysis of cocaine and its metabolites from blood, amniotic fluid, placental and fetal tissues: study of the metabolism and distribution of cocaine in pregnant rats

The ability to simultaneously quantitate cocaine and its 12 metabolites from pregnant rat blood, amniotic fluid, placental and fetal tissue homogenates aids in elucidating the metabolism and distribution of cocaine. An efficient extraction method was developed to simultaneously recover these 13 components using underivatized silica solid-phase extraction (SPE) cartridges. The overall recoveries for cocaine and its metabolites were studied from pregnant rat blood (47–100%), amniotic fluid (61–100%), placental homogenate (31–83%), and fetal homogenate (39–87%). Extraction of the samples using silica is not classical SPE, but rather allows for the concentration of the sample into a small volume prior to injection and the removal of the proteins due to their strong interaction with the active silica surface. A positive ion mode electrospray ionization liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was used and validated to simultaneously quantitate cocaine and 12 metabolites from these four biological matrices. A gradient elution method with a Zorbax XDB C₈ reversed-phase column was used to separate the components. Multiple reaction monitoring (MRM) of a product ion arising from the corresponding precursor ion was used in order to enhance the selectivity and sensitivity of the method. Low background noise was observed from the complex biological matrices due to efficient SPE and the selectivity of the MRM mode. Linear calibration curves were generated from 0.01 to 2.50 ppm. The method also showed high intra-day (n=3) and inter-day (n=9) precision (% RSD) and accuracy (% error) for all components. The limits of detection (LODs) for the method ranged from 0.15 to 10 ppb. The LODs of cocaine and its major metabolites were less than 1 ppb from all four biological matrices. This method was applied to the study of the metabolism and distribution of cocaine in pregnant rats following intravenous infusion to a steady state plasma drug concentration. The following results were observed in the pregnant rat study: (1) the observations correlated strongly with the previous literature data on cocaine metabolism and distribution, (2) cocaine and norcocaine accumulated in the placenta, (3) arylhydroxylation of cocaine was a major metabolic pathway, (4) para-arylhydroxylation of cocaine was favored over meta-arylhydroxylation in rats and (5) accumulation of cocaine and its major metabolites was observed in the amniotic fluid.