Oestradiol 17 beta modifies fowl pituitary prolactin and growth hormone secretion in vitro

Chicken pituitary glands were incubated in medium containing oestradiol 17 beta (E2), alone or together with single whole hypothalami. E2 stimulated prolactin release from the pituitary and increased the prolactin releasing activity of the hypothalamus, but did not affect growth hormone release. Preincubation of pituitaries with E2 dramatically stimulated subsequent prolactin release. Pituitaries primed with E2 were more responsive to the prolactin-stimulating effects of hypothalamic extract (HE) and thyrotrophin-releasing hormone (TRH) and more sensitive to the prolactin-inhibiting effect of dopamine. E2-primed pituitaries were much less sensitive to the growth hormone releasing activity of TRH and HE. These results show that E2 may regulate pituitary function by direct effects on hormone release by modifying pituitary sensitivity to stimulatory or inhibitory influences and by altering hypothalamic releasing activity.     

Age-related changes in prolactin and growth hormone release from pituitary glands in vitro

The basal release of prolactin from cockerel anterior pituitary glands in vitro declined between 1 and 7 weeks of age, to a level less than that released by pituitary glands from 18 week old (adult) cockerels and hens. Basal growth hormone (GH) release increased between 1 and 7 weeks of age but had declined in adults to a level similar to that released from 4 weeks old cockerels. The responsiveness of the pituitary gland to hypothalamic stimulation, using hypothalami from 8 week old broiler fowl, was also age-related. Prolactin release was considerably higher from pituitaries of 1 week old cockerels compared to the other age groups. Stimulation of GH release by the hypothalamus was higher from pituitaries of both 1 and 7 week old cockerels compared to the other groups of birds. The increase in release of prolactin following incubation with thyrotrophin releasing hormone (TRH) declined between 1 and 7 weeks, but increased slightly in adult birds, whereas the increase in release of GH following TRH was higher from pituitaries of both 1 and 7 week old cockerels. Hypothalamic prolactin (Prl) releasing activity, measured as the ability of the hypothalamus to stimulate hormone release from 8 week old broiler fowl anterior pituitary glands, declined with the age of the donor cockerels. The hypothalami from adult hens secreted significantly more Prl releasing activity than did adult cockerel hypothalami. The secretion of GH releasing activity decreased markedly with the age of the donor bird. These results suggest that maturational patterns of hormone secretion in fowl are partly due to changes in autonomous hormone release, to changing patterns of hypothalamic activity and to differences in pituitary responsiveness to provocative stimuli.     

Mechanism of serotonin effects on prolactin and growth hormone secretion in domestic fowl

Brain serotonin levels were increased in immature chickens by ip injection of pargyline (75 mg/kg) and clorgyline (5 mg/kg) and by L-tryptophan (100 mg/kg) and imipramine (10 mg/kg) treatment. These treatments increased the circulating prolactin level and reduced the concentration of plasma growth hormone (GH). Treatment with para-chlorophenylalanine (PCPA, 100 mg/kg) reduced the brain serotonin content and the level of plasma prolactin. Treatment with these drugs in vivo similarly affected the basal level of prolactin release from pituitary glands in vitro, although it did not affect the basal level of GH release. The in vitro responsiveness of the pituitary gland to hypothalamic stimuli eliciting prolactin secretion was increased by in vivo pargyline and combined tryptophan: imipramine treatment but reduced by PCPA administration. The in vitro GH response to hypothalamic stimulation was reduced after the in vivo injection of pargyline, clorgyline and tryptophan: imipramine. The hypothalami from clorgyline and tryptophan: imipramine treated birds induced a greater stimulation of in vitro prolactin secretion from control pituitary glands than hypothalami from controls birds, whereas the GH releasing activity was reduced. These results suggest that serotonin stimulates prolactin secretion in chickens by increasing pituitary responsiveness to hypothalamic releasing factors and by increasing the prolactin releasing activity of the hypothalamus. Serotonin appears to suppress GH secretion by reducing pituitary sensitivity to releasing factors and by reducing hypothalamic GH releasing activity.     

Hypothalamic control of prolactin and growth hormone secretion in different vertebrate species.

Pituitaries from different vertebrates representing mammals, birds, reptiles, and amphibians, were incubated in vitro with various hypothalamic extracts (HE). Prolactin and growth hormone (GH) in medium and pituitary were measured by densitometry after polyacrylamide gel electrophoretic separation (PAGE). Rat (Rattus norvegicus), chicken (Gallus domesticus), terrapin (Chrysemys picta), and toad (Xenopus laevis) pituitaries were incubated with homologous HE. Rat HE inhibited prolactin release. In the other species the HE stimulated prolactin release. In all four species GH release was stimulated by HE. The effects on prolactin and GH release were proportional to the dose of HE added. Chicken pituitaries were incubated with chicken HE together with rat HE. The rat HE inhibited the chicken HE-stimulated release of prolactin, as measured by radioimmunoassay. Heterologous incubations were used to test HE for prolactin releasing and inhibiting factors and for GH releasing and inhibiting factors. Chicken pituitaries were incubated with HE from the eel (Anguilla anguilla), the cod (Gadus gadus) and the flounder (Pleuronectes flesus) as well as from the other species listed. Both cod and flounder HE marginally inhibited autonomous chicken prolactin release. HE from these species dose-responsively inhibited chicken HE-stimulated prolactin release. Cod HE also inhibited chicken HE-stimulated GH release. HE from the eel, the terrapin and the toad stimulated chicken prolactin release. Hormone release from terrapin and toad pituitaries incubated with heterologous HE was consistent with hypothalamic control via releasing factors in these species.     

Serotonin and acetylcholine affect the release of prolactin and growth hormone from pituitary glands of domestic fowl in vitro in the presence of hypothalamic tissue

Anterior pituitary glands from broiler fowl were incubated alone or with hypothalamic tissue in medium containing either serotonin or serotoninergic drugs, acetylcholine or cholinergic drugs, and the release of prolactin (Prl) and growth hormone (GH) measured by homologous radioimmunoassays. The neurotransmitters and drugs affected the release of hormones from the pituitary gland only when hypothalamic tissue was also present. Serotonin and its agonist quipazine stimulated the release of Prl and inhibited release of GH in a concentration-related manner. The antagonist methysergide blocked the effects of serotonin and quipazine on Prl. Acetylcholine and its agonist pilocarpine also stimulated release of Prl and inhibited release of GH in a concentration-related manner. Atropine blocked these responses. The results show that serotonin and acetylcholine affect pituitary hormone secretion by acting on the hypothalamus. They may stimulate the secretion of a Prl releasing hormone and somatostatin.     

STIMULATION OF ANTERIOR PITUITARY ADENYL CYCLASE ACTIVITY AND ADENOSINE 3′:5′-CYCLIC PHOSPHATE BY HYPOTHALAMIC EXTRACT AND PROSTAGLANDIN E1

Hypothalamic extract, containing the releasing factors for anterior pituitary hormones, within minutes stimulated adenyl cyclase activity and adenosine 3':5'-cyclic phosphate (cyAMP) concentrations in rat anterior pituitary in vitro. Cerebral cortical extract was ineffective and hypothalamic extract had no effect on these parameters in posterior pituitary or thyroid. Prostaglandin E(1) also increased adenyl cyclase activity and cyAMP levels in anterior pituitary tissue. Although NaF augmented adenyl cyclase activity, it did not elevate cyAMP. Epinephrine, norepinephrine, histamine, serotonin, dopamine, and vasopressin did not increase either adenyl cyclase or cyAMP. The increased adenyl cyclase and cyAMP produced by hypothalamic extract was associated with greater luteinizing hormone release from anterior pituitary in vitro.     

Evidence for "short loop" feedback regulation of prolactin and growth hormone secretion in the pigeon

When pigeons were injected with an extract of chicken hypothalamus, the increase in weight of the crop sac showed that prolactin secretion was stimulated. Crop sac weight was no higher after 16 days of injection than it was after 8 days. Pituitary gland content of prolactin and growth hormone was elevated at 8 days, but showed a relative decrease after a further 8 days of injections. Prolactin and growth hormone releasor activity in the hypothalamus was investigated in vitro. A decline during treatment with hypothalamic extract was seen, and by 8 days no releasor activity could be detected. Injection with ovine prolactin also reduced prolactin releasor activity in the hypothalamus. The results suggest that the secretion of prolactin and possibly also of growth hormone is controlled by a "short loop" negative feedback mechanism which acts on hypothalamic hypophysiotrophic hormones.     

Inhibition by testosterone of prolactin and growth hormone release from chicken anterior pituitary glands in vitro

Pituitary glands and hypothalami from broiler fowl were incubated in medium containing testosterone, and prolactin and GH release were determined. Pituitary glands were also preincubated for 20 h in medium containing testosterone, and then in medium containing various secretagogues. Testosterone inhibited the release of prolactin directly from the pituitary gland in a concentration-related manner. The hypothalamus stimulated the release of prolactin, but by a lesser amount in the presence of testosterone. When pituitary glands were preincubated with testosterone, subsequent release of prolactin was inhibited, except with the highest concentration which stimulated prolactin release. Hypothalamic extract (HE) markedly stimulated prolactin release from control pituitary glands although testosterone-primed glands were less responsive. The stimulation of prolactin release by thyrotrophin releasing hormone (TRH) and prostaglandin E2 (PGE2) was also reduced by preincubation of the pituitary glands with testosterone. Priming with testosterone did not affect the release of GH from pituitary glands alone, but reduced the TRH-, HE- and PGE2-stimulated release of GH. These results demonstrate that testosterone directly inhibits prolactin secretion and reduces the sensitivity of pituitary lactotrophs and somatotrophs to provocative stimuli.     

Growth hormone and prolactin secretion in water-deprived chickens

The deprivation of water for 12 or 24 hr increased the prolactin concentration in the plasma of immature chickens but had no effect on the circulating growth hormone (GH) level. The increase in plasma prolactin level reflected an increase in the basal rate of prolactin release from incubated hemipituitary glands and an increase in the responsiveness of the pituitary gland to hypothalamic releasing factors. The deprivation of water had no effect on basal level of pituitary GH release in vitro but abolished the stimulatory effect of the hypothalamus on in vitro GH secretion.     

Control of prolactin and growth hormone secretion in the eel Anguilla anguilla

Freshwater eels were placed in seawater, and changes in pituitary prolactin and growth hormone were determined. The content of both hormones declined initially but returned to the original values after 8 weeks. Whole glands were not seen to differ in their prolactin release in vitro from pituitary fragments consisting of the rostral pars distalis only. Release of prolactin and growth hormone in vitro was directly proportional to the amount of hypothalamic extract added. During adaptation to seawater the amount of prolactin- and growth hormone-stimulating ability in the hypothalamus decreased, but the pituitaries of SW-adapted eels were still capable of responding to HE from FW eels.     

Development of radioimmunoassay for bullfrog thyroid-stimulating hormone (TSH): effects of hypothalamic releasing hormones on the release of TSH from the pituitary in vitro

A bullfrog (Rana catesbeiana) thyroid-stimulating hormone (TSH) beta-subunit (TSHbeta) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHbeta cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHbeta corresponded to those positively stained with an antiserum against human (h) TSHbeta. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75+/-0.07ng TSH/100microl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose-response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and alpha-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHbeta coupled with the alpha-subunit by an immunoprecipitation experiment using the TSHbeta antiserum and an alpha-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro.The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.     

Mechanisms of release of prolactin from fowl anterior pituitary glands incubated in vitro: effects of calcium and cyclic adenosine monophosphate

Fowl anterior pituitary glands were bisected and each half was pretreated in either Medium 199 or medium containing EGTA to deplete endogenous calcium (Ca2+) stores, after which they were incubated in Medium 199, or Ca2+-free medium, containing prolactin release-stimulating agents and verapamil, a Ca2+ channel blocker. High K+ concentrations, hypothalamic extract, synthetic thyrotrophin-releasing hormone (TRH) and dibutyryl cyclic AMP (dbcAMP) all stimulated release of prolactin from control (non EGTA-treated) hemianterior pituitary glands. The effects of TRH and dbcAMP were not additive, but the response to submaximal concentrations of TRH was augmented by theophylline, a phosphodiesterase inhibitor. Reduction of Ca2+ availability with EGTA or verapamil reduced basal release of prolactin, prevented the prolactin-stimulating effects of high K+ concentrations and TRH, and markedly attenuated responses to hypothalamic extract and dbcAMP, EGTA being more effective than verapamil. Increasing the Ca2+ concentration of the medium did not augment basal or stimulated release of prolactin. These results suggest that both Ca2+ and cyclic AMP may act as intracellular mediators in the release of prolactin. Both basal and stimulated release of prolactin depend upon the presence of Ca2+. Although influx from the medium may be the major source of Ca2+, endogenous stores of Ca2+, perhaps mobilized by dbcAMP, may be able to maintain some release of prolactin. The prolactin-stimulating effects of TRH may be mediated by cyclic AMP.     

Hypothalamic catecholamine biosynthesis and pituitary gonadotropin secretion in vitro: Effect of hyperprolactinemia

The effect of hyperprolactinemia on central catecholamine biosynthesis and anterior pituitary hormone release was studied using an in vitro methodology. The incorporation of [³H]tyrosine into hypothalamic and neurohypophyseal catecholamines was determined using a new method which combines high performance liquid chromatography (HPLC) with amperometric detection (LCEC). Elevated plasma prolactin levels, induced by pituitary transplants, resulted in increased in vitro biosynthesis of medial basal hypothalamic (MBH) dopamine (DA), but not norepinephrine (NE). Neurohypophyseal DA biosynthesis (including the intermediate lobe) was not affected. Plasma LH levels were depressed by hyperprolactinemia although the content of hypothalamic luteinizing hormone-releasing hormone (LHRH) was not changed. In parallel studies, the anterior pituitaries from these animals were incubated in vitro using a paired-half technique and LH and PRL release measured. While the basal release of prolactin was not altered by hyperprolactinemia, LH release was significantly decreased. Gonadotroph responsiveness to LHRH was significantly increased, while the inhibition of prolactin by dopamine was not altered. There was a decrease in pituitary prolactin content with normal LH levels. These experiments confirm several in vivo reports which show that hypothalamic dopaminergic but not noradrenergic activity is increased by prolactin. This action is specifically localized in the tuberoinfundibular dopaminergic neurons. Furthermore, these experiments suggest that these central changes result in alterations in both gonadotroph and mammotroph function.     

Hypothalamic stimulation of prolactin release in a freshwater catfish, Heteropneustes fossilis.

Hypothalamic extract has been found to contain certain factor(s) which triggers the release of prolactin from the pituitary gland. In assay recipients, hypothalamic extract injection was as effective as perphenazine-HCl treatment in triggering the release of prolactin from pituitary gland. Rise in prolactin concentration of serum and resultant decrease in pituitary prolactin level in response to hypothalamic extract administration suggests that hypothalamus in H. fossilis has prolactin releasing factor(s). This releasing effect of hypothalamus seems to be specific since cortical extract injections were ineffective in producing the similar response.     

Hypothalamic dopamine and catechol oestrogens in rats with spontaneous pituitary tumours

Dopamine concentration within the hypothalamus and its depletion after the administration of alpha-methyl-para-tyrosine were measured in young rats and compared with values obtained in aged animals with and without spontaneously occurring pituitary tumours. Old rats had significantly reduced hypothalamic dopamine concentrations and there was less depletion of dopamine compared with young animals but there were no differences between tumorous and non-tumorous animals. Hyperprolactinaemia induced in young animals caused a much greater depletion of hypothalamic dopamine than in old tumorous rats with comparable plasma prolactin concentrations. The catechol oestrogen 2-hydroxyoestradiol inhibited the release of prolactin from normal rat pituitary glands in vitro but measurement of catechol oestrogens in the hypothalamus showed no differences between young and old tumorous or non-tumorous rats. It is concluded that reduced dopamine concentration and an impaired response to hyperprolactinaemia in old rats may facilitate the growth of prolactin-secreting tumours arising in the pituitary gland.     

Pineal removal or denervation: effects on hypothalamic PRF activity in the rat.

Medial basal hypothalamic (MBH) extracts from intact, blind-anosmic, blind-anosmic-pinealectomized or nervi conarii transected female rats stimulated the release of prolactin from anterior pituitary glands in vitro. Additionally, a lower tissue level of prolactin was measured in the pituitaries incubated with the extracts. These data indicate that the activity of a prolactin-releasing factor(s) (PRF) was being assayed in the MBH extracts. Additionally, it was observed that MBH extracts from blind-anosmic-pinealectomized or nervi conarii transected rats contained significantly higher levels of PRF activity than the extracts from the intact or blind-anosmic groups. The results support the concept that the pineal control of pituitary prolactin may involve hypothalamic PRF.     

Effect of reserpine on the inhibition of prolactin released from different pituitary constructs in vitro by dopamine, bromocriptine and apomorphine.

Hourly release of Prolactin by pituitary constructs 1 whole pituitary (PI), adenohypophysis (P-N) and pituitary-hypothalamus co-incubate (PHC) were compared. Adenohypophysis secreted significantly more prolactin than PI and PHC, while PHC secreted significantly less than PI. Co-incubation of (P-N) with posterior pituitary reduced the elevated secretion of prolactin. Addition of dopamine (10(-7) M), bromocriptine (10(-7) M) and apomorphine (5 x 10(-8) M) to these constructs did not affect the release of prolactin from PI but inhibited the same from (P-N) and PHC. Treatment with reserpine increased serum prolactin levels but intrapituitary prolactin contents were decreased. Hourly release of prolactin from pituitary constructs derived from reserpine-treated rats was significantly reduced as compared to ascorbic acid--treated controls. Inclusion of dopamine (10(-7) M), bromocriptine (10(-7) M) and apomorphine (5 x 10(-8) M) in these constructs inhibited prolactin secretion further. In vitro addition of perphenazine stimulated the release of prolactin by PHC but was without any effect on PI and (P-N). The data are interpreted to suggest that dopamine in posterior pituitary may be an important determinant of hypothalamic modulation of prolactin secretion.     

Sex differences in circadian control of LH secretion

We have studied the secretion of TSH and prolactin from perifused rat anterior pituitary glands in vitro in response to single pulses of thyrotrophin releasing hormone (TRH) and KCl after prior exposure to TRH. Anterior pituitary fragments were incubated in normal medium or in medium containing 28 nmol TRH/1 for 20 h before perifusion. Thyrotrophin releasing hormone (28 nmol/l), administered as a 3-min pulse, stimulated TSH and prolactin release from control tissue to a peak value four or five times that of basal. After exposure of the pituitary tissue to TRH for 20 h, the subsequent response of TSH to a 3-min pulse of TRH was, however, markedly reduced; in contrast, the prolactin response was not significantly reduced. In a similar series of experiments KCl (60 mmol/l) was administered to both control and TRH-'treated' pituitary tissue as a 3-min pulse; no significant differences in TSH responses or prolactin responses were observed. These data indicate that TRH desensitizes the pituitary thyrotroph to a subsequent TRH stimulus but has very little effect on prolactin secretion.     

Prolactin release in vitro and in vivo in the pigeon and the domestic fowl following administration of synthetic thyrotrophin-releasing factor (TRF)

Chicken and pigeon pituitaries were incubated in vitro in the presence of different concentrations of hypothalamic extract and of synthetic thyrotrophin-releasing factor (TRF). It was found that prolactin, measured by pigeon crop-sac bioassay, was released in both species. The amount of prolactin released from the chicken pituitaries was increased at the higher doses of TRF, the increase occurring at a dose which corresponded to the decrease in the biphasic TSH secretion. In the pigeon, prolactin and growth hormone release in vitro were measured by an electrophoretic-densitometric method. Both prolactin synthesis and release showed a biphasic response to the dose level of TRF, as did growth hormone synthesis.Thyrotrophin-releasing factor administered in vivo in pigeons resulted in a significant cropsac response, which was biphasic with respect to dose of TRF. There were also corresponding changes in pituitary prolactin content.