Daniel J. Haisenleder

Regulation of Gonadotropin Subunit Gene Transcription by Gonadotropin-Releasing Hormone: Measurement of Primary Transcript Ribonucleic Acids by Quantitative Reverse Transcription-Polymerase Chain Reaction Assays1

GnRH regulates the synthesis and secretion of the pituitary gonadotropins LH and FSH. One of the actions of GnRH on the gonadotropin subunit genes (α, LHβ, and FSHβ) is the regulation of transcription [messenger RNA (mRNA) synthesis]. Gonadotropin subunit transcription rates increase after gonadectomy and following exogenous GnRH pulses. However, prior studies of subunit mRNA synthesis were limited by the available methodology that did not allow simultaneous measurement of gene transcription and mature mRNA concentrations. The purpose of the current studies was to: 1) develop a reliable and sensitive method for assessing transcription rates by measuring gonadotropin subunit primary transcript RNAs (PT, RNA before intron splicing); 2) investigate the PT responses to GnRH following castration or exogenous GnRH pulses; 3) characterize the half-disappearance time for the three PT species after GnRH withdrawal; and 4) correlate changes in PT concentration with steady-state gonadotropin subunit mRNA levels meas...

Restraint Stress Decreases Afternoon Plasma Prolactin Levels in Female Rats. Influence of Neural Antagonists and Agonists on Restraint-Induced Changes in Plasma Prolactin and Corticosterone

Female Sprague-Dawley rats were ovariectomized, given estrogen, and blood samples were obtained via an atrial catheter in the afternoon during the prolactin (PRL) surge. Restraint stress applied at 16.00 h and continued for 3 h resulted in marked decrease in plasma prolactin (PRL) and an increase in plasma corticosterone (B). The neural mechanism(s) involved in the plasma PRL decrease to restraint stress in the afternoon were examined using neural agonists and antagonists. The administration of pimozide, a dopamine antagonist, increased plasma PRL and completely prevented the restraint-induced decrease in PRL. This result suggested that an increase in dopamine secretion mediated the stress-induced decrease of PRL in the afternoon. In unrestrained animals, the intravenous administration of atropine (a muscarinic cholinergic antagonist), arecoline (a muscarinic cholinergic agonist), propranolol (a beta-adrenergic antagonist) and morphine (a beta-endorphin agonist) at 16.00 h decreased plasma PRL from that of vehicle-injected animals. Bicuculline (a GABAergic antagonist) had no effect while phentolamine (an alpha-adrenergic antagonist) and phenoxybenzamine (an alpha-adrenergic antagonist) initially increased and then decreased plasma PRL. Naloxone (a beta-endorphin antagonist) initially decreased and then increased plasma PRL in unrestrained animals. In restrained animals, the intravenous administration of atropine and naloxone had no effect on the decrease in plasma PRL. Bicuculline and propranolol decreased plasma PRL below that observed for restrained animals alone, while phentolamine and morphine slightly retarded the course of the decrease. Arecoline did not alter the PRL decrease to restraint in the early sample periods but was followed by a rebound increase at later times.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effects of Transient Dopamine Antagonism on Thyrotropin-Releasing Hormone-Induced Prolactin Release in Pseudopregnant Rats*

The effect of transient dopamine (DA) antagonism on the sensitivity of pituitary lactotrophs to the PRL-releasing effect of TRH was investigated in rats on days 3, 9, 15, and 21 of pregnancy. Each animal, bearing an indwelling intraatrial catheter, received injections of either the DA antagonist domperidone (0.01 mg/rat, iv) or saline at 0930 h on the day of the experiment. Five minutes later, all animals were given the DA agonist 2-bromo-alpha-ergocryptine maleate (CB-154; 0.5 mg/rat, iv), followed 60 min later by the administration of TRH (1.0 microgram/rat iv). Plasma samples obtained during the experiment were assayed by RIA for PRL and progesterone (P). The results showed that transient DA antagonism increased the sensitivity to TRH as a PRL-releasing stimulus on the morning of day 3 of pregnancy, but not on days 9 and 15. However, the response was present on day 9 in animals that were hysterectomized (HS) on day 6 of pregnancy. The increase in sensitivity of lactotrophs to TRH after DA blockade was observed on day 21 of pregnancy. Plasma levels of P were high on days 3, 9, and 15, but decreased markedly by day 21. In a second experiment, the anterior pituitary (AP) PRL content was determined on days 3, 9, 15, and 21 of pregnancy. The results demonstrated that AP PRL significantly decreased between days 3 and 9 of pregnancy in both intact and HS animals. However, AP PRL concentrations in animals killed on days 15 and 21 were significantly greater than that on day 9 but were not different from that observed on day 3 of pregnancy. We conclude that the ability to transform AP PRL to a TRH-releasable pool by the transient blockade of DA is present in early and late pregnancy, but is absent in midpregnancy. Since this secretory mechanism is retained on day 9 after hysterectomy on day 6 of pregnancy, it appears that the secretory products of the uterine-placental unit are inhibitory to transformation. Further, this inhibitory effect at midpregnancy cannot simply be the result of decreased AP PRL content or changes in plasma P. Finally, the return of the transformation mechanism on the day before parturition (day 21) may be due to the increase in estrogen secretion that occurs in late pregnancy, since we have previously shown that estrogen can induce this AP secretory mechanism.

Stress-induced decrease of the afternoon prolactin surge the influence of the adrenal gland☆

Blood sampling initiated during the afternoon in ovariectomized, estrogen-treated rats resulted in a suppression of the prolactin (PRL) surge. The administration of 2.5 micrograms or 25 micrograms of corticosterone (B) immediately after the first blood sample did not alter the course of the PRL suppression. Plasma B levels of adrenal intact controls were not acutely altered by blood sampling and the normal afternoon increase was evident. Similar treatment of adrenalectomized rats resulted in a similar pattern of PRL suppression. The data indicated that adrenal secretions were not responsible for the decrease in plasma PRL induced by blood sampling initiated in the afternoon.

Are both the nocturnal and diurnal prolactin surges necessary to maintain pseudopregnancy in the rat

Experiments were performed to determine whether both prolactin (PRL) surges are required to maintain pseudopregnancy (PSP) in the rat. Vaginal cycles were followed and animals that exhibited 2-3 normal cycles were cervically stimulated electromechanically to induce PSP. In one set of experiments the afternoon PRL surge was blocked by i.p. injection of 100 micrograms of 2Br-alpha-ergocryptine (CB-154) at 1200 and 1600h while the nocturnal surge was blocked by injecting 100 micrograms of CB-154 at 2400 and 0400h immediately after cervical stimulation. Pseudopregnancy was maintained only in those animals receiving CB-154 injections in the afternoon. In another series of experiments the initial CB-154 injection, given to block either surge, was delayed for one day. Once again only the nocturnal surge was capable of maintaining PSP in all animals. For those animals who had an initial nocturnal surge but all subsequent nocturnal surges were blocked, the afternoon surge was able to support PSP in only 50% of the animals. In those animals whose afternoon PRL surge was blocked, the duration of the nocturnal surge was extended so that it provided 85% of the total PRL produced by control PSP animals. In those animals whose nocturnal surge was blocked, the duration of the afternoon surge was also extended, however, in this case the extended surge only accounted for 58% of the total PRL produced by control PSP animals. The results indicated that when the diurnal PRL surge was blocked the nocturnal surge was extended so that the amount of PRL released was nearly equivalent to the amount released by both surges, however, when the nocturnal surge was blocked the extension of the diurnal surge did not provide adequate PRL to maintain PSP in all animals.

The influence of oral corticosterone replacement on plasma prolactin levels of adrenalectomized female rats.

Abstract The administration of 80 μg of corticosterone/ml of drinking solution to adrenalectomized (ADX) rats resulted in a 24 hour serum corticosterone pattern similar to that of intact animals except that the magnitude of the afternoon-nocturnal surge was one third. Basal plasma prolactin levels and the estrogen-induced afternoon prolactin surge were similar for intact and for ADX animals receiving corticosterone in the drinking solution. Adrenalectomized animals receiving 0.9% NaCl to drink, however, had an afternoon prolactin surge that was significantly lower than that of intact animals while basal levels were similar.

The Effect of Dopamine Antagonists and/or VIP on TRH- or VIP-Induced Prolactin Release in Estrogen- and Progesterone-Treated Ovariectomized Rats

Abstract These experiments were conducted to test the hypothesis that the effectiveness of VIP in releasing prolactin is, like TRH, enhanced when preceded by a short period of dopamine receptor antagonism. Chronically catheterized, ovariectomized rats pretreated with estradiol benzoate and progesterone to mimic early pregnancy were used throughout these studies. In the first experiment, animals were injected either with the dopamine (DA) antagonist domperidone (DOM, 0.01 mg/rat, iv) or with vehicle (acetic acid in saline). Five minutes later, all animals were treated with the DA agonist 2-Br-α-ergocryptine maleate (CB-154,0.5 mg/rat, iv) followed 60 min later by the administration of thyrotropin-releasing hormone (TRH, 1.0 μg/rat iv) or vasoactive intestinal peptide (VIP, 25 μg/rat, iv). The injection of TRH following DOM treatment increased mean plasma PRL levels 100 ng/ml above levels found in vehicle-injected rats. VIP administration, however, increased PRL levels in the blood in DOM-treated rats only 6 ng/ml above the levels in vehicle-injected animals. The same treatment protocol was used in the second experiment except that the DA antagonist, sulpiride (0.01 mg/rat, iv) was administered instead of DOM, and CB-154 was not given. In this experiment both TRH and VIP released PRL. The response to TRH, but not to VIP, was significantly greater following sulpiride than in animals treated with sulpiride vehicle. In the third experiment animals were treated with DOM, VIP, DOM plus VIP, or vehicle. Five minutes later all rats received CB-154 injections, followed 60 min later by TRH administration. The final experiment was a replicate of the third except that sulpiride was substituted for domperidone and no CB-154 was given. The resulting data revealed that (1) dopamine antagonism enhanced the effectiveness of TRH but not VIP and (2) that VIP augmented the effectiveness of DA blockade on PRL release and was additive with domperidone (but not sulpiride) on increasing the responsivenss to TRH. However, VIP administration without concurrent administration of domperidone or sulpiride did not increase the effectiveness of TRH compared to vehicle-injected animals. From these data we concluded that VIP is a PRL-releasing hormone the effect of which is not affected by interruption in dopamine tone as is observed for TRH. Second, VIP can potentiate the stimulatory actions of at least one DA receptor antagonist and TRH on PRL release. This later finding suggests that VIP may play a modulatory role in the neuroendocrine regulation of PRL secretion in the female rat.

The effects of dopaminergic antagonism by sulpiride on TRH and VIP-induced prolactin release in nonsuckled lactating rats☆

Prolactin (PRL) release was studied in female rats during midlactation using pharmacologic manipulations designed to mimic the hypothalamic effects of suckling. In the first experiment pituitary dopamine (DA) receptors were blocked by sulpiride (10 micrograms/rat i.v.). One hour later, thyrotropin-releasing hormone (TRH, 1.0 micrograms/rat i.v.) was given to induce PRL release. TRH released significantly more PRL following DA antagonism than when no DA antagonism was produced, suggesting that DA receptor blockade increased the sensitivity of the AP to TRH. In a second experiment, VIP (25 micrograms/rat) increased plasma prolactin 3-4 fold but this effect was not enhanced significantly by prior dopamine antagonism with sulpiride. We conclude that dopamine antagonism enhances the PRL releasing effect of TRH but not VIP in lactating rats.

A comparison of the effects of suckling or transient dopamine antagonism on thyrotropin-releasing hormone and suckling induced prolactin release in lactating rats☆

Prolactin (PRL) release was studied in mid-lactational female rats by comparing the stimulatory influence of suckling to a drug protocol that mimics the effect of suckling on the anterior pituitary (AP). Animals that nursed pups for 15 minutes and were allowed to suckle again 60 minutes later for 10 minutes, released PRL effectively during both nursing episodes; however, in animals that received the dopamine (DA) agonist 2-Br-alpha-ergocryptine maleate (CB-154, 0.5 mg/rat i.v.) at the end of the first nursing period did not show an increase in plasma PRL to a second suckling stimulation by the pups. When thyrotropin releasing hormone (TRH) was substituted for the second suckling period in CB-154 treated rats, a slight increase in plasma PRL occurred 5 minutes after the injection. In a third study we transiently blocked the action of DA at the AP by injecting the DA antagonist domperidone (0.01 mg/rat i.v.), followed 5 minutes later by the administration of CB-154. One hour later animals were either allowed to suckle pups for 10 minutes or were injected with TRH. Treatment with TRH resulted in an 11 fold increase in plasma PRL but suckling was completely ineffective in inducing PRL release. These data suggest that the lack of PRL release to suckling in CB-154 treated rats was due to inhibitory effects of CB-154 on neural mechanisms which link nursing to PRL release. In addition, the data show that pharmacologic DA antagonism affects TRH releasable PRL more than does suckling. This may be due to a reduction, by suckling, of the pool of PRL that is available to be released by TRH administration.

Influence of blinding, olfactory bulbectomy and pinealectomy on plasma prolactin levels in the neonatally androgenized female rat

Female rats were neonatally androgenized (NA) by administering 1.25 mg of testosterone propionate at 3 days of age. At 21-23 days of age, groups of 12 animals each were blinded (BLD), olfactory bulbectomized (ANOS) and pinealectomized (PX) either alone or in combination. At 11 weeks of age all animals were ovariectomized, implanted with atrial catheters and given 0.5 mg polyestradiol phosphate. At 12 weeks of age, animals were attached to catheter extensions and blood samples obtained at 09.00, 11.00, 13.00, 15.00, 17.00, 19.00 and 21.00 h. The plasma prolactin (PRL) pattern of NA control animals consisted of elevated basal values at 11.00 and 13.00 h with an afternoon peak at 17.00 h. Anosmic and PX animals had a PRL pattern similar to that of the control. Animals BLD or BLD + ANOS had significantly lower plasma PRL values than sham-operated animals. Animals BLD + PX had PRL values comparable to PX alone. The diurnal PRL pattern observed for NA control animals was still evident in all experimental animals. Blinding + ANOS resulted in a significant decrease in ovarian and oviductal weights and attenuated the estrogen-induced increase in uterine weight. The evidence indicated that the elevated morning PRL levels observed in NA rats was not due to an altered function of the pineal gland and that BLD + ANOS blunted the action of exogenous estrogen on uterine weight.