James L. Voogt

Progesterone reverses the estradiol-induced decrease in tyrosine hydroxylase mRNA levels in the arcuate nucleus

Estradiol (E2) and progesterone (P4) interact to influence tuberoinfundibular dopaminergic neuronal activity and contribute to the control of prolactin (PRL) release. This study examined tyrosine hydroxylase mRNA signal levels in the arcuate nucleus of the hypothalamus and tyrosine hydroxylase activity in the stalk-median eminence after 1 week of steroid treatment and related these to circulating PRL levels. Ovariectomized rats were untreated (control) or were implanted with E2, P4 or both E2 and P4 pellets and were sacrificed after 7 days at either 10:00 or 18:00 h. Some E2 + P4-treated rats were injected with either RU 486 or its vehicle at 12-hour intervals for the last 3 of the 7 days of steroid treatment. Tyrosine hydroxylase mRNA signal levels in the arcuate nucleus were decreased by 70% at both 10:00 and 18:00 h in the E2-treated rats compared to control rats. P4 alone had no effect on tyrosine hydroxylase mRNA levels, but reversed the E2-induced decrease so that mRNA levels in the E2 + P4-treated rats were similar to control levels. The progesterone antagonist RU 486 blocked this effect of P4, supporting the observation of decreased mRNA levels in E2-treated rats. Steroid treatment had no effect on tyrosine hydroxylase mRNA levels in the medial zona incerta. Tyrosine hydroxylase activity in the stalk-median eminence was similar at 10:00 and 18:00 h in control rats, and was decreased by 25 and 36% at 10:00 and 18:00 h, respectively, in E2-treated rats. P4 alone had no effect on tyrosine hydroxylase activity, but reversed the E2-induced decrease in enzyme activity to control levels at both 10:00 and 18:00 h. In contrast to the effect of RU 486 on tyrosine hydroxylase mRNA, tyrosine hydroxylase activity in E2 + P4-treated rats was not significantly altered by RU 486 at either 10:00 or 18:00 h. Circulating PRL levels were elevated in the E2-treated and E2 + P4-treated rats. A diurnal PRL rise was evident at 18:00 h in E2-treated rats, but was abolished by concomitant treatment with P4. The diurnal PRL surge was re-established in E2 + P4-treated rats after administration of RU 486, whereas basal circulating PRL levels were not altered by RU 486. These data indicate that P4 antagonizes the profound inhibitory effect or E2 on tyrosine hydroxylase mRNA content in the tuberoinfundibular dopaminergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Serotonin on Vasopressin Release: A Comparison to Corticosterone, Prolactin and Renin

Previously we reported that 5 min after intracerebroventricular (i.c.v.) injection, serotonin (5-HT, 2.5 micrograms) produced increases in blood pressure and decreases in heart rate in conscious rats that were blocked by LY 53857 (a selective 5-HT2/1C antagonist) and were sensitive to vasopressin antagonism. The present studies were performed to determine if this dose of 5-HT acts similarly to increase plasma vasopressin levels. In addition, the vasopressin responses were compared to prolactin, corticosterone, and plasma renin activity, three other neuroendocrine systems regulated in part by 5-HT. The administration of 5-HT (2.5 micrograms i.c.v.) produced a rapid (maximum response in less than 5 min) and brief (return to baseline by 15 min) increase in plasma vasopressin levels. The response was eliminated by the centrally acting 5-HT2/1C antagonist LY 53857 (100 micrograms/kg i.v.), but only attenuated by xylamidine (100 micrograms/kg i.v.), a 5-HT2/1C antagonist that reportedly does not cross the blood-brain barrier. 5-HT also increased plasma prolactin and corticosterone levels, but neither LY 53857 nor xylamidine altered these responses. In rats rendered chronically baroreceptor deficient by sinoaortic deafferentation, the vasopressin response to 5-HT was reduced, whereas the prolactin response was normal. 5-HT did not increase plasma renin activity in intact or baroreceptor-deficient rats, in contrast to the other neuroendocrine systems studied. Thus, the data demonstrate that vasopressin levels are elevated briefly following 5-HT i.c.v., consistent with the pharmacologic profile of the early cardiovascular response.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of prolactin-releasing peptide mRNA in the rat brain.

In order to identify the distribution of prolactin-releasing peptide (PrRP) mRNA in the rat brain, we independently cloned cDNA of PrRP. Brains were removed from three adult males, and brains from three females each at 0200 and 1400 h on day 7 of pregnancy were obtained. By the nonradioactive in situ hybridization method, the location of PrRP mRNA was detected in very restricted brain areas. The distribution of PrRP mRNA signals was very similar in both sexes. In the hypothalamus, only the ventral part of the caudal dorsomedial nucleus had PrRP mRNA signals. Other forebrain areas did not show any positive signals. In the medulla oblongata, two discrete areas contained PrRP mRNA signals. No positive signal was found in the rostral part of the medulla oblongata extending to the anterior part of the area postrema. The caudal part of the nucleus of the solitary tract (NTS) had neurons with very strong signals of PrRP mRNA. The reticular nucleus showed a few PrRP mRNA positive neurons. The number of PrRP mRNA positive cells in the NTS was not different between experimental groups, although plasma prolactin levels in these animals were different. This anatomical information on the location of PrRP mRNA in the brain provides the framework to understand the physiological functions of PrRP in vivo.

Ontogeny of tyrosine hydroxylase mRNA signal levels in central dopaminergic neurons: development of a gender difference in the arcuate nuclei

Using in situ hybridization and immunocytochemistry, this study examined the tyrosine hydroxylase (TH) mRNA signal levels and immunostaining in the arcuate nuclei of the hypothalamus, zona incerta and substantia nigra of male and female rats during neonatal, peripubertal and adult life. The catalytic activity of TH in the stalk-median eminence was also investigated using the in vitro accumulation of 3,4 dihydroxyphenylalanine (DOPA) after inhibiting aromatic amino acid decarboxylase activity. In the arcuate nuclei, TH mRNA levels increased 3.5-fold between 5 and 15 days of age and remained at a steady level between 15 and 35 days of age in both male and female rats. Similar to TH mRNA levels in the arcuate nuclei, TH activity in the stalk-median eminence increased 2-3 fold between days 10 and 15 of age and remained at a steady level between 15 and 35 days of age in both sexes. A later increase in TH mRNA levels in the arcuate nuclei and catalytic activity in the stalk-median eminence was observed between 35 and 40 days in females, but not males. During adulthood, TH mRNA levels and enzyme activity were 2.7-fold higher in the arcuate nuclei and stalk-median eminence, respectively, of diestrous females vs males. These data suggest that the changes in TH mRNA in the arcuate nuclei may contribute to the developmental alterations, as well as the adult gender differences, in enzyme activity in the stalk-median eminence. Circulating progesterone levels were low (1-10 ng/ml) between days 5 and 25 of age and increased 6-fold between 25 and 35 days of age in both males and females. Progesterone levels increased 2-fold in females, but not males, between days 35 and 40 and were 4-fold higher in diestrous females as compared to adult males. Circulating prolactin levels were low (2-3 ng/ml) between days 5 and 15, increased 15-fold between days 15 and 25 and increased an additional 2- to 3-fold between days 35 and 70 in both males and females. TH mRNA signal levels increased between days 5 and 15 of age in dopaminergic perikarya in the zona incerta and the substantia nigra of both female and male rats. The TH mRNA levels remained constant between days 15 and 70 in the zona incerta, whereas TH mRNA levels declined with age in the substantia nigra.(ABSTRACT TRUNCATED AT 400 WORDS)

Chapter 12 Regulation of prolactin secretion during pregnancy and lactation

Abstract Prolactin plays major roles in maintaining the corpora lutea of pregnancy and in the synthesis of milk during lactation. The hypothalamic mechanisms involved in these functions have been investigated. Mating leads to a surge of prolactin and programs daily surges during early pregnancy. The expression of Fos-immunoreactivity shows that mating activates several hypothalamic nuclei, particularly the arcuate nucleus and medial preoptic area. In the arcuate nucleus, mating is associated with Fos expression in β-endorphin neurons, and infusion of naloxone blocks both mating-induced and diurnal prolactin surges. Tyrosine hydroxylase-immunoreactive dopamine neurons appear not to participate in surge generation. However, after day 10 of gestation the secretion of placental lactogens suppresses prolactin secretion via activation of dopamine neurons without involvement of β-endorphin neurons. Intracerebroventricular implantation of placental lactogen-secreting cells will block pregnancy prolactin surges, increase Fos expression in dopamine neurons, and increase tyrosine hydroxylase activity. During lactation the mechanisms regulating dopamine and β-endorphin neurons are further modified. In early lactation a prolactin-induced increase in tyrosine hydroxylase activity leads to negative feedback, but this effect is lost by mid-lactation. Overriding this negative feedback is the inhibitory effect that suckling has on dopaminergic activity. This may involve β-endorphin-mediated inhibition of dopamine neurons, as naloxone causes a marked increase in tyrosine hydroxylase activity and suppression of circulating prolactin. However, removal of tonic dopamine inhibition is not sufficient to account for the high levels of prolactin attained during lactation, and additional releasing factors are probably involved. In situ hybrization histochemistry for the most recent candidate, prolactin-releasing peptide, suggests that this may involve brain stem neurons that co-localize noradrenaline. Thus, prolactin secretion during pregnancy and lactation involve complex interactions of regulatory factors and plasticity of neuronal responsiveness.

Tyrosine hydroxylase messenger RNA in the hypothalamus, substantia nigra and adrenal medulla of old female rats

The effects of aging in the female rat were analyzed in terms of tyrosine hydroxylase (TH) gene expression and serum prolactin levels. The number of tuberoinfundibular dopaminergic (TIDA) neurons and the concentration of TH mRNA per cell was greater in 16- to 18-month-old rats than in 25-month-old rats. The amount of TH immunostaining was more intense in the median eminence of the 18-month-old rats compared to either younger or older rats. Plasma prolactin levels were moderately elevated in 18-month-old rats compared to 4-month-old rats, and extremely elevated in 25-month-old rats due to the occurrence of pituitary prolactinomas. There were no detectable changes in TH mRNA levels in the substantia nigra with age, whereas adrenal TH mRNA increased with age. We propose that prolactin initially exerts a stimulatory effect on the TIDA neurons as the rat ages, but eventually causes a loss in neuronal number and neuronal function as the pituitary prolactinoma secretes increased amounts of prolactin.

Promoter usage and estrogen regulation of prolactin receptor gene in the brain of the female rat

Mechanisms underlying hormonal regulation of prolactin receptor (PRL-R) gene in the brain are unknown. The 5’-untranslated region of PRL-R mRNA in peripheral tissues contains at least three alternative first exons (1A, B, C) that are expressed as tissue-specific, suggesting the differential usage of PRL-R gene promoters. The present study aimed to investigate: (1) the possible regulation of PRL-R mRNA levels by estrogen in in vitro and in vivo tissues; (2) which exon (1A, or 1B, or 1C)-containing PRL-R mRNA transcript is expressed in the brain, and (3) how the specific exon 1-containing mRNA is affected by estrogen by using RT-PCR, Southern blot and 5’Race PCR techniques. The RT-PCR results showed that PRL-R mRNA was detected in the cerebral cortex and pons medulla in addition to the choroid plexus and hypothalamus in the female rat. The expression of PRL-R mRNA was up-regulated by estrogen treatment in the rat brain tissue and in the GT1-7 cell culture. Both exon 1A- and 1C-containing transcripts were expressed in all four regions, suggesting that promoters 1A and 1C for the PRL-R gene are utilized in the rat brain. Exon 1A-containing transcript was up-regulated by estrogen treatment in all four brain regions, whereas Exon 1C-containing transcript was up-regulated by estrogen treatment in 3 of the 4 brain regions, cerebral cortex being the exception. Exon 1B-containing transcript was neither detectable nor induced by estrogen treatment in any of the brain regions examined. The RT-PCR results were confirmed by partial isolation of 5′-untranslated regions of exon 1A- and 1C-containing PRL-R mRNA transcripts from brain tissue by using 5’Race PCR. The present result confirms the expression of PRL-R mRNA in the cerebral cortex and pons medulla in the female rat. The levels of PRL-R mRNA were up-regulated by estrogen in rat brain tissue and GT1-7 cell cultures. Detection of exon 1A- and 1C-containing transcripts implies that the promoter 1A and 1C are active in the female rat brain. Estrogen differentially regulates expression of the PRL-R mRNA in the different brain regions by increasing the utilization of PRL-R gene promoters 1A and 1C in the female rat.

Lactogenic Hormones of the Placenta and Pituitary Inhibit Suckling-Induced Prolactin (PRL) Release but Not the Ante-Partum PRL Surge

Abstract Prolactin (PRL) and other lactogenic hormones feed back at the hypothalamus to inhibit PRL release. At midpregnancy, high circulating levels of placental lactogens (PL) terminate the mating-induced biphasic PRL surges in female rats. In the dark period preceding parturition, however, an ante-partum PRL surge occurs despite continuously high levels of PL. This study examined whether the lactogenic hormone negative feedback loop is altered during the ante-partum surge using two models: (i) pregnant rats given a hypothalamic implant of albumin, ovine PRL, or recombinant rat PL-I on Day 19 or 20 of pregnancy; and (ii) pregnant rats bearing a transplant of a rat choriocarcinoma cell line, Rcho-1 (PL-secreting), or HRP-1 (non–PL-secreting). Serial blood samples were taken via carotid cannula from all rats. Although lactogenic hormones placed in the hypothalamus reduced suckling-induced PRL release by 89%, hypothalamic implants of oPRL or recombinant rPL-I did not attenuate the ante-partum PRL surge. Rcho-transplanted rats also did not have a significantly reduced ante-partum PRL surge (peak PRL level, 131 ng/ml) compared with HRP-bearing rats (peak PRL level, 107 ng/ml). Northern blot analysis revealed that the Rcho-1 tumors expressed both PL-I and PL-II, while the HRP-1 tumors did not express either PL. The inability of the Rcho-1 transplants to inhibit the ante-partum PRL surge suggests that lactogenic hormone negative feedback is disrupted during the antepartum period, possibly by the changing steroid profile associated with parturition.

In vivo Release of Dopamine, Luteinizing Hormone-Releasing Hormone and Thyrotropin-Releasing Hormone in Male Rats Bearing a Prolactin-Secreting Tumor

The present study was concerned with the effects of a transplantable prolactin-secreting pituitary tumor (7315b) on the hypothalamic release of dopamine, luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) in gonadectomized, adrenalectomized male rats bearing subcutaneously a testosterone capsule and a corticosterone pellet. Similar male rats not inoculated with tumor served as controls. The rats were studied 3-4 weeks after tumor inoculation, while they were anesthetized with urethane. Compared to the controls, prolactin levels in the tumor-bearing rats had increased 70-fold, whereas the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) decreased to 20 and 27%, respectively. In tumor-bearing rats, the secretion of dopamine into hypophysial stalk plasma increased from 2.3 to 4.9 ng/h (p less than 0.025), whereas that of LHRH decreased from 127 to 52 ph/h (p less than 0.005). Since the use of urethane anesthesia may change quantitatively and qualitatively the effects of hyperprolactinemia, it was decided to study these effects on the in vivo release of LHRH, dopamine and TRH in conscious rats by a push-pull perfusion of the median eminence-arcuate nucleus area. Using this technique, it was found that in tumor-bearing rats the secretion of LHRH decreased from 20.0 to 9.8 pg/15 min (p less than 0.005), whereas that of dopamine increased from 118 to 246 pg/15 min (p less than 0.025). The secretion of TRH was not altered by hyperprolactinemia (4.1 vs. 4.4 pg/15 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in estrogen receptor-α expression in hypothalamic dopaminergic neurons during proestrous prolactin surge

A surge of prolactin (PRL) occurs in female rats during proestrus in response to elevated estradiol and progesterone levels. Dopamine is the primary hypothalamic inhibitor of PRL secretion from the pituitary. Using double-label immunocytochemistry, we investigated the pattern of estrogen receptor-α (ER-α) immunoreactivity in dopaminergic neurons in the arcuate nucleus (ARC) and the periventricular nucleus (PeVN) during the proestrous PRL surge and compared it to that during diestrus, when PRL levels are constantly low. Our results showed that during diestrus >80% of dopaminergic neurons in the ARC were also positive for ER-α, and this colocalization percentage decreased significantly during proestrus. By contrast, <15% of dopaminergic neurons in the PeVN expressed ER-α, and the low percentage of ER-α expression was unchanged throughout proestrus and diestrus. Results from estrogen plus progesterone-treated ovariectomized rats showed similar patterns of ER-α expression within the ARC and the PeVN and, once again, compared with the control group, had a significant reduction in ER-α immunoreactivity in dopaminergic neurons in the ARC, but not in the PeVN. These results provide an anatomic basis that dopaminergic neurons in the ARC and the PeVN are functionally different regarding to ER-α expression. Our study also supports the hypothesis that dopaminergic neurons in the ARC are an important neuronal population responsive to estrogen by changing the expression of ER-α in those neurons. This modification of sensitivity of dopaminergic neurons in the ARC in response to ovarian steroids may be an important molecular mechanism involved in PRL regulation, including the regulation of the proestrous surge of PRL.