Thyroid hormone specifically inhibits prolactin synthesis and decreases prolactin messenger ribonucleic acid levels in cultured pituitary cells

Electrophoretic analysis of soluble proteins from pituitary cells pulse labeled with [35S]methionine demonstrated that 10 nM T3 inhibited PRL synthesis, but did not affect the synthesis of most other pituitary proteins. The effects of T3 were somewhat slow, requiring about 3 days for a 50% reduction in PRL synthesis. PRL synthesis slowly returned toward control levels after the removal of T3 from the culture medium. In serum-free medium, a concentration of about 0.6 nM T3 was required for half-maximal inhibition of PRL synthesis. In medium containing 5% fetal calf serum, only slightly higher concentrations of T3 were required to inhibit PRL synthesis. The Kd for the binding of [125]T3 to pituitary cell nuclei was 0.2 nM. Analysis of PRL mRNA levels by hybridization of total cellular RNA to PRL cDNA demonstrated that there was a good correspondence between T3 effects on PRL synthesis and PRL mRNA. These findings demonstrate that T3 can specifically inhibit PRL synthesis and PRL mRNA levels in cultured pituitary cells and suggest that T3 may have a physiological role in the regulation of PRL synthesis.     

Inhibitory effect of androgen on estrogen-induced prolactin messenger ribonucleic acid accumulation in the male rat anterior pituitary gland

Estrogens are known to exert specific stimulatory effects on basal and dopamine-inhibited PRL secretion and synthesis as well as on PRL gene expression. However, dihydrotestosterone (DHT) and progesterone (P), although inactive alone, can reverse the effect of 17 beta-estradiol (E2) on PRL release both in vivo and in vitro. Using castrated male rats, we have studied the effect of E2 (0.25 micrograms), P (2 mg), or DHT (100 micrograms) administered twice daily for 14 days alone or in combination on pituitary PRL mRNA levels measured by quantitative in situ hybridization. Treatment with E2 increased the accumulation of PRL mRNA by about 2.6-fold. Administration of P or DHT alone failed to modify PRL mRNA concentrations. However, DHT could prevent by 80% the stimulatory effect of E2 on PRL mRNA levels. Similar results were obtained by dot blot hybridization assay. The effects of sex steroids on PRL mRNA were closely paralleled to pituitary PRL content measured by RIA. The present data demonstrate that the effect of sex steroids on immunodetectable PRL result from a modulation of PRL mRNA accumulation. The sexual dimorphism observed in pituitary PRL content results from a 3.5-fold greater accumulation of PRL mRNA in intact females than in male rats. These results also clearly show that quantitative in situ hybridization is a powerful tool in the investigation of the regulation of gene expression in addition to providing valuable information on the localization of specific mRNA.     

Prolactin messenger ribonucleic acid concentrations in 4-day cycling rats and during the prolactin surge

Pituitary PRL mRNA concentrations were measured during the 4-day rat estrous cycle. Adult female Sprague-Dawley rats were killed at 3-h intervals throughout the cycle and hourly between 1000 and 2400 h on proestrus (n = 5-12). Serum PRL was increased on the afternoons of proestrus (P) and estrus (E), with peak concentrations at 1700 h (P, 624 +/- 126; E, 261 +/- 107 ng/ml). PRL mRNA concentrations were elevated during the evening on P and E (2300 h: P, 14.4 +/- 1.5; E, 16.1 +/- 1.3 ng cDNA bound/100 micrograms pituitary DNA) to values 2-fold higher than those at 0800 h on each respective day. On diestrus (D) PRL mRNA levels decreased abruptly during the morning (1100 h, 1.7 +/- 0.3 ng cDNA bound), followed by a 6- to 7-fold increase between 1700 and 2000 h on the same evening. In contrast, PRL mRNA levels were elevated at 0800 h on metestrus (M). The changes in PRL mRNA concentrations obtained on M and D were not associated with increased PRL secretion. A more detailed examination of P revealed that PRL mRNA levels increased during the morning (1000 h, 9.9 +/- 2.6 ng cDNA bound), then decreased abruptly at 1100 h (4.9 +/- 1.2). The morning rise in mRNA concentrations was followed by a 2-fold rise in pituitary PRL content. As serum PRL rose during the afternoon surge, a coincident decrease in pituitary PRL content and an increase in PRL mRNA levels were observed. The relationship between PRL secretion and gene expression was further examined in ovariectomized estradiol-replaced rats receiving either bromocriptine (1.2 mg/day, sc) or vehicle control sc. The vehicle-treated group expressed a characteristic afternoon PRL surge between 1500 and 2100 h. Pituitary PRL decreased during the surge to 10% of morning values, and PRL mRNA levels increased 2-fold beginning 2 h after initiation of the surge. These changes in serum PRL, pituitary PRL, and PRL mRNA levels were abolished by bromocriptine administration. These data reveal that alterations in PRL mRNA concentrations occur on a daily basis during the rat estrous cycle. Increases occur during the evenings of P and E at the time of the increase in PRL secretory activity. The effect of blocking the PRL surge in ovariectomized estradiol-replaced rats suggests a regulatory interaction between secretion and gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoregulation of pituitary growth hormone messenger ribonucleic acid levels in rats bearing transplantable mammosomatotrophic pituitary tumors

Female Wistar-Furth rats were implanted sc with GH3 rat pituitary tumor cells. Tumors were palpable by 4 weeks, and animals were killed periodically from 5-9 weeks. Tumor-bearing rats (n = 10) were heavier than their respective controls, reaching a weight of 372 +/- 3 by 9 weeks vs. 195 +/- 5 g in controls (mean +/- SE). Circulating serum GH levels increased in tumor-bearing animals from 218 +/- 50 to 9067 +/- 962 ng/ml. Serum insulin-like growth factor I (IGF-I) levels were elevated 3-fold in tumor-bearing rats. After death, pituitary glands were excised, and their total RNA was extracted. GH mRNA was assayed by dot hybridization of immobilized pituitary RNA with [32P]cDNA for rat GH. The hybridization signal was quantified by densitometry of autoradiographs. Pituitary rat GH mRNA levels were suppressed 50% in tumor-bearing animals after 5 weeks. By the end of the 9-week period, pituitary GH mRNA levels were undetectable in tumor-bearing animals. The results show that GH tumor-bearing animals exhibit high levels of circulating GH and IGF-I and suppressed endogenous pituitary GH mRNA levels. This may be caused by autoregulation of pituitary GH gene expression either at the level of the hypothalamus or by a direct effect of GH on the pituitary. Alternatively, the elevated levels of IGF-I may be responsible for the suppression of pituitary GH gene expression .     

Insulin-induced hypoglycemia increases proopiomelanocortin messenger ribonucleic acid levels in rat anterior pituitary gland

To study the effect of acute stress on ACTH secretion and synthesis in rat pituitary and hypothalamus, ACTH content and POMC mRNA levels (measured by use of Northern blot analysis) in these tissues as well as the levels of ACTH in plasma and those of CRF in the hypothalamus were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min. ACTH levels in the anterior pituitary lobe (AP) decreased at 30 min, and then returned to control levels at 60 min. No change was seen in the intermediate-posterior pituitary (IP) or the hypothalamus after insulin injection. CRF levels decreased at 30 and 60 min, then returned to control levels at 90 min in the medial basal hypothalamus, including the median eminence. Hybridization with a cDNA probe revealed a single size class of POMC mRNA in AP, IP, and hypothalamus, and the size of POMC mRNA in these tissues did not change during the experimental period. POMC mRNA levels in AP increased at 60 min and reached a peak at 120 min, but those in IP and hypothalamus did not change. These results suggest that 1) insulin-induced hypoglycemia stimulates both secretion and synthesis of ACTH (at least by increasing POMC mRNA levels) in the AP, and 2) the levels of ACTH and POMC mRNA in the IP and hypothalamus are not affected by insulin-induced hypoglycemia.     

Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus

Polyadenylated RNA, extracted from rat hypothalami, cross-hybridized with a RNA probe complementary in sequence to rat PTH (rPTH) messenger RNA (mRNA). Amplification of complementary DNA (cDNA) by the polymerase chain reaction also demonstrated the presence of rPTH mRNA in the rat hypothalamus and parathyroid gland. rPTH mRNA was localized by in situ hybridization in the paraventricular and supraoptic nuclei of the rat hypothalamus. These results demonstrate the expression of the PTH gene in the central nervous system of the rat in areas which suggest roles for PTH in neuroendocrine function.     

High plasma growth hormone (GH) levels inhibit expression of GH secretagogue receptor messenger ribonucleic acid levels in the rat pituitary

Synthetic GH secretagogues (GHSs) act via a receptor (GHS-R) distinct from that of GH-releasing hormone. The GHS-R has been cloned from the pituitary and is expressed not only in the pituitary but also in specific areas of the brain, including the hypothalamus. Recent studies suggest that hypothalamic GHS-R expression is regulated by GH. This study was designed to investigate whether pituitary GHS-R expression is modulated by GH. Female Wistar-Furth rats were injected sc with either saline (control) or GC tumor cells (GC) that secrete rat GH. The tumors were allowed to develop for 1-4 weeks. At weeks 1-4, control (n = 4-8) and GC rats (n = 3-8) were killed. Pituitary GHS-R messenger RNA (mRNA) was measured by a quantitative competitive PCR assay. The endogenous GHS-R mRNA levels were measured by determining the amount of competitive template RNA required to produce equimolar amounts of native and competitive template PCR products. The mean log plasma GH levels were significantly greater in the GC rat group than in the control group at weeks 2, 3, and 4. At these times, the mean log pituitary GHS-R mRNA contents were significantly lower in the GC rat group than in the control group. No relationship could be established between log estradiol levels and GHS-R levels. These data indicate that pituitary GHS-R expression is modulated by GH.     

Pituitary alpha-subunit messenger ribonucleic acid remains elevated during the latter stages of the preovulatory luteinizing hormone surge

In this study, pituitary alpha-subunit mRNA amounts were assessed during the latter stages of the preovulatory LH surge in the normal cycling ewe. The times chosen were 15 and 25 h after the onset of behavioral estrus, designated E+15 and E+25. These mRNA values were correlated with pituitary and serum LH concentrations to assess the possible role of subunit synthesis during these times of the cycle, and then compared to values obtained from hybridizations of RNAs from animals in other groups representing defined stages of the cycle. One of these groups included animals during the midluteal phase (day 12), whose amounts were assigned a value of 1. It was observed that the amounts of alpha-subunit mRNA in E+15 and E+25 groups, as measured by translations and cDNA hybridizations, were approximately 10-fold higher than the day 12 values and were roughly similar to those observed in a group examined 5 h after estrus. Thus, it is evident that the alpha mRNA levels are not only elevated during the LH surge, but also remain high at a time when both serum and pituitary LH concentrations have fallen dramatically, suggesting the involvement of changing mRNA amounts in the feedback systems associated with the maintenance of pituitary and serum gonadotropin amounts.     

Follicle-stimulating hormone beta subunit messenger ribonucleic acid concentrations during the rat estrous cycle

Serum follicle-stimulating hormone (FSH), pituitary FSH content and FSH beta subunit mRNA concentrations were measured at 1 to 3h intervals throughout the 4 day estrous cycle in rats. Serum FSH was stable (range 200-320 ng/ml) apart from the biphasic proestrus surge (5 fold elevation) which was present from 1800 h of proestrus through 0800 h on estrus. Basal FSH beta mRNA concentrations from late metestrus through the afternoon of proestrus were 0.10 +/- 0.04 f mol cDNA bound/100 micrograms pituitary DNA. The major increase in FSH beta mRNA began at 2000 h on proestrus, 2 h after the initial rise in serum FSH and peak mRNA concentrations (0.43 +/- 0.08 f mol cDNA bound) occurred at 0200 h on estrus. FSH beta subunit mRNA concentrations were again increased at 2300 h on estrus (peak 0.24 f mol cDNA bound) and remained elevated through 1700 h on metestrus. Pituitary FSH content was transiently increased during metestrus and diestrus, but was elevated at 1000 h through 1900 h on proestrus (peak 5-fold increase). FSH content fell rapidly at 2000 h and remained low until 1400 h on estrus when values again rose. These data show that FSH beta mRNA is increased 4-5 fold during the proestrus FSH surge, and a smaller increase occurs on metestrus in the absence of elevated FSH secretion. The increased concentrations of FSH beta mRNA occurred at different times to the previously reported changes in alpha and LH beta mRNAs. Therefore, the data suggest that different mechanisms are involved in the regulation of LH and FSH beta subunit gene expression during the 4-day estrous cycle in rats.     

Follicle-stimulating hormone beta subunit messenger ribonucleic acid concentrations during the rat estrous cycle

Serum follicle-stimulating hormone (FSH), pituitary FSH content and FSH beta subunit mRNA concentrations were measured at 1 to 3h intervals throughout the 4 day estrous cycle in rats. Serum FSH was stable (range 200-320 ng/ml) apart from the biphasic proestrus surge (5 fold elevation) which was present from 1800h of proestrus through 0800 h on estrus. Basal FSH beta mRNA concentrations from late metestrus through the afternoon of proestrus were 0.10 +/- 0.04 f mol cDNA bound/100 micrograms pituitary DNA. The major increase in FSH beta mRNA began at 2000 h on proestrus, 2 h after the initial rise in serum FSH and peak mRNA concentrations (0.43 +/- 0.08 f mol cDNA bound) occurred at 0200 h on estrus. FSH beta subunit mRNA concentrations were again increased at 2300 h on estrus (peak 0.24 f mol cDNA bound) and remained elevated through 1700 h on metestrus. Pituitary FSH content was transiently increased during metestrus and diestrus, but was elevated at 1000 h through 1900 h on proestrus (peak 5-fold increase). FSH content fell rapidly at 2000h and remained low until 1400 h on estrus when values again rose. These data show that FSH beta mRNA is increased 4-5 fold during the proestrus FSH surge, and a smaller increase occurs on metestrus in the absence of elevated FSH secretion. The increased concentrations of FSH beta mRNA occurred at different times to the previously reported changes in alpha and LH beta mRNAs. Therefore, the data suggest that different mechanisms are involved in the regulation of LH and FSH beta subunit gene expression during the 4-day estrous cycle in rats.     

Dynamic regulation of follicle-stimulating hormone-beta messenger ribonucleic acid levels by activin and gonadotropin-releasing hormone in perifused rat pituitary cells.

Maintenance of FSH biosynthesis requires ongoing exposure to pulsatile GnRH. Recent data demonstrate that activin also stimulates FSH biosynthesis. We used a perifused pituitary system to examine regulation of FSH beta mRNA levels by pulsatile GnRH and activin. Hourly pulses of 10 nM GnRH increased FSH beta mRNA levels by 3-fold. In the same experiment, continuous infusion of 50 ng/ml activin elicited a 50-fold increase in FSH beta mRNA. This magnitude of response to activin in perifusion was unexpected, as only a 2.7-fold increase in FSH beta mRNA was observed when activin was administered to pituitary cells that were cultured in dishes. Since perifusion columns, unlike culture dishes, are exposed to a continuous supply of fresh medium, we examined the possibility that endogenous factors produced by pituitary cells cultured in dishes were stimulating the cells in a paracrine fashion, thereby precluding the full response to exogenously added activin. The kinetics of FSH beta mRNA expression were examined immediately after pituitary dispersion and at different times after culturing the cells in plates. FSH beta mRNA levels fell rapidly after dispersion to 8% of initial levels and remained low over 8 h. Thereafter, FSH beta mRNA levels increased slowly and exceeded initial levels by the second day of culture. In a parallel set of experiments, when medium conditioned by exposure to plated cells was applied to the perifusion system, FSH beta mRNA levels were selectively stimulated (6-fold). These data suggest the removal during dispersion and subsequent accumulation in culture of pituitary-derived factors that are important for the maintenance of FSH beta mRNA levels. We conclude that activin plays a greater role in the regulation of FSH beta mRNA levels than was suggested by previous experiments employing static culture systems in which autocrine or paracrine stimulation may have obscured the effects of exogenously added activin.     

The effect of altered thyroid status on pituitary hormone messenger ribonucleic acid concentrations in the rat

To study the effects of altered thyroid status on pretranslational control of pituitary hormones, adult male rats were given propylthiouracil for 6 weeks and underwent the following studies. 1) Rats were injected with T3 at 10 micrograms/100 g BW daily for 10 days. 2) Rats were given T3 injections at 0, 0.01, 0.1, 1.0, or 10 micrograms/100 g BW for 10 days. 3) Rats were killed 0, 1, 6, or 24 h after a single injection of T3 at 10 micrograms/100 g BW or after 5 or 10 days of daily T3 injections. Pituitary mRNA concentrations of TSH beta, alpha-subunit, PRL, GH, POMC, FSH beta, and LH beta were determined for individual animals. Marked increases in TSH beta and alpha-subunit mRNAs occurred after PTU treatment, and these changes were reversed by 1.0 microgram/100 g BW T3 and within 24 h of a single T3 injection of 10 micrograms/100 g BW. Further increases in the dose or time course of T3 administration led to a relatively greater suppression of TSH beta mRNA levels than alpha-subunit mRNA levels. In contrast, GH and PRL mRNA levels were low in hypothyroid animals, and both rose toward control levels with 0.1 microgram/100 g BW T3 and by 24 h after a single T3 dose. Induction of hyperthyroidism did not further increase GH mRNA levels above control, but increased PRL mRNA levels 2-fold over control. No changes were seen in FSH beta, LH beta, or POMC mRNA levels with any treatment. Thus, studies of altered thyroid status in the rat reveal dose-response and time-course variability in the pretranslational control of TSH beta, alpha-subunit, GH, and PRL by thyroid hormone.     

Gonadotropin-releasing hormone messenger ribonucleic acid levels are unaltered with changes in the gonadal hormone milieu of the adult male rat

Testicular function is regulated by the negative feedback effect of sex hormones acting at the brain and pituitary to inhibit the secretion of LH and FSH. An important component of this feedback axis is presumed to involve regulation of secretion and possibly synthesis of GnRH by the brain. We tested the hypothesis that the castration-induced increase in gonadotropin secretion is subserved, at least in part, by increased synthesis of GnRH. Using in situ hybridization and an oligonucleotide probe to pro-GnRH messenger RNA (GnRH mRNA), we compared the level of cellular GnRH mRNA and the relative number of GnRH mRNA-containing neurons between intact and 21-day castrate adult male rats. To derive estimates of the number of GnRH cells and the cellular GnRH mRNA content, coronal sections from each animal were anatomically matched between intact and castrate groups. All identifiable cells within these sections were counted and analyzed with the aid of a computerized image analysis system, by an observer unaware of the animal's experimental group and were assigned an anatomical location for reference. In an initial experiment, we observed no difference in cellular GnRH mRNA signal level between intact (n = 4) and castrate (n = 5) animals (129 +/- 8 vs. 139 +/- 5 grains per cell); however, we did find a statistical difference between the intact and castrated groups in the relative number of GnRH mRNA-containing cells (intact: 212 +/- 15 vs. castrate: 320 +/- 18). To confirm this observation, we repeated the experiment by again comparing the number of GnRH mRNA-positive cells between intact (n = 4) and castrate (n = 4) rats. In this second experiment, we found no difference in the number of identifiable GnRH mRNA-containing cells between intact and castrate animals (272 +/- 14 vs. 274 +/- 36, respectively); this was the case for the total cell count as well as when the data were analyzed by anatomical region. To clarify the conflicting results on cell counts of Exps 1 and 2, we repeated the experiment a third time, again comparing both the number of GnRH mRNA-containing cells and the cellular content of GnRH mRNA. In this experiment, we observed that neither cell number nor content of GnRH mRNA differed between the intact and castrate groups. Again, this was the case for total cell count, as well as when the data were analyzed by anatomical region.(ABSTRACT TRUNCATED AT 400 WORDS)