Effect of gonadotropin-releasing hormone pulse frequency on gonadotropin secretion and subunit messenger ribonucleic acids in perifused pituitary cells.

Slow frequency GnRH pulses have been proposed to preferentially increase circulating FSH levels by increasing FSH synthesis and pulsatile release. Examination of this proposal using various in vivo models, however, has produced conflicting results. To examine directly the effects of GnRH pulse frequency on the pituitary, we compared the effects of 2.5-nM GnRH pulses administered every 1 h or every 4 h vs. no GnRH, using perifused rat pituitary cells. FSH secretion (total area under the response curve) was 2-fold greater (P less than 0.01) with every hour than with every 4 h GnRH pulses. This difference resulted from the increased number of GnRH pulses and increased (P less than 0.05) interpulse FSH secretion, whereas FSH pulse amplitude was unchanged. FSH beta mRNA levels at the completion of the 11-h perifusion were increased 4.5-fold by GnRH every h (P less than 0.01) and 3.3-fold by GnRH every 4 h (P less than 0.05) above levels in untreated cells. FSH beta mRNA levels were greater (P less than 0.05) at the faster GnRH pulse frequency. Because more frequent stimulation delivered more GnRH during the study, cells were next stimulated with 2.5 nM GnRH every 1 h for nine pulses, 7.5 nM GnRH every 4 h for three pulses to equalize the GnRH dose, or 2.5 nM GnRH every 4 h for three pulses. Interpulse FSH secretion and FSH beta mRNA levels were again greater (P less than 0.05) with every hour than every 4 h GnRH pulses. Interpulse LH secretion, FSH and LH pulse amplitude, and LH beta and alpha-subunit mRNA levels were not different between the groups. GnRH doses of 0.1-10 nM every hour increased FSH and LH pulsatile secretion dose-dependently, but FSH beta, LH beta, and alpha-subunit mRNA levels were similar. In conclusion, our data reveal that reducing the frequency of GnRH pulses from every hour to every 4 h reduces both FSH beta mRNA levels and FSH interpulse secretion, but does not change GnRH-stimulated FSH pulsatile release. We suggest that the finding by others that slow frequency GnRH pulses increase circulating FSH levels under certain experimental conditions in vivo may instead be explained by complex hormonal interactions or changes in FSH clearance.     

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.     

Gonadal steroids modulate pulsatile luteinizing hormone secretion by perifused rat anterior pituitary cells

Recent studies have shown that LH secretion is pulsatile and that LH pulse characteristics are affected by the prevailing steroid environment in both male and female rats. In the present study, a cell perifusion system was used to examine the effects of testosterone (T) and 17 beta-estradiol (E) on LHRH-stimulated pulsatile LH secretion. T inhibited LH secretion, increasing the EC50 for LHRH, while E stimulated secretion, lowering the EC50. Steroid effects were independent of both LHRH pulse amplitude and frequency. E also affected the pattern of LH secretion by facilitating both LHRH self-priming and desensitization to LHRH. These results show that steroids can affect pulsatile LH secretion by actions exerted at the pituitary level and that steroids can induce both quantitative and qualitative changes in LH secretion in the presence of an invariant LHRH stimulus. These results help to elucidate the mechanisms underlying steroid feedback in vivo, since reduction in pituitary responsiveness to LHRH may play an important role in T feedback, while facilitation by E of both self-priming and desensitization may serve to increase the magnitude and shorten the duration of the proestrous LH surge.     

Effects of gonadal steroids on gonadotropin secretion in males: studies with perifused rat pituitary cells

The feedback effects of testosterone (T) and estradiol (E2) on FSH and LH secretion were compared in dispersed pituitary cells from adult male rats perifused with pulses of GnRH. Cells were stimulated with 10 nM GnRH for 2 min every 1 h. T (10 nM) pretreatment for 24 h reduced the amplitude of FSH and LH pulses to 77 +/- 4% (mean +/- SE) and 47 +/- 3% of control values, respectively (P less than 0.01), whereas 6-h T treatment was without effect. By contrast, interpulse secretion of FSH was increased after 24 h T to 184 +/- 7% of the control value (P less than 0.01), but interpulse LH release was unchanged (104 +/- 5%). E2 (0.075 nM) treatment of pituitary cells reduced GnRH-stimulated FSH and LH release within 2 h to 75 +/- 2% and 73 +/- 3% of control values, respectively (P less than 0.01). E2 pretreatment for 24 h stimulated (P less than 0.025) GnRH-induced FSH (136 +/- 10%) and LH (145 +/- 8%) release and also increased (P less than 0.01) interpulse FSH (127 +/- 5%) and LH (145 +/- 8%) secretion. These data indicate that the suppression of FSH and LH secretion by T in males is due in part to a direct effect on the pituitary. The findings that T suppresses GnRH-stimulated FSH less than LH, and that T stimulates interpulse FSH, but not LH, provide evidence for differential regulation of FSH and LH secretion by T. The dissimilar actions of T on GnRH-stimulated pulses and interpulse gonadotropin secretion suggest that interpulse secretion is unrelated to stimulation by GnRH, although its physiological significance is unknown. Since E2, in physiological levels for males, increased pituitary FSH and LH secretion, the suppression of gonadotropin secretion by E2 in vivo in males may result from an effect on the hypothalamic pulse generator; however, additional studies are needed before extending these conclusions to higher mammals and men.     

Differential expression of growth hormone messenger ribonucleic acid by somatotropes and gonadotropes in male and cycling female rats

Past studies have reported the appearance of cells sharing phenotypic characteristics of gonadotropes and GH cells. During diestrus and early proestrus, a subset of somatotropes (40-60%) expressed both GH antigens and gonadotropin (LH-beta, LHbeta, or FSH-beta) messenger RNAs (mRNAs) or GnRH receptors. More recently, we reported that subsets of gonadotropes identified by LHbeta or FSHbeta antigens expressed GH- releasing hormone (GHRH) binding sites. The present studies were designed to learn if these putative multipotential cells also expressed GH mRNA. Biotinylated sense and antisense oligonucleotide probes were developed and cytochemical in situ hybridization tests were optimized for the detection of GH mRNA with GH, LHbeta, and FSHbeta antigens. RNase protection assays were developed with a complementary RNA probe that detected a 380-bp region at the 5' end of the GH mRNA. Both the in situ hybridization and RNase protection assays detected changes in expression of GH mRNA during the estrous cycle with the lowest expression occurring during metestrus and peak expression occurring on the morning of proestrus. Cell counts confirmed the results of the RNase protection assays showing that increases in mRNA levels seen from metestrus to proestrus reflected increased percentages of GH mRNA-bearing cells. In addition, densitometric analyses demonstrated that the higher GH mRNA levels assayed from diestrus to proestrus reflected increased area and density of label per cell. Both types of assays showed sex differences in expression of GH mRNA; male rat cell populations had higher values than female rats in metestrus, diestrus, or estrus. However, percentages of GH cells in male rats were equal to those from proestrous female rats and levels of GH mRNA were lower in male rats than proestrous females. Dual labeling experiments showed that, in male rats and diestrous, proestrous, or estrous females, GH mRNA was expressed in over 70% of GH cells. Expression of GH mRNA was also found in 50-57% of cells with LHbeta or FSHbeta antigens in the same groups. The lowest expression was seen in the metestrous groups (30-40% of GH cells or gonadotropes expressed GH mRNA). Expression of GH mRNA was first increased from metestrus to diestrous largely in GH cells, and slightly in cells with LHbeta antigens. Further increases were seen in GH and LH cells by the morning of proestrus. In contrast, FSH gonadotropes did not show an increased expression of GH mRNA until the morning of proestrus (reaching the same peak reached by LH cells). These data confirm the working hypothesis that a multihormonal cell type develops during diestrus to support both the somatotrope and gonadotrope populations. Collectively, our studies suggest that this multihormonal cell may function to help support the regulatory functions of the gonadotrope during the periovulatory period. In addition, the appearance of significant levels of expression of GH mRNA by male rat gonadotropes suggests that this multihormonal cell may play a role in regulation of the male reproductive system as well.     

Mechanisms of age-related changes in gonadotropin-releasing hormone receptor messenger ribonucleic acid content in the anterior pituitary of male rats.

To determine the mechanism(s) of age-related changes in gonadotropin release from pituitary gonadotrophs in male rats, we measured the number of GnRH (gonadotropin-releasing hormone) receptor containing cells and expression of GnRH receptor mRNA per cell in the anterior pituitary. An in situ hybridization procedure was performed using young (six months) and old (24-25 months) Wistar rats. An image analysis system was employed for the autoradiographic analysis. The number of pituitary cells increased during aging (approximately 45%, p < 0.01). On the other hand, the number of GnRH receptor mRNA-containing cells decreased (approximately 25%, p < 0.05). The percentage of these cells in old rats decreased to less than a half of that in young animals (p < 0.01). GnRH receptor mRNA per cell in old rats was only 7% lower than in young (p < 0.01). These results suggest that loss of pituitary gonadotroph GnRH receptors and response is primarily due to the loss of gonadotrophs, and that the death mechanism(s) are responsible for decreased stimulation of Gn release during aging.     

Insulin-like growth factor I regulates growth hormone secretion and messenger ribonucleic acid levels in human pituitary tumor cells

GH secretion and mRNA levels were measured in cultured human GH adenoma cells incubated in serum-free medium for up to 48 h. A human recombinant insulin-like growth factor I (IGF-I) analog, Thr-59-IGF-I (6.5 nM), inhibited basal GH secretion by up to 60% in tumor cell cultures. The 30-50% stimulation of GH secretion by GH-releasing hormone (GHRH) was prevented by simultaneous exposure of the cells to IGF-I (6.5 nM). Gel electrophoresis of total RNA derived from GH cell adenoma tissue, followed by transfer and hybridization with 32P-labeled human GH cDNA, revealed a distinct mRNA species of about 1.0 kilobases. Using cytoplasmic dot blot hybridization, IGF-I inhibited the levels of human GH mRNA sequences in these cells and also prevented the GHRH-induced stimulation of GH mRNA. A monoclonal antibody to the type I IGF-I receptor (alpha IR3) prevented the inhibitory effects of IGF-I on basal and GHRH-stimulated GH secretion. This antibody also prevented the IGF-I-induced suppression of GH mRNA sequences. PRL secretion in these cells was not altered by IGF-I. Furthermore, relative levels of beta-actin mRNA were unaltered by IGF-I. Thus, IGF-I suppresses basal and GHRH-stimulated GH secretion and GH mRNA levels in pituitary adenoma cells, indicating that IGF-I acts selectively on the somatotroph to directly regulate GH gene expression.     

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 .     

Regulation of follicle-stimulating hormone beta and common alpha-subunit messenger ribonucleic acid by gonadotropin-releasing hormone and estrogen in the sheep pituitary

The effect of gonadotropin-releasing hormone (GnRH) and/or estradiol (E2) on pituitary messenger ribonucleic acid (mRNA) levels of luteinizing hormone beta (LH beta), follicle-stimulating hormone beta (FSH beta) and the common alpha-subunit were determined in anterior pituitary glands from ovariectomized (OVX) ewes. Hypothalamo-pituitary disconnected (HPD) ewes receiving appropriate hormonal treatment were used to assess the relative roles of GnRH and E2 in directly regulating FSH beta and alpha-subunit mRNA levels. Levels of LH beta mRNA were increased in OVX animals compared with intact controls, and E2 treatment of OVX animals significantly reduced mRNA levels of LH beta and FSH beta. HPD substantially reduced FSH beta and alpha-subunit mRNA levels. Treatment of OVX/HPD animals with pulses of GnRH (250 ng/2 h) for 1 week restored FSH beta and alpha-subunit mRNA to OVX levels. Combined GnRH and E2 treatment significantly lowered FSH beta mRNA levels, but resulted in a rise in alpha-subunit mRNA levels. Treatment of OVX/HPD ewes with E2 alone had no effect on FSH beta and alpha-subunit mRNA levels. These findings indicate that E2 acts directly on the pituitary to negatively regulate FSH beta mRNA levels, and to positively regulate alpha-subunit mRNA levels in the presence of GnRH.     

Regulation of growth hormone action by gonadal steroids.

Sex steroids modulate growth hormone (GH) secretion and action. Estrogen attenuates GH action in a dose- and route-dependent manner by inhibiting GH-regulated endocrine function of the liver. Testosterone amplifies the metabolic action of GH while exhibiting similar but independent effects of its own. The strong modulatory effect of gonadal steroids on GH responsiveness provides insights into the biologic basis of sexual dimorphism in growth, development, and body composition and practical information for the clinical endocrinologist in the treatment of hypopituitary patients.     

Gonadotropin-releasing hormone-stimulated luteinizing hormone secretion by perifused pituitary cells from normal, gonadectomized, and testicular feminized rats

To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King x Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P less than 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P less than 0.01) than that by cells from intact animals, but lower (P less than 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-dependent LH release. Pituitary cells from intact female rats showed an overall greater (P less than 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P less than 0.0001) or intact female (P less than 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRH-stimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P less than 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact females rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens.     

Identification of human growth hormone messenger ribonucleic acid in pituitary adenoma cells by in situ hybridization

The technique of in situ hybridization was used to examine human GH gene expression in human GH-secreting pituitary adenoma cells. Five somatotroph adenoma specimens obtained at surgery were dispersed into single cell suspensions. Hybridization experiments were performed on cells immediately after dispersal or on cells cultured for 48-72 h in a defined serum-free medium. Tritiated GH cDNA was used to probe fixed cells, and hybridization was determined by liquid autoradiography. Of the freshly dispersed adenoma cells probed with GH cDNA, more than 70% contained GH mRNA, as determined by counting silver grains per cell. Significant cellular grain counts were obtained for GH cDNA-probed cells from all five tumors, while negative controls showed negligible silver grain counts. In cultured cells derived from three of five tumors, an average of 40% contained detectable GH mRNA. Therefore, quantitative in situ hybridization is a useful technique to demonstrate the expression of GH mRNA in human pituitary adenoma cells.     

Regulation of insulin-like growth factor-I messenger ribonucleic acid expression in Leydig cells

In the present study, we evaluated insulin-like growth factor-I (IGF-I) messenger RNA expression in the rat testis. Crude interstitial cells were separated into three distinct bands on 15-60% Percoll density gradients. IGF-I mRNA was mainly localized in the Leydig cell-enriched fraction (band 3), while band 1 and band 2 cells did not contain significant amounts of IGF-I mRNA. Leydig cell IGF-I mRNA consisted of multiple species varying from 0.8 to 7.5 kb and was present in rat Leydig cells all ages examined, from 25 to 55 days old. To further document that IGF-I mRNAs are present in Leydig cells, the method of Klinefelter et al. (Biol. Reprod. (1987) 36, 769-783) was used to isolate highly purified (greater than 98% pure) Leydig cells. Most of the IGF-I mRNA was localized in these Leydig cells, while there was no detectable IGF-I mRNA in the whole testis or other interstitial cells. Furthermore, IGF-I mRNA in Leydig cells was increased more than 2-fold by growth hormone (GH) administration in vivo. This suggests that IGF-I mRNA in Leydig cells is also GH dependent. Interstitial IGF-I produced in Leydig cells may have both autocrine and paracrine effects in the testis.