Gonadal hormones and gonadotropin-releasing hormone (GnRH) alter messenger ribonucleic acid levels for GnRH receptors in sheep

GnRH regulates the synthesis and secretion of pituitary gonadotropins. The number of receptors for GnRH (GnRH-rec) can vary from 500 to 15,000-20,000/gonadotrope in ovine pituitary cultures after treatment with physiologically relevant combinations of gonadal hormones. This large range suggests that regulation of GnRH-rec expression may be an important control point in GnRH action at the pituitary level. Reported here are the changes in GnRH-rec mRNA associated with pituitary treatments (48 h) of 17 beta-estradiol (E), progesterone (P), and an enriched preparation of porcine follicular inhibin (IN). Northern blot analysis was used to detect 3 species of GnRH-rec mRNA in primary ovine pituitary culture [5.5 kilobases (kb; 32%), 3.6 kb (51%), and 1.4 kb (17%)]; all were changed in parallel by E, P, and IN. GnRH-rec mRNAs were increased 190% over control levels after treatment with either E or IN, and 400% with E and IN combined; when E and IN were added along with P, the increase was only 50% (P caused an 87% inhibition of E plus IN induction). The addition of P in the absence of any other treatment reduced levels of GnRH-rec mRNA by 50%. Studies were also conducted with GnRH agonists (GnRH-A) due to their widespread clinical use for down-regulating reproductive function in men and women. The addition of GnRH-A to cultures was as effective as P in blocking E plus IN induction of GnRH-rec mRNA. In vivo studies in wethers showed that 7 days of chronic treatment with GnRH-A decreased all sizes of ovine GnRH-rec mRNA by 84-89%. These data indicate that E, P, and IN change GnRH-rec levels at least in part by changing levels of GnRH-rec mRNAs. They also show that GnRH-A can almost entirely block E plus IN induction of GnRH-rec mRNA in vitro and decrease levels of GnRH-rec mRNA in vivo in wethers.     

Gonadotropin-releasing hormone (GnRH) in ancient teleosts, the bonytongue fishes: putative origin of salmon GnRH

PURPOSE: To measure the compressive forces of the haptics of 28 intraocular lens (IOL) models for different modes of compression and compare the results of two types of measurements. SETTING: Department of Ophthalmology, Central Hospital of Central Finland, Jyv?skyl?, Finland. METHODS: The haptics of 28 types of IOLs were compressed to a diameter of 9.0 mm between curved anvils. The compression forces in the plane of compression (i.e., in the plane of the optics) were measured at 0.5 mm intervals. During compression, the optics and the haptics were free to rotate with respect to the anvils. The results were compared with those of earlier measurements in which the optics were held fixed during compression. Perpendicular forces were measured at 0.4 mm intervals. RESULTS: The measured forces in the plane of the optics varied between 114 and 659 mg at a diameter of 10.0 mm and 192 and 1047 mg at a diameter of 9.0 mm. When compressed to 10.0 mm in diameter, the forces were 1 to 75% lower than when lens rotation was not possible. The forces perpendicular to the optic varied between 0 and 96 mg at a 10.0 mm diameter and correlated with the forces in the plane of the optic. CONCLUSION: The compression forces of the lens haptics were generally lower when the lenses were allowed to rotate during compression. The orders of stiffness of the haptics in these two measurements were similar. The perpendicular forces were generally small and correlated significantly with the forces measured in the plane of the optic.     

Cyclin D1 and estrogen receptor messenger RNA levels are positively correlated in primary breast cancer

The CCND1 gene, encoding the cell cycle regulatory protein cyclin D1, maps to chromosome 11q13, a locus that is amplified in about 13% of breast cancers. Because several studies have indicated a relationship between 11q13 amplification and markers of phenotype including estrogen receptor (ER) status, we tested the relationship between CCND1 and ER gene expression in 364 primary breast cancers using Northern blot analysis. Seventy-three % of samples were positive for ER mRNA, and cyclin D1 mRNA levels in the ER-positive group were significantly higher than those in the ER-negative group (P = 0.0001). When the samples were divided into quartiles of cyclin D1 expression, 58% of samples were ER positive in the lowest quartile and 87% in the highest quartile. The tumors expressing the highest levels of cyclin D1 (7%) were all ER positive. Furthermore, ER mRNA levels in the half with lower cyclin D1 mRNA were significantly less than in the half with higher cyclin D1 levels (P = 0.0001). Using simple regression analysis, there was a significant positive correlation between cyclin D1 and ER mRNA levels in the total population (P = 0.0001). This study demonstrates that cyclin D1 mRNA and ER mRNA are positively correlated in primary breast cancer, but the functional relationship between these genes remains to be elucidated.     

Gonadotropin-releasing hormone regulation of gonadotropin subunit gene expression in female rats: actions on follicle-stimulating hormone beta messenger ribonucleic acid (mRNA) involve differential expression of pituitary activin (beta-B) and follistatin mRNAs

GnRH is the primary stimulus in the regulation of gonadotropin subunit mRNA expression. Additionally, local (pituitary) production of activin and follistatin appear to modulate the expression of FSH beta mRNA. The current studies aimed to determine whether GnRH regulation of pituitary activin (beta-B) and follistatin mRNAs could play a role in the differential actions of GnRH pulse pattern on gonadotropin mRNA expression in female rats. In response to altered GnRH pulse amplitude, the expression of FSH beta and follistatin mRNAs followed an inverse pattern. Only high dose GnRH increased expression of follistatin whereas, in contrast, beta-B and FSH beta expression were increased following lower doses of GnRH. To determine whether increased follistatin mRNA expression was correlated with FSH beta mRNA responses, we examined their temporal relationship following high dose GnRH. Both FSH beta and follistatin mRNAs were increased within 2 h and remained increased through 6 h. However, by 12 h FSH beta mRNA levels returned to values seen in controls, suggesting that increased follistatin requires 6-12 h to reduce FSH beta mRNA. In response to altered GnRH pulse frequency, FSH beta expression was increased at all pulse intervals (8-240 min) examined. Rapid GnRH pulse frequencies (8-min intervals) increased follistatin expression, whereas beta-B mRNA was only increased after 30-min pulse intervals, which also resulted in maximal FSH beta mRNA concentrations. These results suggest that changes in pituitary activin (beta-B) and follistatin mRNA expression may be important components of gonadotrope responses to pulsatile GnRH, and potentially imply that GnRH stimulation of activin and follistatin peptide production provides regulatory control over the production of FSH.     

Expression of gonadotropin-releasing hormone (GnRH) gene in human uterine endometrial tissue

OBJECTIVE: To determine if adolescent mothers who request early removal of levonorgestrel implants differ from those who do not in ways that might predispose them to repeated conceptions and in their concerns about adverse effects. We hypothesized that adolescent mothers who request removal of levonorgestrel implants within 2 years of insertion have more risk factors for repeated pregnancy than those who do not. METHODS: We studied the prevalence of 21 characteristics associated with repeated adolescent pregnancy and 16 adverse effects of levonorgestrel implants in 181 postpartum, adolescent levonorgestrel implant recipients, 66 (36%) of whom had the levonorgestrel implants removed within 20 months of insertion (hereafter, removers). RESULTS: Removers (n = 66) had significantly more risk factors for repeated pregnancy and reported significantly more adverse effects than did those who continued to use levonorgestrel implants (hereafter, users) (n = 115). Concerns about adverse effects rose in tandem with risk factors for repeated pregnancy (r = 0.26; P = .001) and were the most important determinant of levonorgestrel implant removal (relative risk, 9.72; 95% confidence interval, 4.62-19.49). However, the number of risk factors for repeated pregnancy was also a significant, independent predictor of levonorgestrel implant removal (relative risk, 2.34; 95% confidence interval, 1.10-4.66). Following removal, contraceptive use was poor and conception occurred rapidly; 24 (37%) of the removers conceived again within 2 years of the index delivery. CONCLUSIONS: The study hypothesis was supported. Our findings that concerns about the adverse effects of levonorgestrel implants rise in tandem with risk factors for repeated pregnancy suggest that the efficacy of counseling before and after levonorgestrel implant insertion could be improved by addressing those aspects of the user's life that undermine the motivation to use contraception.     

Thyroid hormone and follicle-stimulating hormone regulate Müllerian-inhibiting substance messenger ribonucleic acid expression in cultured neonatal rat Sertoli cells

Thyroid hormone is a major regulator of Sertoli cell development, and the present study sought to determine the role of T3 in Müllerian-inhibiting substance (MIS) messenger RNA (mRNA) expression. MIS, a Sertoli cell secretory protein that induces Müllerian duct regression and also may be critical for germ and Leydig cell development, is maximal perinatally, then decreases as Sertoli cells mature. The fall in MIS mRNA expression is delayed by hypothyroidism in vivo, indicating that T3 could regulate MIS mRNA. However, understanding of the hormonal regulation of MIS has been limited due partly to the lack of a primary Sertoli cell culture system in which sustained expression of MIS or its mRNA can be obtained. We have developed a Sertoli cell culture system for examining hormonal regulation of MIS mRNA. We then tested the effects of T3 and/or FSH treatment on MIS mRNA levels in this new system. Initial studies indicated that MIS mRNA production by 5-day-old rat Sertoli cells was minimal in vitro. Therefore, Sertoli cells from 2-day-old rats were cultured for 2 or 4 days. After 2 days in vitro, steady state MIS mRNA levels were decreased to 36% of the levels seen in freshly isolated Sertoli cells from 2-day-old rats. However, by day 4 of culture, steady state MIS mRNA production had recovered to 67% of that seen in freshly isolated 2-day-old Sertoli cells, which closely paralleled the decrease seen in MIS production in vivo from days 2-6. MIS mRNA levels were decreased 53%, 64%, and 86% in cultures treated with 0.01, 0.1, and 1.0 nM T3 (P < 0.05), respectively. This decrease in Sertoli cell MIS mRNA did not reflect a nonspecific effect on cell viability and/or activity, as shown by a dose-responsive increase in inhibin-alpha mRNA in these same cultures. FSH (2.5-100 ng/ml) also produced a dose-responsive decrease in MIS mRNA levels, and FSH and T3 together had an additive inhibitory effect on MIS mRNA levels, indicating that these hormones may act through distinct mechanisms. In summary, this is the first primary culture system in which sustained MIS mRNA production can be demonstrated, and it should prove useful for understanding the regulation of MIS in developing Sertoli cells. In addition, T3 and FSH are major regulators of the postnatal decrease in MIS production by the rat Sertoli cell, and these hormones may act through separate pathways.     

Effects of bovine follicular fluid and passive immunization against gonadotropin-releasing hormone (GnRH) on messenger ribonucleic acid for GnRH receptor and gonadotropin subunits in ovariectomized ewes

In cultured ovine pituitary cells, inhibin increases concentrations of mRNA encoding GnRH receptor and numbers of GnRH receptors. The objective of this study was to test the hypothesis that inhibin increases concentrations of ovine GnRH receptor mRNA in vivo. Ovariectomized ewes were used to eliminate effects of endogenous ovarian hormones, and passive immunization against GnRH was employed to avoid possible confounding influences of GnRH on GnRH receptor gene expression. Two groups of ewes (n = 5/group) were treated with 50 ml GnRH antiserum on Days 0 and 3 of the experiment. One group of immunized ewes received 10 ml charcoal-extracted bovine follicular fluid (bFF) as a source of inhibin every 8 h for 48 h on Days 4-6 of the experiment. A third group of ewes was not passively immunized and was treated only with bFF, and control ewes received no treatments. Anterior pituitary glands were collected from all ewes on Day 6. Passive immunization against GnRH, alone or in combination with treatment with bFF, decreased mean concentrations of LH (p < 0.01) and LH pulse amplitude (p < 0.001). In ewes treated only with GnRH antiserum, number of LH pulses was also reduced (p < 0.03). Circulating concentrations of FSH tended to be lower (p = 0.06) in passively immunized ewes compared to controls. Treatment with bFF, alone or in combination with GnRH antiserum, reduced circulating concentrations of FSH (p < 0.02) and amounts of FSHbeta subunit mRNA (p < 0.001) to less than 30% and 10% of control values, respectively. Despite effects of bFF on concentrations of FSHbeta mRNA and secretion of FSH, concentrations of GnRH receptor mRNA were similar among controls, ewes treated with bFF alone, and passively immunized ewes treated with bFF. Passive immunization against GnRH did not affect concentrations of GnRH receptor mRNA but resulted in a reduction (p < 0.05) in amount of LHbeta mRNA. Treatment with bFF did not affect amounts of either alpha subunit or LHbeta subunit mRNA except when combined with treatment with antiserum, when amounts of both alpha and LHbeta subunit mRNA were reduced (p < 0.05). These results do not support the hypothesis that inhibin increases concentrations of GnRH receptor mRNA in the ewe, and they provide evidence that inhibin is not an acute regulator of ovine GnRH receptor gene expression in vivo.     

Tamoxifen down-regulates CD36 messenger RNA levels in normal and neoplastic human breast tissues

Tamoxifen (TAM) exerts a long-term suppressive effect on human breast cancer cell proliferation. To determine whether the effects of TAM are mediated by specific gene activation or repression, normal and tumoral human breast tissues obtained before and during TAM treatment were analyzed by differential display technique. Total RNA for differential display analysis was obtained from breast tissues from two women with the diagnosis of estrogen receptor-positive stage II (T2N1M0) infiltrating ductal carcinoma, made by incisional biopsy, followed by modified radical mastectomy performed after a 30-day treatment with TAM (20 mg/day). One 202-bp cDNA band, AP5-1, was present in normal and tumoral biopsy samples, but was absent in breast tissue obtained during TAM treatment, and was confirmed by Northern hybridization, which showed a 2.7-kb band in both patients. The differentially expressed cDNA fragment showed 99% homology to Homo sapiens CD36 gene, a glycoprotein that acts as a receptor for the extracellular matrix proteins thrombospondin-1, collagen types I and IV, and oxidized low-density lipoprotein. These results indicate that the down-regulation of CD36 induced by TAM might represent alternative or additional mechanisms of action of this drug affecting the functions of thrombospondin-1, which is involved in hematogenous tumor spread, invasion and angiogenesis, and oxidized low-density lipoprotein, playing a role in inhibition of arteriosclerosis. The multiple functions affected by the down-regulation of CD36 by TAM warrant the need for additional studies.     

NGF, cyclic AMP, and phorbol esters regulate oxytocin receptor gene transcription in SK-N-SH and MCF7 cells

In an initial study, the effects of galactose intoxication on nerve laser Doppler blood flow (NLDF) and nerve conduction velocity (NCV) were assessed after 1-16 weeks of galactose feeding in pentobarbital-anesthetized rats. NLDF was not significantly changed at any time point. NCV was significantly reduced after 16, but not 1 or 4, weeks of galactose feeding. In a second study, NLDF was not significantly changed by 4 weeks of galactose intoxication, but streptozotocin-diabetic NLDF was significantly reduced compared to both control (P<0.001) and galactose-intoxicated rats (P<0.05). Compared to control animals, sciatic motor NCV was significantly (P<0.001) reduced in the galactose group, while sciatic and saphenous sensory NCVs were not significantly changed. In the streptozotocin-diabetic rats, motor and sensory NCVs were all significantly reduced (P<0.001). In contrast to the NCV findings, mean caliber of myelinated axons in both the saphenous and sciatic nerves was reduced in galactose-intoxicated, but not streptozotocin-diabetic rats. The observed sequence of changes associated with these two models of diabetic neuropathy is not consistent with the proposed roles of ischemia and axonal dwindling in the reported nerve conduction deficits.     

Effects of ACTH and cytokines on dehydroepiandrosterone sulfotransferase messenger RNA in human adrenal cells

Dehydroepiandrosterone sulfate (DS) is the major adrenal androgen produced in the fetal and adult human; its formation is dependent upon the action of dehydroepiandrosterone sulfotransferase (DST). Since the factors that regulate DST are poorly characterized, we investigated the effects of ACTH, which stimulates DS production, and the cytokines transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) , both of which are inhibitory to adrenal steroidogenesis, on cultured human fetal adrenal cells. Cellular levels of DST mRNA were increased in a dose dependent fashion in response to ACTH; DST mRNA was less responsive to ACTH stimulation than was 17 hydroxylase (CYP 17) mRNA. The stimulatory effects of ACTH on DST mRNA levels were blunted by both TGF-beta and TNF-alpha; the inhibitory effects of TNF-alpha on DST mRNA were more striking than were those on CYP 17 mRNA. These data suggest that DS production can be altered by several agents acting on the DST gene.     

Expression of thyroid hormone receptors alpha and beta-1 messenger RNAs in human endothelial cells. The T3 hormone stimulates the synthesis of the messenger RNA of the intercellular adhesion molecule-1

A study of the effect of the L-3,5,3'-triiodothyronine hormone on the expression of the mRNA of the adhesion molecule ICAM-1 led to the observation that the mRNA level is slightly up-regulated in human umbilical-cord endothelial cells. To analyze this induction at a molecular level, the search of T3 hormone receptors was undertaken. In this paper, we show that ECV 304 endothelial cells express the mRNAs encoding two thyroid hormone receptor isoforms alpha(alpha1 and alpha2) and one beta(beta1). This is, to our knowledge, a first important step towards the demonstration of the involvement of these receptors in the induction of the expression of ICAM-1 by the T3 hormone.     

Platelet ionized magnesium, cyclic AMP, and cyclic GMP levels in migraine and tension-type headache

Decreased serum and intracellular levels of magnesium have been reported in patients with migraine. It has been suggested that magnesium may play an important role in the attacks and pathogenesis of headaches. We measured ionized magnesium, cyclic AMP (adenosine monophosphate), and cyclic GMP (guanosine monophosphate) in platelets of patients with migraine, in patients with tension-type headache, and in healthy controls. The platelet level of ionized magnesium from patients with tension-type headache was significantly lower than the levels from the other two groups. The platelet level of cyclic AMP from patients with migraine was higher than those from the other groups. We found no significant differences in the platelet cyclic GMP levels among the three groups. It is suggested that reduced platelet ionized magnesium in patients with tension-type headache is related to abnormal platelet function, and that increased platelet cyclic AMP in patients with migraine is related to alteration of neurotransmitters in the platelet.     

Distribution of thyrotrophin-releasing hormone receptor messenger RNA in rat pituitary and brain

X-ray diffraction methods have been used to determine the structure of the 8.3 kDa hydrophobic protein from soybean and to refine the atomic co-ordinates to a crystallographic R-factor of 18.7% at 1.8 A resolution. The molecule is a four-helix bundle, which together with the connecting loops and a twisted beta-strand form a spiral. The surface contains 70% apolar atoms, and the crystal packing is dominated by hydrophobic interactions, producing a two-dimensional sheet of protein molecules. Most of the 59 water molecules located are involved in hydrophilic contacts and their structural organization does not seem to be affected by the high hydrophobicity of the molecule. From the protein fold it appears that three of the four disulphide bridges are important for keeping the amino and carboxyl-terminal segments in place in the native form, while the central part of the molecule is stabilized by many hydrophobic interactions. Although the protein function is not known, a number of possibilities can be excluded on experimental grounds and by comparison with other members of the family.     

Luteinizing hormone releasing hormone (LHRH) neurons maintained in nasal explants decrease LHRH messenger ribonucleic acid levels after activation of GABA(A) receptors

Inhibition of the LHRH system appears to play an important role in preventing precocious activation of the hypothalamic-pituitary-gonadal axis. Evidence points to gamma-aminobutyric acid (GABA) as the major negative regulator of postnatal LHRH neuronal activity. Changes in LHRH messenger RNA (mRNA) levels after alterations of GABAergic activity have been reported in vivo. However, the extent to which GABA acts directly on LHRH neurons to effect LHRH mRNA levels has been difficult to ascertain. The present work evaluates the effect of GABAergic activity, via GABA(A) receptors, on LHRH neuropeptide gene expression in LHRH neurons maintained in olfactory explants generated from E11.5 mouse embryos. These explants maintain large numbers of primary LHRH neurons that migrate from bilateral olfactory pits in a directed manner. Using in situ hybridization histochemistry and single cell analysis, we report dramatic alterations in LHRH mRNA levels. Inhibition of spontaneous synaptic activity by GABA(A) antagonists, bicuculline (10(-5) M) or picrotoxin (10(-4) M), or of electrical activity by tetrodotoxin (TTX, 10(-6) M) significantly increased LHRH mRNA levels. In contrast, LHRH mRNA levels decreased in explants cultured with the GABA(A) receptor agonist, muscimol (10(-4) M), or KCl (50 mM). The observed responses suggest that LHRH neurons possess functional pathways linking GABA(A) receptors to repression of neuropeptide gene expression and indicate that gene expression in embryonic LHRH neurons, outside the CNS, is highly responsive to alterations in neuronal activity.     

Immunochemical visualization and quantitation of cyclic AMP in single cells

Adenosine 3':5'-cyclic monophosphate (cAMP) is a key second messenger in signaling pathways governing many cellular processes. To define the subcellular localization and relative abundance of cAMP, we developed a novel immunochemical approach based on acrolein fixation to visualize cAMP within cells. We describe here the fixation and immobilization of cAMP within cells and the production of specific, high titer polyclonal antibodies that recognize cAMP. Relative levels of cAMP immunofluorescence were quantitated in glial cells (oligodendrocytes, astrocytes, Schwann cells, and glioma cells) that were either untreated or treated with activators of endogenous adenylyl cyclase to raise cAMP levels. In treated cells, cAMP immunofluorescence is strongly localized in the perinuclear cytoplasm.