Potent antagonism between estrogens and androgens on GCDFP-15 expression and cell growth in the ZR-75-1 human breast cancer cells.
Explore the source record for details and available documents.
Apolipoproteins↗
Biomedical subjects
J Simard
Publications and source records attributed to J Simard.
Purification, cloning, complementary DNA structure, and predicted amino acid sequence of human estradiol 17 beta-dehydrogenase.
Explore the source record for details and available documents.
17-Hydroxysteroid Dehydrogenases↗
Interactions between estrogens, androgens, progestins, and glucocorticoids in ZR-75-1 human breast cancer cells.
The human breast cancer cell line ZR-75-1 possesses androgen, estrogen, progesterone, and glucocorticoid receptors, thus offering a good model to study the specific role of each class of steroids in the control of breast cancer growth. Although the stimulatory action of classical estrogens (E2 and estrone) is well known, we have found a potent mitogenic effect of the adrenal estrogen androst-5-ene-3 beta,17 beta-diol (delta 5-diol) at concentrations within the range of those found in the serum of adult women, thus suggesting that delta 5-diol might be the most important estrogen in women. Androgens, on the other hand, exert a potent inhibitory effect on basal ZR-75-1 cell growth and completely reverse the stimulatory effect of estrogens on the same parameter. The antiproliferative effect of androgens was completely prevented by the antiandrogen OH-FLU, thus suggesting an action mediated by the androgen receptor. Part of the effect of androgens can be explained by the marked inhibition of estrogen receptor binding and mRNA levels by androgens. The antiproliferative effect of androgens is additive to that exerted by antiestrogens. Progestins, on the other hand, exert a specific antiproliferative effect in the presence of estrogens, the effect of progestins being antagonized by the stimulatory action of insulin on cell growth. Medroxyprogesterone acetate (MPA), a compound frequently used in the treatment of breast cancer in women, exerts its main inhibitory action through an androgen receptor-mediated action, whereas its glucocorticoid-like activity could play an additional role at high concentrations. All four classes of steroids are present, to various extents, as lipophilic esters of long-chain fatty acids. It is of interest to mention that all steroids that inhibit ZR-75-1 breast cancer cell growth (androgens, progestins, and glucocorticoids) stimulate the secretion and mRNA levels of gross cystic disease fluid protein-15 (GCDFP-15), whereas estrogens have the opposite effects, thus suggesting that GCDFP-15 could well be a good marker for monitoring the response to androgens, progestins, and antiestrogens during the course of breast cancer therapy.
Androgens↗
Multihormonal control of pre-pro-somatostatin mRNA levels in the periventricular nucleus of the male and female rat hypothalamus.
The influence of sex steroids as well as the possible involvement of dopaminergic pathways in the modulation of pre-pro-somatostatin (SS) mRNA levels was investigated by quantitative in situ hybridization in the hypothalamic periventricular nucleus (PeN) in adult male and female rats. In situ hybridization was performed using a [35S]-labeled cDNA probe encoding pre-proSS mRNA. Gonadectomy performed 14 days earlier decreased the mean number of silver grains/neuron corresponding to the relative pre-proSS mRNA levels by 22% in male and by 18-28% in female rats. A 14-day treatment with the nonaromatizable androgen dihydrotestosterone (DHT) increased the mean number of silver grains/neuron by 34-40% in gonadectomized animals of both sexes. Moreover, administration of 17 beta-estradiol (E2, 0.25 microgram twice daily) increased pre-proSS mRNA levels by 40% in ovariectomized (OVX) animals. Such treatment with E2 or DHT changed the frequency distribution profile of the hybridization signal intensity, thus increasing the percentage of highly labeled neurons (greater than or equal to 61 grains/neuron) by 10 to 12-fold. A 14-day treatment with the D2 dopamine receptor agonist bromocriptine (BRO) increased pre-proSS mRNA levels by 15 and 28% in intact female and OVX animals, respectively, while the dopaminergic antagonist haloperidol (HAL) decreased the value of this parameter by 20 and 30%. Furthermore, BRO increased pre-proSS mRNA levels by 10 and 20% in intact and castrated male rats, respectively, whereas HAL decreased pre-proSS mRNA levels by 25 and 14% in the same groups of animals. Administration of E2 in combination with HAL in OVX animals increased pre-proSS mRNA levels by 70% compared to those measured in OVX animals treated with HAL alone. In HAL-treated castrated male rats, administration of DHT increased the relative pre-proSS mRNA levels by 35% compared to those measured in castrated animals treated with HAL alone. The present data clearly demonstrate that androgens and estrogens as well as dopamine-mediated mechanisms could play a regulatory role in pre-proSS mRNA levels in somatostatinergic neurons in the hypothalamic PeN in both male and female rats.
Animals↗
Regulation of progesterone-binding breast cyst protein GCDFP-24 secretion by estrogens and androgens in human breast cancer cells: a new marker of steroid action in breast cancer.
We have previously demonstrated that androgens are potent inhibitors of breast cancer cell proliferation under both basal and estrogen-induced incubation conditions, while they suppress expression of the estrogen and progesterone receptors. To better understand the mechanisms responsible for the antagonism between androgens and estrogens in breast cancer and to obtain a new tumor marker for the actions of these two steroids, we have investigated the effects of androgens and estrogens on expression of the major protein found in human breast gross cystic disease fluid, namely GCDFP-24. This study was performed in ZR-75-1 and MCF-7 human breast cancer cells. After a 9-day incubation period, physiological concentrations of 17 beta-estradiol stimulated proliferation of ZR-75-1 and MCF-7 cells by 2- to 3.5-fold while simultaneously exerting a marked 70-90% inhibition of GCDFP-24 secretion. The estrogenic effects on GCDFP-24 secretion and cell proliferation were both competitively blocked by simultaneous incubation with the new steroidal pure antiestrogen EM-139. On the other hand, a maximal concentration (10 nM) of the nonaromatizable androgen dihydrotestosterone decreased by 50% the proliferation of ZR-75-1 cells; the half-maximal inhibitory effect was exerted at 0.01 nM. The androgen exerted a 3- to 4-fold stimulatory effect on GCDFP-24 secretion at an EC50 value of 0.01 nM. The effect of dihydrotestosterone on these parameters was competitively blocked by simultaneous incubation with the pure antiandrogen OH-flutamide. The present data show that the effects of estrogens and androgens in ZR-75-1 cells on GCDFP-24 secretion and cell growth are opposite. Similarly, in MCF-7 cells, estrogens stimulate cell growth, while GCDFP-24 secretion is inhibited. The present data also suggest that GCDFP-24 could well be a good biochemical marker for monitoring the response to androgenic and antiestrogenic compounds in the therapy of advanced breast cancer.
Androgens↗
Localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in rat gonads and adrenal glands by immunocytochemistry and in situ hybridization.
The enzyme complex delta 5-3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD) is involved in the biosynthesis of all classes of steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex steroids. To obtain information on the precise localization of 3 beta HSD in rat gonads and adrenal glands, two complementary cytochemical techniques were used; immunocytochemical localization was achieved with antibodies developed against purified human placental 3 beta HSD, while 3 beta HSD mRNA localization was achieved by in situ hybridization performed with a recently cloned rat 3 beta HSD cDNA. In the testis, specific immunostaining was restricted to the cytoplasm of the interstitial cells, while by in situ hybridization, specific silver grains were also seen over the interstitial cells. In the ovary, immunostaining was found in the cytoplasm of cells of the corpus luteum and theca interna, while the granulosa cells of the follicles showed no positive reaction. By in situ hybridization, a specific hybridization signal was observed over granulosa cells of the corpus luteum, which are mainly responsible for progesterone secretion, and to a lesser extent over theca interna cells, known for their role in secreting C19 androgens. In the adrenals, the three zones of the cortex were equally immunolabeled, whereas no staining could be detected in the medulla. Similarly, by in situ hybridization, silver grains were located over the zona glomerulosa, fasciculata, and reticularis, while no specific autoradiographic reaction could be observed on the chromaffin cells of the medulla. The present study provides new information about the precise cellular localization of 3 beta HSD in the adrenal glands and gonads in the rat, thus providing useful information about the site of action of 3 beta HSD, especially in the gonads. Moreover, the approaches used for localization studies, especially quantitative in situ hybridization, should provide a useful tool for assessing the role of hormones on 3 beta HSD expression in the different compartments of the gonads and adrenal glands.
3-Hydroxysteroid Dehydrogenases↗
Localization of the endogenous benzodiazepine ligand octadecaneuropeptide in the rat testis.
Diazepam binding inhibitor (DBI) is the precursor of a family of peptides, including an octadecaneuropeptide (ODN), which share with DBI the ability to specifically displace benzodiazepines (BZD) from their receptors. BZD receptors have been found not only in the brain, but also in a variety of peripheral tissues, including the testis. To clarify the role of ODN in the testis, we have investigated the localization of ODN in the rat testis using two different cytochemical approaches: immunocytochemistry and in situ hybridization. Immunocytochemical localization was achieved using rabbit antibodies developed against rat ODN. At the light microscopic level, immunostaining was exclusively located in interstitial cells; the seminiferous tubules were totally unlabeled. In the developing rat, immunostaining in the interstitial cells was first detected in an 18-day-old fetus. The immunolabeling increased as a function of age to reach a plateau at 40 days of age. The ultrastructural localization of ODN was achieved by immunogold staining. The gold particles were exclusively found in the cytoplasm of Leydig cells. HPLC analysis performed in adult rat testicular extracts revealed that immunoreactive material was detected in a peak eluted later than synthetic rat ODN. The cellular distribution of ODN was also studied by in situ hybridization using a 35S-labeled single stranded RNA probe complementary to DBI mRNA. Hybridization signal obtained at the light microscopic level was only detected over interstitial cells. The data obtained clearly indicate that in the rat, Leydig cells synthesize ODN and accumulate ODN-like immunoreactivity. Since Leydig cells have been shown to contain BZD receptors, it might be hypothesized that ODN and/or other DBI-related peptides can play a role in Leydig cell regulation.
Animals↗
Regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase expression and activity in the hypophysectomized rat ovary: interactions between the stimulatory effect of human chorionic gonadotropin and the luteolytic effect of prolactin.
The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes an obligatory step in the conversion of pregnenolone and other 5-ene-3 beta-hydroxysteroids into progesterone as well as precursors of all androgens and estrogens in the ovary. Since 3 beta-HSD is likely to be an important target for regulation by pituitary hormones, we have studied the effect of chronic treatment with LH (hCG), FSH, and PRL on ovarian 3 beta-HSD expression and activity in hypophysectomized adult female rats. Human CG (hCG) [10 IU, twice a day (bid)], ovine FSH (0.5 microgram, bid), and ovine PRL (1 mg, bid) were administered, singly or in combination, for a period of 10 days starting 15 days after hypophysectomy. In hypophysectomized rats, PRL exerted a potent inhibitory effect on all the parameters studied. In fact, PRL caused a 81% decrease in ovarian 3 beta-HSD mRNA content accompanied by a similar decrease in 3 beta-HSD activity and protein levels. In addition, ovarian weight decreased by 40% whereas serum progesterone fell dramatically from 1.92 nmol/liter to undetectable levels after treatment with PRL. Whereas hCG alone had only slight stimulatory effects on 3 beta-HSD mRNA, protein content and activity levels, treatment with the gonadotropin partially or completely reversed the potent inhibitory effects of oPRL on all the parameters measured. FSH, on the other hand, had no significant effect on 3 beta-HSD expression and activity. In situ hybridization experiments using the 35S-labeled rat ovary 3 beta-HSD cDNA probe show that the inhibitory effect of PRL is exerted primarily on luteal cell 3 beta-HSD expression and activity. On the other hand, it can be seen that hCG stimulates 3 beta-HSD mRNA accumulation in interstitial cells. The present data show that hCG and PRL exert potent and opposite cell-specific effects on ovarian 3 beta-HSD expression, activity, and content in the rat ovary. Moreover, the present study could suggest that female infertility associated with hyperprolactinemia in women could well be related, at least in part, to the potent inhibitory effect of PRL on ovarian 3 beta-HSD expression and activity.
3-Hydroxysteroid Dehydrogenases↗
Structure and sexual dimorphic expression of a liver-specific rat 3 beta-hydroxysteroid dehydrogenase/isomerase.
The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes the obligatory oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into delta 4-3-ketosteroids which lead to the formation of all classes of steroid hormones. We report the molecular cloning of a third type of cDNA clone encoding rat 3 beta- HSD isolated from a rat liver lambda gt11 cDNA library. The nucleotide sequence of 1955 bp determined from overlapping cDNA clones predicts a protein of 372 amino acids which displays 80% similarity with that of rat type I and type II 3 beta-HSD proteins. RNA blot analysis reveals the presence of mRNA transcripts of 2.1 kb in male liver in contrast to the 1.7 kb mRNA species detected in adrenal and gonad poly(A)+ RNA. Ribonuclease protection assays using type I, type II and type III specific cRNA probes demonstrate the liver-specific expression of type III mRNA while the two others are expressed in adrenals and gonads. The type III mRNA species was below the detection limit in intact female liver while in hypophysectomized females, its accumulation was restored to 55% of the levels measured in intact or hypophysectomized male rats. The present data describe the presence of a third type of 3 beta-HSD mRNA species and its marked sexual dimorphic gene expression in the liver which apparently results from pituitary hormone-induced gene repression in female rat liver tissue.
Amino Acid Sequence↗
Structure of two in tandem human 17 beta-hydroxysteroid dehydrogenase genes.
Two human 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) genes (h17 beta-HSDI and h17 beta-HSDII) included in tandem within an approximately 13 kilobase pair fragment were isolated from a genomic lambda EMBL3 DNA library using cDNA encoding human 17 beta-HSD (hpE2DH216) as probe. We have determined the complete exon and intron sequences of the two genes as well as their 5' and 3'-flanking regions. Human 17 beta-HSDII contains six exons and five short introns for a total length of 3250 base pairs. The exon sequence of h17 beta-HSDII is identical to the previously reported hpE2DH216 cDNA while the overlapping nucleotide sequences of the corresponding exons and introns of h17 beta-HSDI and h17 beta-HSDII show 89% homology. In addition, we have used the hpE2DH216 cDNA to demonstrate the widespread expression of 17 beta-HSD mRNAs in steroidogenic and peripheral target tissues. These new findings provide the basis for a better understanding of the molecular mechanisms involved in 17 beta-HSD deficiency and peripheral sex steroid metabolism.
17-Hydroxysteroid Dehydrogenases↗
Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase.
We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.
3-Hydroxysteroid Dehydrogenases↗
Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17.
Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.
17-Hydroxysteroid Dehydrogenases↗
Antagonism between estrogens and androgens on GCDFP-15 gene expression in ZR-75-1 cells and correlation between GCDFP-15 and estrogen as well as progesterone receptor expression in human breast cancer.
The androgen dihydrotestosterone (DHT) caused a maximal 65% inhibition of proliferation of the human breast cancer cells ZR-75-1 after a 10-day incubation period. The same treatment, on the other hand, stimulated by 25-fold the secretion of the breast marker protein GCDFP-15 (gross cystic disease fluid protein-15). The stimulatory effect of DHT on GCDFP-15 mRNA accumulation was already significant (1.6-fold, P less than 0.01) after a 12 h exposure and reached a maximal 25-fold increase after a 12-day incubation period. On the other hand, a 2-day exposure to 1 nM 17 beta-estradiol (E2) alone decreased by 60% GCDFP-15 mRNA levels while it completely blocked the 2.5-fold stimulation of GCDFP-15 secretion induced by concomitant incubation with DHT. Furthermore, a 10-day incubation with E2 increased by 4-fold the proliferation of ZR-75-1 cells whereas such treatment decreased by about 85% both GCDFP-15 mRNA accumulation and the secretion of the glycoprotein. The presence of GCDFP-15 mRNA in human breast cancer samples was restricted to estrogen receptor positive tumors and was significantly correlated with progesterone receptor expression.
Apolipoproteins↗
Electron microscopic autoradiographic localization of prolactin mRNA in rat pituitary.
Recent immunoelectron microscopic studies have shown that immunoreactive prolactin (PRL) in rat pituitary can be detected not only in typical PRL cells, characterized by large secretory granules, but also in another type of cell, which contains small secretory granules. To determine whether or not these two cell types are involved in PRL biosynthesis, we developed a procedure to investigate PRL gene expression by using in situ hybridization at the ultrastructural level. Rat pituitary was fixed and vibratome sections were incubated with a PRL [35S]-cDNA probe and subsequently flat-embedded in Araldite. Semi-thin and ultra-thin sections were processed for autoradiography. The results indicate that only the two PRL cell types were labeled. When immunolabeling for PRL was applied to ultra-thin sections, only immunopositive cells were seen to contain silver grains. In these cells the silver grains were associated with the rough endoplasmic reticulum and nucleus. When a growth hormone (GH) [35S]-cDNA probe was used as a control, only GH-secreting cells were labeled. This study confirms that the two PRL cell types are involved in biosynthesis of PRL. Moreover, this simple in situ hybridization technique provides a new approach to accurately localize mRNA in complex tissue and to investigate the subcellular distribution of mRNA under differing experimental conditions.
Animals↗
Stimulation of androgen-dependent gene expression by the adrenal precursors dehydroepiandrosterone and androstenedione in the rat ventral prostate.
Androgens play a major role in the development, growth, and function of accessory sexual organs, especially the prostate. However, the testis is not the sole source of circulating androgens in man, since the adrenal gland secretes dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione (delta 4-dione) in large quantities. The aim of the present study was to investigate the effect of plasma concentrations of DHEA and delta 4-dione similar to those found in adult man on sensitive and specific markers of androgen action in the rat ventral prostate. In addition to ventral prostate weight, we have measured the steady state levels of the mRNAs encoding the C1 component of rat prostatic binding protein (PBP-C1) and spermine-binding protein (SBP) using 35S-labeled cDNA probes for in situ hybridization. One week after castration, ventral prostate weight fell 84%, while prostatic 5 alpha-dihydrotestosterone (DHT) and androgen-dependent mRNAs were undetectable. When administered via Silastic implants to castrated adult rats for 1 week, plasma concentrations of 1.37 +/- 0.06 ng/ml DHEA or 0.43 +/- 0.08 ng/ml delta 4-dione independently caused increases in ventral prostate weight to 33% and 65% of normal values, respectively. The same plasma levels of DHEA and delta 4-dione resulted in high intraprostatic levels of DHT to 1.19 +/- 0.34 and 3.66 +/- 0.89 ng/g tissue, respectively. Furthermore, DHEA caused an increase in the steady state levels of PBP-C1 and SBP mRNAs to 50% and 57% of the normal state, respectively, while delta 4-dione caused increases corresponding to 80% and 119% of control values, respectively. Castrated adult rats receiving testosterone at a concentration of 1.66 +/- 0.37 ng/ml plasma maintained normal ventral prostate weight and gene expression levels. The present results demonstrate that circulating levels of the adrenal steroids DHEA and delta 4-dione comparable to those found in man cause an important stimulation of androgen-dependent gene expression in the rat, probably after their conversion to DHT in the prostatic tissue itself.
Adrenal Glands↗
Down-regulation of estrogen receptors by androgens in the ZR-75-1 human breast cancer cell line.
Much clinical evidence indicates that androgens have beneficial effects in the treatment of breast cancer in women. Physiological concentrations of androgens strongly inhibit both basal and estrogen-induced cell proliferation in the human breast cancer cell line ZR-75-1 through their interaction with the androgen receptor. The present study shows that androgens strongly suppress estrogen receptor (ER) and progesterone receptor contents in this model, as measured by radioligand binding and anti-ER monoclonal antibodies. Similar inhibitory effects are observed on the levels of ER messenger RNA (mRNA) measured by ribonuclease protection assay. The androgenic effect is observed at subnanomolar concentrations of the nonaromatizable androgen 5 alpha-dihydrotestosterone, regardless of the presence of estrogens, and is competitively reversed by the antiandrogen hydroxyflutamide. Such data on ER expression provide an explanation for at least part of the antiestrogenic effects of androgens on breast cancer cell growth and moreover suggest that the specific inhibitory effects of androgen therapy could be additive to the standard treatment limited to blockade of estrogens by antiestrogens.
Androgens↗
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.
Androgens↗
Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats.
The fine modulation of gonadotropin gene expression and secretion is well recognized to be regulated by sex steroids through their direct action both at the anterior pituitary level and on the pulsatile pattern of GnRH secretion at the hypothalamic level. Since the influence of sex steroids on hypothalamic GnRH mRNA levels remains to be elucidated, quantitative in situ hybridization was used to study the effect of sex steroids on cellular levels of pro-GnRH mRNA in adult rats of both sexes. The effects of 14-day gonadectomy as well as administration of 17 beta-estradiol (E2, 0.25 micrograms) or dihydrotestosterone (DHT, 100 micrograms) twice a day during 14 days to gonadectomized animals were evaluated. In addition, the effect of progesterone (P, 2 mg, twice daily) alone or in the presence of E2 was also studied in ovariectomized animals. Hybridization was performed using a 35S-labeled cDNA probe encoding rat pro-GnRH and the corresponding mRNA levels were assessed by counting the number of silver grains overlying labeled neurons. In male rats, castration induced a highly significant 65% increase (compared to intact rats) in the mean number of grains per neuron. Administration of E2 or DHT to castrated animals completely prevented the post castration rise in pro-GnRH mRNA levels. In female animals, the effect of ovariectomy was less striking than in the male, a 25% increase (P less than 0.001) being observed. Treatment with E2 or DHT also completely prevented the increase in pro-GnRH mRNA levels induced by ovariectomy. Moreover, treatment with P in ovariectomized animals markedly potentiated the inhibitory effect of E2 on pro-GnRH mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗