Biomedical subjects

H F Zhao

Publications and source records attributed to H F Zhao.

35 records · Page 2Linked to original sources

PubMed1990

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↗

PubMed1990

Changes in 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase messenger ribonucleic acid, activity and protein levels during the estrous cycle in the bovine ovary.

A 1169 base pair fragment of bovine 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) cDNA was used to quantitate 3 beta-HSD messenger RNA (mRNA) levels in the bovine ovary during the estrous cycle. The content of 3 beta-HSD protein was measured by immunoblot analysis using an antiserum developed in rabbits against human 3 beta-HSD, whereas 3 beta-HSD activity was measured using [3H]pregnenolone, [3H] dehydroepiandrosterone, and [3H]androst-5-ene-3 beta,17 beta-diol as substrates. There was a parallel increase in 3 beta-HSD mRNA, protein content, and enzymatic activity levels from days 1-3 after estrus to maximal values at 50-100% above control on days 8-11 after estrus. Thereafter, all values decreased progressively until days 16-17 before a dramatic fall to 5% or less than maximal values on days 18-20 after estrus. Almost superimposable results of enzymatic activity were obtained with the three substrates, thus suggesting a unique 3 beta-HSD or parallel changes in the activity of multiple 3 beta-HSDs. The above-described changes observed during the luteal phase are almost exclusively due to variations in corpora lutea. In fact, 3 beta-HSD activity in ovarian follicles was approximately 10,000 lower than that measured in corpora lutea. The close correlation observed over a wide range of 3 beta-HSD mRNA, protein content, and activity levels suggests that changes of ovarian 3 beta-HSD activity are controlled at the level of 3 beta-HSD gene expression and/or 3 beta-HSD mRNA stability.

Animals↗

PubMed1990

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↗

PubMed1990

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↗

PubMed1989

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↗

PubMed1989

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↗

PubMed1989

Ontogeny of prolactin mRNA in the rat pituitary gland as evaluated by in situ hybridization.

We have studied the ontogeny of prolactin (PRL) messenger RNA (mRNA) in male and female rats. Quantitative in situ hybridization was performed on sections of fixed pituitaries using a 32S-labeled PRL cDNA probe. With this technique, hybridization signal was first detected on day 19 of gestation. The PRL mRNA levels were very low in foetuses and newborn animals. Higher PRL mRNA levels were found in 5-day-old animals. Thereafter, mRNA concentrations regularly increased to reach a plateau at 60 and 90 days of age in males and females, respectively. Sexual dimorphism was first observed in 20-day-old animals, the PRL mRNA levels being higher in the female than in the male. This difference in PRL mRNA became more marked after puberty such that in 90-day-old animals the amounts of PRL mRNA in females were 2.7 times those observed in males. Since sexual dimorphism in PRL mRNA levels occurs well before sexual dimorphism in PRL secretion, which takes place first during puberty, it is suggested that during sexual maturation PRL secretion is regulated translationally as well as transcriptionally.

Animals↗

PubMed1989

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↗

PubMed1989

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↗

PubMed1989

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↗

PubMed1989

Inhibitory effect of estrogens on GCDFP-15 mRNA levels and secretion in ZR-75-1 human breast cancer cells.

In order to better understand the mechanisms responsible for the antagonism between steroids in human breast cancer cells, we have studied the effect of 17 beta-estradiol (E2), dihydrotestosterone (DHT), and dexamethasone (DEX) alone or in combination on the expression of the breast gross cystic disease fluid protein-15 (GCDFP-15) in ZR-75-1 cells. Incubation with E2 markedly decreased basal GCDFP-15 mRNA levels accompanied by a parallel inhibition of the secretion of this tumor marker, the estrogenic effect being exerted at a half-maximal concentration of about 44 pM E2. The inhibitory effect of E2 on GCDFP-15 expression was competitively reversed by the antiestrogen LY156758. In addition, 1 nM E2 inhibited the marked stimulation induced by 1 nM DHT or 300 nM DEX on GCDFP-15 mRNA accumulation and on the secretion of the glycoprotein. However, at the concentration used, E2 reversed by only 65% the stimulation achieved by the combination of DHT and DEX on GCDFP-15 mRNA levels. It is of interest to mention that the effect of DHT, DEX, and E2 on GCDFP-15 expression is opposite to the respective effect of each steroid on ZR-75-1 cell proliferation. The present data on the regulation of GCDFP-15 mRNA demonstrate an estrogen-induced inhibition of mRNA levels under physiological conditions, thus offering a unique opportunity to study the mechanisms involved in the down-regulation of gene expression by estrogens and to achieve a better understanding of the antagonism between estrogens, androgens, glucocorticoids, and progestins in breast cancer cells. Furthermore, GCDFP-15 could well be a good marker for monitoring the response to androgens and antiestrogens during the course of breast cancer therapy.

Apolipoproteins↗

PubMed1989

Characterization of cDNAs for human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17: evidence of two mRNA species with distinct 5'-termini in human placenta.

Human placenta estradiol 17 beta-dehydrogenase (E2DH) cDNA clones were isolated from a lambda gt11 expression library by screening with 33 mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of E2DH and with polyclonal antibodies raised against the enzyme purified from human placenta. Using 32P-labeled fragments from the coding and 5'-untranslated regions, two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kilobases (kb) while a minor one is found at 2.2 kb. Primer extension analysis identifies the major mRNA as starting 9-10 nucleotides upstream from the in-frame ATG initiating codon while the longer mRNA has at least 814 noncoding nucleotides at its 5'-terminus. Sequence analysis of the longest cDNA clone (2092 base pairs) shows that this clone possesses identical coding and noncoding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5'-noncoding fragment hybridized only to the 2.2 kb band, thus providing additional evidence for the existence of two distinct mRNA species which differ only in their 5'-noncoding regions. Using hpE2DH36 cDNA as a probe for in situ hybridization, the human E2DH gene was localized to the q11-q12 region of chromosome 17. The cloned cDNAs encode E2DH, a 327-amino acid protein having a calculated molecular weight of 34,853. Since E2DH is the enzyme required for the formation of 17 beta-estradiol, the availability of the cDNA encoding the enzyme should permit a detailed investigation of the factors regulating the expression and activity of this crucial enzyme, in both normal and malignant tissues, especially breast cancer.

17-Hydroxysteroid Dehydrogenases↗

PubMed1987