Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Specific binding of radiolabelled FSH and LH to rat ovaries was demonstrated at the age of 7 days. However, when the biological response to LH and FSH was monitored by cAMP production in vitro, the FSH response appeared earlier than that of LH, on days 4 and 7, respectively. Cholera toxin stimulated cAMP production even in fetal ovaries, suggesting the presence of functional post-receptor machinery of cAMP production. Hence, the appearance of the functional gonadotropin receptor probably plays a key role in the onset of postnatal ovarian steroidogenesis. To test the effect of gonadotropin suppression during postnatal ovarian development, a potent GnRH antagonist was administered to neonatal animals between days 1-6 or 1-9 of life. The ovarian responsiveness to FSH developed even in the absence of normal gonadotropin levels, but that to LH was suppressed after the longer antagonist treatment. The temporal relationship between the onset of LHR gene expression, i.e. transcription, and translation to functional receptor protein was thereafter investigated using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The measurements revealed the existence only of truncated versions of LHR mRNA in the fetal ovary from day 17 of gestation up to day 7 of postnatal life. With the onset of the receptor function around day 7, also larger mRNA transcripts, corresponding to the full-length receptor protein appeared. Our findings suggest that the LHR gene may be constitutively expressed in the ovary and a change in the alternative splicing pattern may cause the onset of translation of a functional receptor protein.
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PMID:Ontogeny of gonadotropin action in the rat ovary. 134 71

A sensitive assay of multiple mRNAs by reverse transcriptase-polymerase chain reaction was adopted to study the hormonally regulated expression of steroidogenic enzymes in primary rat granulosa cells in culture. As little as 15-60 ng total RNA prepared from cultured cells were reverse transcribed in the presence of pd(T)6, and polymerase chain reaction was conducted in the presence of specific oligonucleotide pairs designed to identify cDNAs of steroidogenic enzymes. In combination with Northern blot analysis of cholesterol side-chain cleavage cytochrome P450 (P450scc) message, it is shown that a novel protein kinase inhibitor, tyrphostin AG18, arrests the FSH-induced accumulation of P450scc mRNA. This inhibition is dose dependent (IC50, 15 microM) and reversible. The addition of 80 microM AG18 to cells containing high levels of P450scc mRNA caused a rapid decline of the cytochrome message (t 1/2, 5 h), similar to the effect of 30 micrograms/ml alpha-amanitin. However, concomitant addition of the two drugs did not accelerate the mRNA degradation process, suggesting that AG18 does not affect message stabilization. Tyrphostin AG18 did not affect mRNA species that are not FSH inducible, such as the ribosomal protein L19, or the constitutively expressed low levels of steroid 5 alpha-reductase mRNA. Moreover, even the extremely high levels of P450scc mRNA in granulosa-lutein cells, being cAMP independent and terminally differentiated a few hours after LH surge, were not affected by the addition of AG18 in culture. In contrast, two additional key and FSH-inducible steroidogenic enzymes, i.e. aromatase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase-I, were inhibited by AG18 at their mRNA levels. These results suggest that an as yet undetermined tyrosine kinase pathway is involved in the cAMP-dependent signal transduction pathway of FSH action, so that the presence of AG18 does not allow FSH induction of gene expression to occur.
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PMID:Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids. 751 96

Hormonal regulation of the expression of mRNA transcripts for cAMP response element-binding protein (CREB) and cAMP response element modulator (CREM) during spermatogenesis was studied in the adult rat testis. Northern analysis of CREB and CREM identified two mRNA transcripts for CREM (2.4 and 1.6 kb) and one transcript for CREB (2.0 kb). Analysis of mRNAs from isolated testicular cells by reverse transcriptase polymerase chain reaction (RT/PCR) showed that CREM mRNAs were expressed by the germ cells but not the Sertoli or interstitial cells, whereas CREB mRNA was located in germ cells, Sertoli cells, and interstitial cells. RNA was isolated and analyzed from the testes of 1) rats treated for 24 h with FSH, 2) rats in which androgen withdrawal had been induced by ethane dimethane sulphonate (EDS) treatment 6 days earlier (EDS-treated), 3) EDS-treated rats supplemented with testosterone (EDS + T), or 4) intratesticular administration or dibutyryl cAMP (dbcAMP) in the preceding 24 h. CREM mRNA transcript expression was found to be decreased after all of these treatments in samples from intact testis and from isolated cells. Expression of the CREB transcript was also decreased by EDS-induced androgen withdrawal, but not by FSH or EDS + T. In situ hybridization of paraffin-embedded testis sections probed with digoxigenin-labeled riboprobes confirmed the localization of CREB and CREM mRNA to the same cell types as found with RT/PCR. No stage-dependent expression of CREM mRNA transcripts could be observed. Hybridization of the CREB probe was highest around the base of stage VII-VIII tubules, and this was shown to be androgen-dependent. The data presented suggest that regulation of the expression of CRE-binding protein mRNAs in Sertoli and germ cells during spermatogenesis is dependent on both androgen and FSH. However, the effects of androgen or FSH on the regulation of CRE-binding protein mRNAs are different.
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PMID:Differential regulation of cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein and cAMP response element modulator messenger ribonucleic acid transcripts by follicle-stimulating hormone and androgen in the adult rat testis. 751 86

In the present work we explored cellular sites of interleukin-6 (IL-6) biosynthesis in human follicular aspirates from patients undergoing in vitro fertilization therapy and the effects of this cytokine on oocyte fertilization and granulosa cell (GC) steroidogenesis. Biological IL-6 activity from 20-40 IU/mL was present in follicular fluids from 22 patients; it was also detected in 10 of 22 supernatants of cultured oocyte-cumulus complexes and in cumulus cell and GC cultures. Biological IL-6 activity in oocyte-cumulus complex cultures was not related to fertilization rates. Total ribonucleic acid was isolated from follicular aspirates and GC-enriched preparations. After reverse transcriptase and polymerase chain reaction cycles using oligonucleotide primers corresponding to known cDNA sequences for IL-6, a 126-basepair band characterized the amplification product of IL-6 transcripts on gel electrophoresis. To localize IL-6 messenger ribonucleic acid, in situ hybridization analysis was performed using a [35S]IL-6 riboprobe. The distribution of transcripts was more dense (15% vs. 3% stained cells) in GC-enriched preparations, which contained more than 95% GCs, than in original follicular preparations, which contained 20-40% viable GCs; it was not significantly modified by the presence of macrophage contaminants. The expression of IL-6 protein was assessed by positive immunohistological stainings. Biological IL-6 activity was higher, and in situ hybridization signals were more dense and more intense in 24-h GC cultures than in 72-h GC cultures, suggesting that IL-6 biosynthesis was transiently induced. Under experimental conditions of low IL-6 endogenous levels in cultures, adding recombinant human IL-6 from 10-200 IU/mL had no effect on progesterone production or aromatase activity in GC cultures free of macrophages, whereas in GC cultures including macrophage contaminants, stimulatory effects on basal and hCG-stimulated progesterone production and on basal and FSH-stimulated aromatase activities were observed. The present study provides strong support for the view that IL-6 is produced by GCs in the preovulatory follicle at the time of ovulation. In addition, we showed that IL-6 might be an intraovarian regulatory factor concerned with steroidogenesis.
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PMID:Interleukin-6 biosynthesis in human preovulatory follicles: some of its potential roles at ovulation. 751 93

The two gonadotropins, LH and FSH, are thought to be synthesized and secreted solely by the anterior pituitary. We present here evidence for expression of the LH beta and common alpha-subunit (C alpha) genes in the rat testis. The LH beta and C alpha-subunit messenger RNAs (mRNAs) were detected by reverse transcriptase-polymerase chain reaction in the rat testis and pituitary with primer pairs producing 247- and 199-base pair complementary DNA (cDNA) fragments, corresponding to nucleotides 154-400 of LH beta and nucleotides 250-448 of C alpha cDNA, respectively. The specificity of the cDNA species generated was verified by Southern hybridization using nested [32P]cDNA or oligonucleotide probes, and identity with the published rat LH beta and C alpha-subunit gene structures was determined by sequencing. The mRNA bands with specific hybridization to complementary RNA (cRNA) probes corresponding to nucleotides 154-368 of the rat LH beta cDNA and nucleotides 250-448 of the rat C alpha cDNA were found in the rat pituitary and testis by Northern hybridization. The major C alpha mRNA had a size of 0.8 kilobases (kb) in the pituitary and testis. The major LH beta transcripts were 0.8 and 2.7 kb in the pituitary and testis, respectively. To further characterize the larger testicular LH beta-subunit transcript, rapid amplification of the 3'-end of cDNA (3'-RACE) was performed using an oligo(deoxythymidine-17) adapter and a specific 5'-primer. Southern hybridization of the 3'-RACE product of rat testicular RNA with a LH beta [32P]cDNA probe had the same size as the 3'-RACE product of pituitary RNA. The pituitary and testicular RNAs were then cut into two segments using oligonucleotide-directed ribonuclease H digestion and subjected to Northern hybridization using a cRNA probe specific to the 5'-end segment. The digested 5'-end segments of the pituitary and testicular mRNAs were 0.4 and 2.3 kb, respectively, indicating that the testicular LH beta mRNA has a 1.9-kb 5'-extension, compared to the cognate pituitary mRNA. This was further verified by Northern hybridization using a cRNA probe corresponding to nucleotides -790 to -10 upstream of the pituitary initiation site of LH beta gene transcription. Specific hybridization of a 2.7-kb mRNA transcript was found in the rat testis, but none in the pituitary. Hence, the 3'-end polyadenalytion site of the LH beta mRNA is the same in rat pituitary and testis, and the different transcript sizes are due to a difference at the 5'-end.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Novel expression of luteinizing hormone subunit genes in the rat testis. 754 May 43

Ovulation rate, serum hormone concentrations, follicular fluid (FFL) concentrations of steroids and IGF, IGF binding protein (IGFBP) activity in FFL, and follicular IGF-I and -II mRNA were compared during the follicular phase among five genotypes of ewes: Finn (F), Composite III (C), 1/2 Booroola Merino (B) x 1/2 F (B x F), 1/2 F x 1/2 C (F x C), 1/2 B x 1/2 C (B x C). Composite III ewes were a Columbia x Suffolk x Hampshire crossbred. Ovulation rates for F (n = 7), C (n = 5), B x F (n = 6), F x C (n = 3), and B x C (n = 8) ewes were 3.1, 1.6, 3.8, 2.9, and 2.9 (Pooled SEM = .5), respectively. Concentrations of IGF-I in FFL were 53% greater (P < .05) in large (> or = 4.1 mm) than in small (< 4.1 mm) follicles but did not differ (P > .10) among genotypes. In contrast, FFL IGF-II concentrations were greater (P < .05) in B x C and B x F ewes than in C or F x C ewes but did not differ between small and large follicles. Ligand blotting revealed that IGFBP activity of three species (34, 27 to 29, and 24 kDa) were lower (P < .05) in FFL of large than in FFL of small follicles but did not differ (P < .10) among genotypes. Follicular wall IGF-I mRNA and IGF-II mRNA was detected in 5 and 32% of the samples from preovulatory follicles, respectively, using reverse transcriptase-PCR and ethidiumbromide staining. Ovarian IGF-I mRNA levels, assessed by Northern analysis, in B x F and B x C ewes were greater (P < .05) than those in C ewes; ovarian IGF-I mRNA levels in F and F x C ewes were intermediate and did not differ (P > .10) from those in C ewes. Small follicles from B x C and B x F ewes had severalfold greater (P < .05) estradiol concentrations than those from F or C ewes, whereas large follicles from B x F ewes had twice (P < .05) the estradiol concentrations of follicles from F or C ewes. Progesterone in FFL did not differ among genotypes. Serum LH, FSH, inhibin, IGF-I, and progesterone did not differ (P > .10) among genotypes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Serum hormones, follicular fluid steroids, insulin-like growth factors and their binding proteins, and ovarian IGF mRNA in sheep with different ovulation rates. 754 85

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist. 758 48

Specific factors involved in the pathogenesis of tumors that stimulate clonal human pituitary adenoma cell proliferation remain unknown. An important question regarding the pathogenesis of human pituitary tumors is whether they synthesize autocrine regulatory factors that regulate both hormone biosynthesis and neoplastic growth. Activin and inhibin are both comprised of inhibin subunits and have diverse regulatory roles as growth and differentiation factors in normal and neoplastic tissue. Activin stimulates FSH beta messenger ribonucleic acid (mRNA) biosynthesis and FSH secretion, and these effects are down-regulated in normal gonadotrophs by the endogenous glycoprotein follistatin. In addition to its effects on gonadotrophs, activin modulates hormone secretion by somatotroph and corticotroph cell lines. It is not known whether human neoplastic pituitary tissue synthesizes inhibin subunits or follistatin or whether their expression is cell type specific. We investigated whether alpha-, beta A-, and beta B-inhibin subunit and follistatin mRNAs could be detected in 27 human pituitary adenomas [clinically nonfunctioning (n = 11), somatotroph (n = 5), corticotroph (n = 5), and lactotroph (n = 6)] using reverse transcriptase-polymerase chain reaction techniques. Twenty-six of the tumors contained mRNAs encoding one or more inhibin subunits. beta B-Inhibin mRNA was the most prevalent (81% of tumors), followed by beta A-inhibin (59% of tumors) and alpha-inhibin (52% of tumors). Endogenous alpha-, beta A-, and beta B-inhibin subunit mRNA synthesis was also examined in normal human pituitary and testicular complementary DNA libraries, and all subunit mRNAs were detected. In contrast to the widespread expression of inhibin subunits in pituitary tumors, follistatin mRNA was detected in a subset of nonfunctioning tumors (54%) as well as in control normal human pituitary and testicular complementary DNA libraries. Tumor-specific follistatin biosynthesis was not observed in other pituitary tumor subtypes. These data are the first to demonstrate that 1) endogenous inhibin subunits are synthesized in human pituitary adenomas of all known secretory phenotypes as well as normal pituitary tissue; and 2) follistatin gene expression in pituitary adenomas is specific to clinically nonfunctioning or gonadotropin subunit-producing tumors. The characterization of inhibin subunit and follistatin biosynthesis by human pituitary tumors will be important in investigating their potential roles in regulating both tumor phenotype and cell proliferation.
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PMID:Human pituitary adenomas express endogenous inhibin subunit and follistatin messenger ribonucleic acids. 782 3

Gonadotropin subunit gene expression is regulated by gonadal, hypothalamic, and locally derived hormones. In particular, activin rapidly (within hours) acts at the gonadotrope to selectively increase the expression of FSH beta messenger RNA (mRNA). A family of activin receptors (ActRI, ActRII, and ActRIIB) has been identified, which is expressed in the pituitary as well as numerous other tissues in which activin is thought to act. As alterations in activin sensitivity could modulate activin action and, thereby, FSH beta mRNA, the purpose of this study was to determine whether ovariectomy (OVX), which results in rapid (< 2 h) increases in FSH beta, is associated with changes in ActRII gene expression. Adult female rats were ovariectomized, and some animals also received a GnRH antagonist from the time of OVX. Animals were killed between 2 h and 7 days later, and ActRII mRNA levels were measured by a quantitative reverse transcriptase-polymerase chain reaction assay. Although levels were unchanged at 2 h, ActRII mRNA levels increased 5- to 6-fold by 8 h and remained increased through 7 days after OVX. These changes were not altered by GnRH blockade. To determine whether ActRII was regulated by gonadal steroids, female rats were ovariectomized, and some animals were replaced with estradiol and progesterone (Silastic implants) for 2 days. Again, ActRII mRNA levels increased significantly after OVX, and gonadal steroid replacement had no effect. Finally, to investigate whether pituitary ActRII mRNAs are regulated by circulating inhibin, intact female rats were treated with an inhibin antiserum or nonimmune sheep serum as a control and killed 12 h later. Despite its action to increase FSH beta mRNA and FSH secretion, selective removal of inhibin did not alter ActRII mRNA levels. Based on these results we conclude the following. 1) Pituitary ActRII mRNAs increase rapidly after OVX, although increases in FSH beta precede changes in ActRII. These data suggest that changes in activin sensitivity may be a factor involved in the regulation of FSH beta. 2) An ovarian factor, other than inhibin, estradiol, and progesterone, acting independently of GnRH maintains an inhibitory tone on pituitary ActRII gene expression in adult rats.
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PMID:Ovarian regulation of pituitary inhibin subunit and activin receptor type II gene expression: evidence for a nonsteroidal inhibitory substance. 807 Mar 90

Receptors for gonadotropin releasing hormone (GnRH), located in the cell membranes of adeno-hypophysial gonadotropes, mediate the action of GnRH to stimulate the secretion of the gonadotropic hormones (LH and FSH). In the present studies, we have isolated a GnRH receptor cDNA from bovine pituitary, determined its primary structure, and studied the regulation of its gene expression. The cDNA is composed of 1326 nucleotides and encodes a protein containing 328 amino acids. The GnRH receptor of cattle, like that in humans and mice, is a seven transmembrane receptor and has structural characteristics homologous with the family of G protein-coupled receptors. It exhibits 91% identity at the amino acid level with the human and 86% identity with mouse and rat receptors. Northern blot analysis of the RNA from bovine pituitary, probed with 32P-labeled bovine GnRH receptor cDNA, revealed the presence of four different transcripts (5.0, 3.5, 2.5 and 1.5 kb) in the pituitary of which the 5.0 kb form was most abundant. Using the reverse transcriptase/PCR technique, we detected expression of GnRH receptor mRNA in the pituitary but not in any other extrapituitary tissues such as the hypothalamus, hippocampus, testis, corpus luteum, ovary (containing follicles), myoendometrium, adrenal, kidney, liver and spleen. Higher levels of GnRH receptor mRNA were found in the pituitaries of steers than in cohort bulls, suggesting regulation of GnRH receptor gene expression by testicular steroids.
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PMID:Molecular cloning, sequencing, and characterizing the bovine receptor for gonadotropin releasing hormone (GnRH). 830 35


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