Biomedical subjects

J A Grootegoed

Publications and source records attributed to J A Grootegoed.

At least 19 recordsLinked to original sources

PubMed1992

Hormone-induced dissociation of the androgen receptor-heat-shock protein complex: use of a new monoclonal antibody to distinguish transformed from nontransformed receptors.

The hormone-induced transformation process of the androgen receptor in the androgen-responsive human prostatic carcinoma cell line LNCaP was studied. Immunoprecipitation of the nontransformed cytosolic receptor (8S on sucrose gradients) with a specific monoclonal antibody (F39.4.1) resulted in coprecipitation of three heat-shock proteins (hsp90, hsp70, and hsp56). Upon incubation of the cells with the synthetic androgen R1881, the sedimentation value of the receptor complex decreased to an intermediate form of 6S, and an almost complete loss of coprecipitating heat-shock proteins was observed. After a 2-h incubation, the receptor was recovered in considerable part from the nuclear fraction (extraction with high salt; 4.6S form). By use of the bifunctional cross-linker dimethyl pimelimidate, dissociation of the 8S complex, but not of the 6S complex, was blocked. A newly developed monoclonal antibody (F52.24.4), directed against the C-terminal part of the DNA-binding domain of the androgen receptor, specifically recognized both the 4.6S and the 6S forms of the receptor but did not react with the nontransformed 8S form. It is concluded that the unoccupied androgen receptor is associated with several heat-shock proteins and that transformation of the receptor to the tight nuclear-binding form is a multistep process that involves the dissociation of heat-shock proteins from the receptor.

Amino Acid Sequence↗

PubMed1992

Synthetic peptides based upon a three-dimensional model for the receptor recognition site of follicle-stimulating hormone exhibit antagonistic or agonistic activity at low concentrations.

Follicle-stimulating hormone (follitropin, FSH) belongs to a group of closely related glycoprotein hormones that contain two noncovalently linked dissimilar subunits designated alpha and beta. By using synthetic peptides, several receptor interaction sites in these hormones have been identified; however, the peptides have a reduced potency (lowest effective concentration of 10(-4) to 10(-5) M) relative to the hormone itself (10(-8) to 10(-11) M). This suggests that the peptides represent only a portion of a larger recognition site in the intact hormone that comprises parts of both the beta and the alpha chains. To develop peptides that exhibit FSH-antagonistic activity at low concentrations, we have constructed a three-dimensional model for FSH, which is based on an alignment of both the beta and the alpha chains of glycoprotein hormones with thioredoxin, for which x-ray diffraction data are available. This model resulted in the prediction of a conformational receptor-binding site in FSH, in which (parts of) three earlier proposed binding regions on the FSH molecule [namely, the regions FSH alpha-(34-37), with the amino acid sequence SRAY; FSH beta-(40-43), with the amino acid sequence TRDL; and FSH beta-(87-94), the "determinant loop" with the amino acid sequence CDSDSTDC] are located within 10 A of one another. On the basis of this model, peptides have been synthesized in which two of these binding regions are linked by a synthetic amino acid whose length was derived from the model, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH2 and Ac-SRAY-NH-(CH2)4-CO-TRDL-NH2. Both peptides inhibited FSH-induced cAMP production in Sertoli cells at 1000-fold lower concentrations (10(-7) M) than the peptides Ac-TRDL-NH2, Ac-SRAY-NH2, or Ac-TDSDS-NH2. In another peptide, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH-(CH2)4-CO-TRDL-NH2, all three binding regions have been linked. This peptide appeared to be a strong agonist of FSH action, as measured by the ability to stimulate cAMP production, at concentrations as low as 10(-7) M. The observation that a synthetic peptide, in which (parts of) three earlier described receptor interaction sites are combined according to the three-dimensional model, can mimic the action of FSH, at 10(-7) M, shows that this model is useful to predict a conformational receptor-binding site in FSH and that combination of only a few amino acid residues from the alpha and beta chains of FSH in a small synthetic peptide is sufficient to transduce a signal upon binding to the receptor.

Amino Acid Sequence↗

PubMed1992

Anti-androgens and the mutated androgen receptor of LNCaP cells: differential effects on binding affinity, heat-shock protein interaction, and transcription activation.

Previous studies from this laboratory have described that LNCaP prostate tumor cells contain an androgen receptor (AR) with a point mutation in the steroid-binding domain (codon 868, Thr to Ala). This defect leads to a change in specificity of the AR. Estrogens, progestagens, and some anti-androgens (e.g., cyproterone acetate, hydroxyflutamide, nilutamide) stimulate LNCaP cell growth rate through the AR. The present studies indicate that not all anti-androgens showed agonistic effects with the mutated receptor. The growth rate of LNCaP cells did not increase with the anti-androgen ICI 176334, nor could this compound increase transcription activation of the reporter gene construct via the mutant receptor in a cotransfection system [HeLa cell cotransfection system with an androgen-regulated reporter gene construct (pG29G-tk-CAT) and the mutant receptor as trans-vector]. Interaction of the AR of LNCaP cells with heat-shock proteins was studied by isolation of the receptor with a specific monoclonal antibody and characterization of associated proteins. Hsp90, hsp70, and hsp56 were found to coprecipitate with the AR. Incubation of the cells at 37 degrees C with androgen (R1881, 10 nM) or the anti-androgen hydroxyflutamide, prior to receptor isolation, resulted in dissociation of the AR-heat-shock protein complex. This dissociation is paralleled by the transformation to a tight nuclear-binding form of the AR. In contrast, ICI 176334 could not induce a release of heat-shock proteins and did not increase nuclear binding, but inhibited the transformation process induced by R1881.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen Antagonists↗

PubMed1992

The progression of spermatogenesis in the developing rat testis followed by 31P MR spectroscopy.

To evaluate the use of human testicular 31P MR spectroscopy as a diagnostic tool to differentiate between several stages of male infertility, we have studied the testicular levels of several phosphorus containing compounds in the rat in relation to the condition of spermatogenesis and the cell types present in the seminiferous tubules of the testis. During testicular maturation several characteristic changes occur in the 31P MR spectrum of the testis of male Wistar rats. The phosphomonoester/adenosine triphosphate (PM/ATP) ratio shows a decline from 1.61 to 1.02 between the age of 3 and 12 weeks, whereas the phosphodiester (PD)/ATP ratio increases from 0 to 0.72. The testicular pH increases in the same time from 7.06 to 7.32. Testicular MR data obtained after 12 weeks of age onward do not show significant change anymore. The high PM/ATP ratio is associated by a relative high amount of proliferating spermatogonia and spermatocytes during meiosis in the testis, whereas the PD peak seems to be correlated with the release and maintenance of spermatozoa. The MR spectra show a specific fingerprint in all developmental stages of the rat testis as a result of the different cell types in the testis.

Adenosine Triphosphate↗

PubMed1992

Transcriptional regulation of androgen receptor gene expression in Sertoli cells and other cell types.

Regulation of androgen receptor (AR) mRNA expression was studied in Sertoli cells and peritubular myoid cells isolated from immature rat testis, and in the lymph node carcinoma cell line derived from a human prostate (LNCaP). Addition of dibutyryl-cyclic AMP (dbcAMP) to Sertoli cell cultures resulted in a rapid transient decrease in AR mRNA expression (5 h), which was followed by a gradual increase in AR mRNA expression (24-72 h). This effect of dbcAMP mimicked follicle-stimulating hormone (FSH) action. In peritubular myoid cells, there was only a moderate but prolonged decrease during incubation in the presence of dbcAMP, and in LNCaP cells no effect of dbcAMP on AR mRNA expression was observed. When Sertoli cells or peritubular myoid cells were cultured in the presence of androgens, AR mRNA expression in these cell types did not change. This is in contrast to LNCaP cells, that showed a marked reduction of AR mRNA expression during androgen treatment. In the present experiments, transcriptional regulation of AR gene expression in Sertoli cells and LNCaP cells was also examined. Freshly isolated Sertoli cell clusters were transfected with a series of luciferase reporter gene constructs, driven by the AR promoter. It was found that addition of dbcAMP to the transfected Sertoli cells resulted in a small but consistent increase in reporter gene expression (which was interpreted as resulting from AR promoter activity); a construct that only contained the AR 5' untranslated region of the cDNA sequence did not show such a regulation. The same constructs, transfected into LNCaP cells, did not show any transcriptional down-regulation when the synthetic androgen R1881 was added to the cell cultures. A nuclear transcription elongation experiment (run-on), however, demonstrated that androgen-induced AR mRNA down-regulation in LNCaP cells resulted from an inhibition of AR gene transcription. The present results indicate that in Sertoli cells and LNCaP cells, hormonal effects on AR gene transcription play a role in regulation of AR expression. However, AR gene transcription in these cells is differentially regulated.

Animals↗

PubMed1992

Activin receptor mRNA expression in rat testicular cell types.

cDNA encoding the extracellular domain of the rat activin receptor was cloned using the polymerase chain reaction (PCR). This cDNA is highly homologous to cDNA encoding the extracellular domain of the mouse activin receptor, whereas at the protein level the extracellular domains of both receptors are identical. Employing this cDNA as a probe in Northern blot analysis, expression of two activin receptor mRNAs (6 kb and 4 kb) was observed, in testes of immature and mature rats. Between day 21 and 28 of postnatal development, a large increase in testicular expression of the 4 kb mRNA was found, suggesting expression of this activin receptor mRNA in germ cells. The 4 kb mRNA was indeed present in isolated pachytene spermatocytes and round spermatids, but was absent in elongating spermatids. Sertoli cells obtained from immature and mature rats expressed both the 6 kb and 4 kb mRNAs, whereas the expression of these mRNAs in Leydig cell preparations was very low. These results may imply that activin has multiple actions in the control of testicular function.

Activin Receptors↗

PubMed1992

Regulation of inhibin beta B-subunit mRNA expression in rat Sertoli cells: consequences for the production of bioactive and immunoreactive inhibin.

In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the alpha-subunit of inhibin, and the production of immunoreactive inhibin are stimulated by follicle-stimulating hormone (FSH). In contrast, the amount of beta B-subunit mRNA is not increased after FSH treatment of the cells, and the ratio between bioactive and immunoactive inhibin decreases after stimulation with FSH. These data suggest that the beta B-subunit is the limiting factor in the production of bioactive inhibin. The aim of the present experiments was to investigate the effect of changes in the amount of beta B-subunit mRNA on the production of bioactive and immunoreactive inhibin. During early postnatal testicular development, the relative amounts of the 4.2 kb and 3.5 kb mRNAs encoding the beta B-subunit of inhibin changed markedly. The meaning of this changing ratio between beta B-subunit mRNAs is not clear, since both mRNAs are actively translated, as demonstrated by polysomal analysis. The total amount of beta B-subunit mRNA correlated with the in vitro production of bioactive inhibin as published earlier. Prolonged stimulation of cultured Sertoli cells from 14-day-old rats with 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a decreased expression of the beta B-subunit mRNAs, presumably by down-regulation of protein kinase C. A similar effect was obtained after addition of the calcium ionophore A23187. Concomitantly, a decreased production of bioactive inhibin was observed. Furthermore, Western blotting revealed that secretion of the 32 kDa inhibin alpha beta-dimer was decreased, whereas secretion of the combination of the C-terminal part with the pro-region of the alpha-subunit was increased. It is concluded that the level of the beta B-subunit of inhibin is rate-limiting for the production of bioactive inhibin in cultured Sertoli cells, and that its expression can be influenced by modulation of protein kinase C, and/or intracellular calcium levels.

Alkaloids↗

PubMed1992

Effect of testosterone deprivation on expression of the androgen receptor in rat prostate, epididymis and testis.

Adult rats were treated with ethane dimethane sulphonate (EDS) to eliminate the Leydig cells. This treatment resulted in very low levels of testosterone in the blood and in the testis. Furthermore, histological evaluation of spermatogenesis showed no marked differences between control and EDS-treated animals. In the ventral prostate, 5 days after EDS-treatment, a 4.0 +/- 0.3-fold up-regulation of androgen receptor (AR) mRNA was observed, together with a 2.2 +/- 0.2-fold increase in actin mRNA. In the epididymis, a 2.0 +/- 0.5-fold increase in AR mRNA level was observed, without a change in actin mRNA level. In the testes of EDS-treated rats, the AR mRNA level was not changed (1.02 +/- 0.17-fold of controls), and there was also no change in actin mRNA level at 5 days after EDS-treatment. These results indicate that AR mRNA expression in the ventral prostate and epididymis is regulated differentially by testosterone when compared to regulation in the testis. Testicular androgen binding sites were assayed by Scatchard analysis of the binding of 3H-R1881 to a nuclear fraction, that was isolated by a method which involved the use of liquid nitrogen and high sucrose buffer. The number of specific binding sites per testis in EDS-treated rats with testosterone-implants, remained unaltered compared to control rats (9.1 +/- 1.4 pmol/testis). In these rats, 20% of the normal testicular testosterone level was sufficient to maintain the androgen receptor in a tight nuclear binding (transformed) form. In testes from EDS-treated rats without testosterone-implants, the AR did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals was close to control levels, as measured by nuclear 3H-R1881 binding after receptor transformation through injection of a high dose of testosterone (10 mg) 2 h before killing the rats (testosterone pulse). In the different experimental groups, FSH was not required to maintain the total testicular AR content (ligand binding). Immunoprecipitation and Western blotting of the testicular AR using specific monoclonal and polyclonal antibodies indicated that the total testicular amount of immunodetectable AR protein in long-term testosterone deprived rats was very low when compared to that in control rats or rats with testosterone-implants. This is in disagreement with results obtained in the ligand binding assay, and may point to a structural modification of the AR in the testis that possibly occurs in the prolonged absence of androgens.

Animals↗

PubMed1992

Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein.

Cooperative actions of FSH and androgens on initiation, maintenance, and restoration of spermatogenesis have been described. In the present experiments the regulatory effects of FSH on androgen receptor (AR) gene expression in Sertoli cells were studied. In immature rats injection of FSH (1 microgram/g BW, ip) resulted in a rapid down-regulation of testicular AR mRNA expression (4 h), followed by recovery to the control level (10 h). Using cultured immature Sertoli cells, a similar transient effect on AR mRNA expression was observed after the addition of FSH (500 ng/ml) or (Bu)2cAMP (0.5 mM). Cycloheximide treatment of the cells did not prevent the rapid FSH-induced down-regulation of AR mRNA expression, indicating that de novo protein synthesis is not required for this effect. Furthermore, using a transcriptional run-on assay, no marked decrease in the rate of AR gene transcription was found upon treatment of the cultured Sertoli cells with FSH for 2 or 4 h. This demonstrates that the short term effect of FSH or AR mRNA expression reflects a change in mRNA stability. The AR protein level was not markedly affected by the transient decrease in AR mRNA expression. When immature Sertoli cells were incubated with FSH for longer time periods (24-72 h), both AR mRNA and protein expression were increased. In Sertoli cells isolated from 15-day-old rats, this increase was higher (mRNA, 2- to 3-fold; protein, 2-fold) than in Sertoli cells isolated from 25-day-old animals. The results indicate that FSH plays a complex role in the regulation of AR expression in immature rat Sertoli cells.

Aging↗

PubMed1991

Magainins affect respiratory control, membrane potential and motility of hamster spermatozoa.

The hypothesis was tested that the magainin peptides, known to compromise bacterial and mitochondrial energetics, are highly active against spermatozoa. A mixture of magainin A and PGLa (1:1) caused a 50% reduction in motility of hamster spermatozoa at 4 micrograms/ml total peptide concentration. All motility was lost at 8 micrograms/ml. At this concentration, respiratory control was released and respiration in the presence of uncoupler was inhibited. Uptake of the lipophilic cation tetraphenyl phosphonium was largely abolished by addition of magainin A and PGLa showed synergism with respect to release of respiratory control.

Amino Acid Sequence↗

PubMed1991

Characterization of the human androgen receptor transcription unit.

A full length human androgen receptor (hAR) cDNA was constructed from cDNA and genomic clones. Structurally the 10.6-kilobase (kb) hAR cDNA consists of a long 5'-untranslated region (5'-UTR, 1.1 kb), a previously described open reading frame (ORF, 2.7 kb) (Trapman, J., Klaassen, P., Kuiper, G. G. J. M., van der Korput, J. A. G. M., Faber, P. W., van Rooij, H. C. J., Geurts van Kessel, A., Voorhorst, M. M., Mulder, E., and Brinkmann, A. O. (1988) Biochem. Biophys. Res. Commun. 153, 241-248; Faber, P. W., Kuiper, G. G. J. M., van Rooij, H. C. J., van der Korput, J. A. G. M., Brinkmann, A. O., and Trapman, J. (1989) Mol. Cell. Endocrinol. 61, 257-262), and a very long 3'-untranslated region (3'-UTR, 6.8 kb). The complete 5'- and 3'-UTRs were found to be encoded by the previously reported first and eight protein coding exons of the hAR gene, respectively (Kuiper, G. G. J. M., Faber, P. W., van Rooij, H. C. J., van der Korput, J. A. G. M., Ris-Stalpers, C., Klaassen, P., Trapman, J., and Brinkmann, A. O. (1989) J. Mol. Endocrinol. 2, R1-R4). Two major sites of transcription initiation were identified in a 13-base pair region. DNA fragments spanning these transcription initiation sites conferred promoter activity upon a promoterless chloramphenicol acetyltransferase reporter gene construct. Two equally effective, functional polyadenylation signals (ATTAAA and CATAAA) at a mutual distance of 221 base pairs were detected. The ATTAAA hexamer sequence gave rise to multiple sites of poly(A) addition, whereas only one position was used following the CATAAA hexamer. In LNCaP prostatic carcinoma cells an alternatively spliced hAR mRNA species was identified which lacks 3 kb of the 3'-UTR.

Base Sequence↗

PubMed1991

Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation.

An immunochemical method has been used to detect quantitatively DNA damage caused by ionizing radiation in germ cells. With this method, DNA strand breaks as well as lesions converted into breaks in alkaline medium are measured as a function of controlled partial unwinding of the DNA, a time-dependent process starting at each breakage site, followed by the determination of the relative amount of single-stranded regions by use of a single-strand specific monoclonal antibody. With this method the induction and repair of DNA damage in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids) of the hamster were investigated. Germ cells were irradiated in vitro with 60Co-gamma-rays, at doses between 0 and 5 Gy. A linear dose-response relationship was observed. Spermatocytes and round spermatids had normal, fast repair of the lesions when compared with the repair of these sites in cultured V79 or CHO cells and human lymphocytes. The elongated spermatids, however, showed hardly any repair. Similar results were obtained after the in vivo gamma-irradiation of hamsters with doses of 0. 4, and 8 Gy and subsequent isolation of germ cells. The damage was still detectable in the elongated spermatids at 24 h after exposure. The results of the experiments show substantial differences in repair capacity between different stages of germ cell development. Because DNA is the major target for mutation induction, this assay may be useful for assessment of the genetic risk of exposure of male germ cells to ionizing radiation, in relation to the stage of development.

Animals↗

PubMed1991

Follitropin receptor down-regulation involves a cAMP-dependent post-transcriptional decrease of receptor mRNA expression.

The regulation by FSH (follitropin; follicle-stimulating hormone) of FSH receptor mRNA and protein (FSH binding) was studied using cultured Sertoli cells isolated from 21-day-old rats. FSH induced a dose-dependent and almost complete down-regulation of receptor mRNA at 4 h after addition of the hormone. At subsequent time points (16 h and later) the FSH receptor mRNA levels had returned close to control values. The effect of FSH was mimicked by dibutyryl cyclic AMP (dbcAMP) and forskolin, and the phosphodiesterase inhibitor methyl-isobutylxanthine (MIX) prolonged the FSH action. These findings indicate that the effect of FSH on its receptor mRNA was mediated by cAMP. A down-regulatory effect of FSH and dbcAMP on FSH receptor mRNA was also observed in the presence of the protein synthesis inhibitor cycloheximide, suggesting a direct effect of FSH/dbcAMP on the expression of the FSH receptor gene. Transcriptional run-on experiments revealed that FSH did not inhibit initiation of the FSH receptor gene; hence a post-transcriptional mechanism is involved. Binding of 125I-FSH to the cultured Sertoli cells was rapidly (4 h) decreased when the cells were incubated with FSH or FSH in combination with MIX. This effect can be explained by ligand-induced receptor sequestration. In contrast, incubation of Sertoli cells with dbcAMP had no effect on binding of 125I-FSH after 4 h, but resulted in a 60% loss of FSH binding sites after 24 h, probably caused by decreased mRNA expression. In conclusion, FSH receptor down-regulation in Sertoli cells is effected not only by the well-documented ligand-induced loss of receptors from the plasma membrane, but also involves a cAMP-mediated decrease of FSH receptor mRNA through a post-transcriptional mechanism.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Synthesis and post-translational modification of the androgen receptor in LNCaP cells.

Androgen receptor synthesis and modification were studied in the human LNCaP cell line. Immunoblotting with a specific polyclonal antibody showed that the androgen receptor migrated as a closely spaced 110-112 kDa doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Most of the receptor protein is present in the higher molecular mass form. Pulse labelling experiments with [35S]methionine showed that the androgen receptor is synthesized as a single 110 kDa protein which is rapidly converted to a 112 kDa protein. Alkaline phosphatase treatment of cytosols from [35S]methionine pulse labelled cells caused a gradual elimination of the 112 kDa isoform with a concomitant increase of the 110 kDa isoform. This indicates that the observed 110 to 112 kDa upshift of the newly synthesized androgen receptor reflects receptor phosphorylation. Both isoforms can bind hormone and can undergo a hormone dependent transformation to a tight nuclear binding form, indicating that the 110 to 112 kDa conversion is not an obligatory step for hormone binding or receptor transformation.

Humans↗

PubMed1991

Regulation of androgen receptor mRNA and protein in the rat testis by testosterone.

Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration. In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment. In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered. Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens.

Androgens↗

PubMed1991

Characterization of the testicular cell types present in the rat by in vivo 31P magnetic resonance spectroscopy.

Testes of vitamin A-deficient Wistar rats before and after vitamin A replacement, of rats irradiated in utero, and of control rats were investigated by in vivo 31P magnetic resonance (MR) spectroscopy. The testicular phosphomonoester/ATP (PM/ATP) ratio ranged from 0.79 +/- 0.05 for testes that contained only interstitial tissue and Sertoli cells to 1.64 +/- 0.04 for testes in which spermatocytes were the most advanced cell types present. When new generations of spermatids entered the seminiferous epithelium, this ratio decreased. The testicular phosphodiester/ATP (PD/ATP) ratio amounted to 0.16 +/- 0.06 for testes in which Sertoli cells, spermatogonia, or spermatocytes were the most advanced cell type present. When new generations of spermatids entered the seminiferous epithelium, the PD/ATP ratio rapidly increased and finally reached a value of 0.71 +/- 0.06 for fully developed testes. Taken together, specific patterns of the PM/ATP ratio, the PD/ATP ratio, and pH were obtained that were correlated to the presence of spermatogonia, spermatocytes, round spermatids, and elongated spermatids or to the absence of spermatogenic cells. Hence, a good impression of the status of the seminiferous epithelium in the rat can be obtained by in vivo 31P MR spectroscopy.

Adenosine Triphosphate↗

PubMed1990

The rat androgen receptor gene promoter.

The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was cloned from a genomic library and the promoter was identified. S1-nuclease protection analysis showed two major transcription start sites, located between 1010 and 1023 bp upstream from the translation initiation codon. The area surrounding these start sites was cloned in both orientations in a CAT reporter plasmid. Upon transfection of the constructs into COS cells, part of the promoter stimulated transcription in an orientation-independent manner, but the full promoter showed a higher and unidirectional activity. In the promoter/reporter gene constructs, transcription initiated from the same positions as in the native gene. Sequence analysis showed that the promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but one SP1 site is located at about 60 bp upstream from the major start site of transcription. Other possible promoter elements are TGTYCT sequences at positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505, resembling half-sites of the glucocorticoid-responsive element (GRE). Furthermore, a homopurine stretch containing a total of 8 GGGGA elements and similar to sequences that are present in several other GC-rich promoters, is located between -89 and -146 bp upstream from the major start site of transcription.

Animals↗

PubMed1990

Regulation of inhibin alpha- and beta B-subunit mRNA levels in rat Sertoli cells.

The hormonal regulation of the expression of the inhibin alpha-subunit and beta B-subunit genes was studied in cultured rat Sertoli cells. The alpha-subunit mRNA level increased during incubation of the cells in the presence of follicle-stimulating hormone (FSH), reaching maximal levels within 1.5 h. This stimulation was mimicked by addition of dibutyryl-cyclic AMP, indicating that FSH action on the alpha-subunit gene is exerted via cyclic AMP. Inhibition of translation by cycloheximide (CX) caused upregulation of the alpha-subunit mRNA, and did not block the effect of FSH on the level of this mRNA. In FSH-stimulated cells, the half-life of the alpha-subunit mRNA was 6 h, and this half-life was prolonged by inhibition of transcription using actinomycin D (AD). It is concluded that the effect of FSH on alpha-subunit mRNA expression represents a direct effect on the alpha-subunit gene, and that alpha-subunit mRNA levels are influenced by a short-lived mRNA destabilizing protein. The levels of two beta B-subunit mRNAs (4.2 kb and 3.5 kb) were not affected by FSH or dbcAMP. However, these mRNAs were also upregulated by CX treatment. Experiments using AD showed that the 4.2 kb mRNA is less stable than the 3.5 kb mRNA. The differential regulation of the inhibin alpha- and beta B-subunit mRNAs is discussed.

Animals↗