Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105, 000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1, 2-³h]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M ? 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1, 2-α-methylene-6-chloro-pregn-4, 6-diene-17α-o1–3, 20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105,000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1,2-³H]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M — 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1,2-α-methylene-6-chloro-pregn-4, 6-diene-17α-ol-3,20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

Androgen nuclear exchange activity in rat testis.

Androgen nuclear exchange activity has been measured in the rat testis. Exchange activity was increased 2-10-fold by pretreatment of the tissue with testosterone and persisted for at least 21 days after hypophysectomy in mature animals. Competition for exchange activity using 3H-testosterone or 3H-dihydrotestosterone was significant for a 500-fold excess of testosterone, dihydrotestosterone, progesterone, and cyproterone acetate. The activity was found in tubules and in isolated germ cells from mature rat testes. These results suggest that the molecular mechanism of androgen action in the testis may be quite complex and may involve components both in the more mature germ cells as well.     

Androgen binding to subcellular particles of rat testis

Androgen binding activity was evaluated in different subcellular particulate fractions obtained by differential centrifugation of 32-day-old rat seminiferous tubules homogenates. After eliminating heavy particles by centrifugation at 4300 g during 4 min in 0.25 M sucrose buffer, a 27,000 g pellet was obtained and layered on 1.05 M sucrose buffer. The relatively light particulate interface (LPF) formed during centrifugation at 27,000 g 60 min, showed the highest androgen binding activity among particulate fractions.This binding was associated with the plasma membrane marker 5'-nucleotidase and it did not follow any of six other subcellular structure markers: DNA for nuclei, succinate dehydrogenase for mitochondria, acid phosphatase for lysosomes, NADPH-cytochrome C reductase for smooth endoplasmic reticulum, RNA for rough endoplasmic reticulum and lactate dehydrogenase for cytosol.In LPF, concentrations of sites were estimated to be 328 ± 54 fmol per mg proteins and affinity constant 0.78 ± 0.23 10⁹ M⁻¹. Heat stability, steroid specificity, affinity constant and rate of dissociation were similar to the well known androgen binding protein, ABP. Presence of ABP or a similar protein in subcellular particles might play a role in the mechanism of action of androgens in seminiferous tubules of maturing rats.     

Androgen receptor complex binding in murine skeletal muscle nuclei

Examination of binding of androgen-receptor complexes from murine skeletal muscle cytosol was performed by modified nuclear retention assay and modified nuclear acceptor assay. The experiments showed the binding of androgen-receptor complexes to the nuclear acceptor sites to be a cooperative process. Hill analysis of the data obtained resulted in a Hill coefficient of 3,6. The apparent dissociation constant for binding of cytosolic [³H]-testosterone-receptor complexes to nuclei was found to be in the range of K_D = 6 ? 8 × 10^?11 M. The nuclear matrix was able to bind androgen-receptor complexes in a saturable way, too.     

Diisopropylfluorophosphate-interacting proteinases of nuclei of rat testis cells

Nuclei and chromatin of seminiferous epithelial cells of rat testis contain acid-extractable and non-extractable proteins which interact readily with [³H]DFP (diisopropylfluorophosphate). Proteinase activity is closely associated with these DFP-interacting proteins, and the proteinase activities are inhibited by DFP and PMSF. DFP-interacting proteins of testis chromatin increase greatly in amount at 26–32 days after birth when spermatids are appearing in increasing numbers. In nuclei separated by zonal centrifugation on sucrose gradients, the DFP-labeled proteins are highest in activity in the elongated spermatids at the stage in spermiogenesis at which histones are being replaced by testis-specific proteins and protamines. Electrophoresis in SDS-polyacrylamide gels reveals the presence of three species of DFP-interacting proteins in nuclei of seminiferous epithelial cells of the testis. The chromatin of epididymal spermatozoa of the rat contains three or four species of DFP-interacting proteins by SDS-polyacrylamide electrophoresis and some of these labeled proteins co-migrate with two of the three basic proteins which are observed during electrophoresis on polyacrylamide gels in Triton-urea.     

Androgen receptor in rat Harderian and submandibular glands

Summary Androgens regulate the development and sexual dimorphism of rodent Harderian and submandibular glands. This effect is believed to be mediated by the androgen receptor. Immunohistochemistry and immunoblotting were carried out to study the receptor in normal, castrated and dihydrotestosterone-supplemented rat Harderian and submandibular glands. Immunohistochemically, the most intense nuclear staining was observed in the acinar cells of the submandibular glands, followed by intercalated duct cells. The granular convoluted tubules showed weak immunostaining and the striated ducts were negative. In the Harderian gland, nuclear staining was seen in both type I and II secretory cells. Castration and treatment had no effect on the expression of the androgen receptor protein in either gland. A 110 K androgen receptor signal was detected by immunoblotting in the Harderian gland but not in the submandibular gland. An experiment was designed to explore the possible effect of proteinases on the receptor protein in the homogenate of submandibular gland. Our results demonstrate the cell-specific location of the receptor in Harderian and submandibular glands, and show that the expression of the receptor protein is androgen-independent.     

Chromatin organization in rat testis nuclei. Flow cytometric detection of the morphological compaction

The unusual histone composition of testicular cells generates changes in chromatin organization in order to allow the chromosomal pairing necessary for genetic recombination. Accessibility of testis nuclear DNA was determined by flow cytometry. The observed differences in staining between testis and liver nuclear chromatin, as well as the differences of perpendicular light scatter signal, correlate with alterations in protein composition with the chromatin reorganization.     

Androgen receptor immunoreactivity in rat occipital cortex after callosotomy

Gonadal steroidogenesis can be influenced by direct neural links between the central nervous system and the gonads. It is known that androgen receptor (AR) is expressed in many areas of the rat brain involved in neuroendocrine control of reproduction,such as the cerebral cortex.It has been recently shown that the occipital cortex exerts an inhibitory effect on testicular stereoidogenesis by a pituitary-independent neural mechanism. Moreover, the complete transection of the corpus callosum leads to an increase in testosterone (T) secretion of hemigonadectomized rats. The present study was undertaken to analyze the possible corticocortical influences regulating male reproductive activities. Adult male Wistar rats were divided into 4 groups: 1) intact animals as control; 2) rats undergoing sham callosotomy; 3) posterior callosotomy; 4) gonadectomy and posterior callosotomy. Western blot analysis showed no remarkable variations in cortical AR expression in any of the groups except in group I where a significant decrease in AR levels was found. Similarly, both immunocytochemical study and cell count estimation showed a lower AR immunoreactivity in occipital cortex of callosotomized rats than in other groups. In addition, there was no difference in serum T and LH concentration between sham-callosotomized and callosotomized rats. In conclusion, our results showthat posterior callosotomy led to a reduction in AR in the right occipital cortex suggesting a putative inhibiting effect of the contralateral cortical area.     

Androgen binding by skeletal muscle nuclei

A study was made of 3H-19-nortestosterone binding by isolated nuclei and 0.4 M KCl nuclear extract of the rat skeletal muscle. Binding specificity was ascertained by incubation in the presence of various unlabeled steroids. The Kd values were measured for nuclei and 0.4 M KCl nuclear extract (11.6 +/- 2.5 nM and 9.9 +/- 1.6 nM, respectively). The amount of binding sites was 24.1 +/- 1.7 fmol/mg DNA or 13.7 +/- 1.0 fmol/g tissue. Enzymatic treatment with pronase and DNase shows that nuclear androgen receptors are proteins. DNA was noted to have a stabilizing effect. DNase treatment of nuclei during extraction with 0.4 M KCl was shown to significantly increase the amount of specifically bound radioactivity in the extract.     

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.     

A1 adenosine receptor of rat testis membranes. Purification and partial characterization

Purification of an A1 adenosine receptor of rat testes was performed using a newly developed affinity chromatography system (Nakata, H. (1989) Mol. Pharmacol. 35, 780-786). The A1 adenosine receptor was solubilized with digitonin from rat testicular membranes and then purified more than 25,000-fold by sequential use of affinity chromatography on xanthine amine congener-immobilized agarose, hydroxylapatite chromatography, re-affinity chromatography on xanthine amine congener-agarose, and finally gel permeation chromatography on TSK-3000SW. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation showed a single broad band of Mr 41,000 by autoradiography after radioiodination. This Mr 41,000 peptide was also specifically labeled with an A1 adenosine receptor affinity labeling reagent. A high affinity A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, bound saturably to the purified receptor with a KD of approximately 1.4 nM. The purified receptor also showed essentially the same specificity for adenosine agonists and antagonists as the unpurified receptor preparations, although the affinities of the purified adenosine receptor for agonists were significantly low compared to those of unpurified receptor preparations indicating that the purified A1 adenosine receptor exists as a low agonist-high antagonist affinity state. Deglycosylation of the purified testis adenosine A1 receptors with endoglycosidase F produced an increase in the mobility of the receptor protein to an apparent Mr 30,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to that of deglycosylated A1 adenosine receptors of rat brain membranes. Peptide maps of the purified testis and brain A1 adenosine receptors using trypsin and V8 protease suggest that these receptors show some structural homologies.