Nuclear androgen binding sites in the male rat. III. Late spermatids and spermatozoa in the testis, with an introduction to epididymal spermatozoa.
Nuclear androgen binding sites were examined in late spermatids (stages 12-19) which resisted sonication of homogenized testes of mature male rats. The measurement of unoccupied binding sites in salt extract of purified spermatid heads by nuclear exchange at -10 degrees C was developed and validated. As in the prostate, unoccupied nuclear androgen binding sites in sonicated testes were in low concentration, were not artefactual, and could be occupied both in vivo and in vitro by exogenous androgens, and uniquely in hemicastrated rats by endogenously compensated androgens in the remaining testis. The properties of occupied binding sites in salt extract of purified spermatid heads (measured by nuclear exchange at 4 degrees C for 48 or more hours with 5 nM [3H]dihydrotestosterone) were almost identical to those of occupied binding sites in nuclei of the ventral prostate, except for their concentration. However, levels of specific binding activity approaching 50 fmol/mg DNA could be expected in salt extract of spermatid pellets, by use of a sulfhydryl reducing agent (dithiothreitol) prior to salt extraction, a protease inhibitor (phenylmethylsulfonyl fluoride) in all buffers, and optimization of the sonication protocol. Nuclear androgen binding sites of sonicated epididymal spermatozoa, collected by retrograde perfusion of the cauda epididymidis, were found to be completely salt-resistant. These binding proteins could be extracted by 0.4 M KCl if dithiothreitol and dihydrotestosterone were incorporated into the sonication buffer, if phenylmethylsulfonyl fluoride was added to all buffers, and if the purified epididymal sperm pellet was treated with sarkosyl, a non-ionic detergent, just before salt extraction. The salt extract of epididymal spermatozoa which were treated as described above contained two binding components: a soluble form which was eluted from hydroxylapatite by increasing concentrations of phosphate buffers, and a non-soluble form, free of DNA, which remained in the hydroxylapatite column, and which contained most of the androgen binding sites. Affinity (Kd) of dihydrotestosterone to the soluble and insoluble fractions of the steroid-binding protein complex was determined to be 0.7 and 0.1 nM, respectively. Salt-resistance of binding proteins in germ cells was shown to develop significantly in the last stages of spermiogenesis.
D000818:Animals; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000378:metabolism*; D013196:Dihydrotestosterone / Q000378:metabolism; D004822:Epididymis / Q000502:physiology; D007700:Kinetics; D008297:Male; D011467:Prostate / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains; D011944:Receptors, Androgen / Q000302:isolation & purification / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D013087:Spermatids / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000502:physiology
D011944:Receptors, Androgen; D011987:Receptors, Steroid; D013196:Dihydrotestosterone