SULFOCONJUGATION OF STEROIDS AND THE VASCULAR PATHWAY OF COMMUNICATION IN DOGFISH TESTIS

The zonal testis of the dogfish (Squalus acanthias) has proven advantageous to study biochemical changes in relation to stage of spermatogenesis, including information on steroidogenic enzymes and steroid receptors. To investigate whether sulfotransferase is part of a mechanism regulating the availability of biologically active hormone in close proximity to receptors, we measured in vitro conversion of [H-3]estrone (E1) to sulfoconjugated metabolites in cytosolic subfractions of testes grossly dissected according to germ cell composition (premeiotic-PrM, meiotic-M, and postmeiotic-PoM) stages). Assays were carried out in the presence of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) at 22-degrees-C and optimized for time (60 min) and protein (500 mug/ml). Michaelis-Menten kinetics and saturation analysis gave the following reaction constants for [H-3]E1:Km = 0.33 muM, Vmax = 2.5 pmol/min/mg; and for PAPS: Km = 33 muM, Vmax = 1.1 pmol/min/mg; competition studies carried out in the absence or presence of 1- or 5-fold excess radioinert steroids indicated that estrogen (E2 > E1) as well as androgens (T = DHEA > 5alpha dihydrotestosterone, DHT) were effective inhibitors. Sulfotransferase activity was found to be stage-related, being highest in PoM regions (2.31 +/- 0.24 pmol/min/mg protein) when compared to M and PrM regions (1.22 +/- 0.22 and 1.28 +/- 0.21 pmol/min/mg protein, respectively). Sulfoconjugation and the intratesticular distribution of steroid sulfates were also measured in vivo by perfusion of the intact testis with [H-3]androgen or -estrogen. The pathway of blood flow via the genital artery was epigonal organ --> PoM --> M --> PrM (mature --> immature). Perfused [H-3]E2, T, and DHT were all extensively metabolized in a one-pass, 1 hr perfusion, less than 10% of perfused [H-3] steroid being recovered from testicular tissues as unchanged steroid. In general, recovery of polar metabolites was greater than non-polar metabolites from all three substrates. Sequential hydrolysis with glucuronidase and glusulase indicated that sulfoconjugation is a minor component (< 20%) of several "inactivating" pathways, which include glucuronide conjugation, 17-ketosteroid synthesis, and pathways leading to unidentified polar metabolites. No consistent stage-related distribution patterns were observed for any of the metabolite subfractions; however, total recovered radioactive steroid (polar plus non-polar) formed a decreasing concentration gradient from point of entry of perfusate (PoM region) to point of exit (PrM region). These data support the conclusion that access to receptors by steroid ligands may be controlled by a balance between activating and inactivating pathways. Furthermore, the intratesticular blood flow pattern identified here suggests that sulfoconjugation may be one of several mechanisms for screening out hormonal signals reaching the testis via the general circulation. The parallel-antiparallel arrangement of blood flow and the spermatogenic progression provides an anatomical basis for paracrine communication between mature --> immature stages by which the orderly progression of spermatogenesis and testicular zonation is maintained. Finally, given the obvious advantages of the shark testis model it should be possible to elucidate more precisely the role of free steroids versus steroid conjugates as intratesticular paracrine regulators.