Role of Steroid Sulfonation as a Mechanism Controlling the Flow of Substrates through the Cascade of Steroidogenic Enzymes in the Porcine Testis
Final Report Abstract
An intriguing feature of boars is their exceptionally broad spectrum of sulfonated steroids originating from testicular steroidogenesis including estrogen sulfates and numerous sulfonated C19- and C21-steroids related to the synthesis or metabolism of estrogens, androgens and steroidal pheromones. However, their synthetic pathways and the biological role of the excessive steroid sulfonation in the porcine testis are still elusive. Moreover, porcine Leydig cells have been demonstrated to highly express steroid sulfatase (StS). The aims of our project were to investigate the suitability of the porcine testicularepididymal compartment as a model for the study of steroid sulfonation and physiological sulfatase pathways in general and to obtain new information on porcine testicular steroidogenesis for a better understanding of its unique features. Secretion patterns (spontaneous and hCG-stimulated) of 9 sulfonated steroids were comprehensively characterized applying LC-MS/MS. Long-term profiles (6-7 h) are consistent with a wave-like pattern closely associated with testosterone pulses (3-5/day). Measurements in afferent and efferent testicular blood vessels provided new information concerning a predominant testicular sulfonation or major contributions from extratesticular tissues. However, no evidence for time shifts between profiles or other forms of decoupling indicative of differential transport (e.g. blood, lymph fluid, tubular fluid) or metabolism were observed. In addition to the sulfonation of free steroids by sulfotransferases (SULTs), sulfonated steroids could also arise from the direct conversions of sulfonated precursors. This "sulfate pathway" of steroidogenesis has been previously suggested in various tissues including the porcine testis. However, its productivity and physiological relevance have never been investigated in detail. In boars the existence of a sulfate pathway would provide an explanation for the high StS expression in Leydig cells, as the enzyme could control the flow of sulfonated precursors into the pool of free steroids. To test this hypothesis, the utilization of pregnenolone sulfate (PregS) by the steroidogenic key enzyme CYP17A1 was investigated. PregS (sulfate pathway), pregnenolone (∆5-pathway) and progesterone (∆4-pathway) were incubated in parallel with a recombinant enzyme system (porcine CYP17A1, bovine cytochrome P450 oxidoreductase, bovine cytochrome b5) or porcine testicular microsomal fractions as a source of the enzyme system. The results clearly show that PregS is not an efficient substrate of porcine CYP17A1. These observations show that in the porcine testis a productive sulfate pathway for the synthesis of androgens and estrogens does not exist. In order to obtain new information on the sulfonation of free steroids in the porcine testicular-epididymal compartment, the expression of relevant sulfotransferases (SULTs) (estrogens: SULT1E1, neutral hydroxysteroids: SULTs 2A1 and 2B1) was characterized on the mRNA and protein level in the testis and 8 defined localizations of the epididymis. Moreover, SULT activities were measured using estrone, DHEA and pregnenolone as substrates. In the testis, only SULT2A1 was readily detectable. In the epididymis, all three SULTs were found with head, body and tail being the major sites of expression for SULT1E1, SULT2A1 and SULT2B1, respectively. The results provide clear evidence that the multispecific SULT2A1 highly expressed in the Leydig cells is the predominant source of sulfonated testicular steroids in boars including estrogen sulfates. Possibly, SULT2B1 highly expressed in the epididymal tail serves the production of sulfonated oxysterols, which are considered as stabilizers of sperm membranes.
Publications
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(2014) Free and sulfated steroids secretion in postpubertal boars (Sus scrofa domestica). Reproduction 148(3):303-314
Schuler G, Dezhkam Y, Bingsohn L, Hoffmann B, Failing K, Galuska CE, Hartmann MF, Sánchez- Guijo A, Wudy SA
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(2017) Efficiency of the sulfate pathway in comparison to the ∆4- and ∆5-pathway of steroidogenesis in the porcine testis. J Steroid Biochem Mol Biol
Klymiuk MC, Neunzig J, Bernhardt R, Sánchez-Guijo A, Hartmann MF, Wudy SA, Schuler G
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(2017) Simultaneous profiles of sulfonated androgens, sulfonated estrogens and sulfonated progestogens in postpubertal boars (sus scrofa domestica) measured by LC-MS/MS. J Steroid Biochem Mol Biol
Schuler G, Sánchez-Guijo A, Hartmann MF, Wudy SA
