SULFATION PATHWAYS: Expression of SULT2A1, SULT2B1 and HSD3B1 in the porcine testis and epididymis.

In the porcine testis, in addition to estrogen sulfates, the formation of numerous sulfonated neutral hydroxysteroids has been observed. However, their functions and the underlying synthetic pathways are still widely unclear. To obtain further information on their formation in postpubertal boars, the expression of sulfotransferases considered relevant for neutral hydroxysteroids (SULT2A1, SULT2B1) was investigated in the testis and defined segments of the epididymis applying real-time RT-qPCR, Western blot and immunohistochemistry (IHC). Sulfotransferase activities were assessed in tissue homogenates or cytosolic preparations applying dehydroepiandrosterone and pregnenolone as substrates. A high SULT2A1 expression was confirmed in the testis and localized in Leydig cells by IHC. In the epididymis, SULT2A1 expression was virtually confined to the body. SULT2B1 expression was absent or low in the testis but increased significantly along the epididymis. Immunohistochemical observations indicate that both enzymes are secreted into the ductal lumen via an apocrine mechanism. The results from the characterization of expression patterns and activity measurements suggest that SULT2A1 is the prevailing enzyme for the sulfonation of hydroxysteroids in the testis, whereas SULT2B1 may catalyze the formation of sterol sulfates in the epididymis. In order to obtain information on the overall steroidogenic capacity of the porcine epididymis, the expression of important steroidogenic enzymes (CYP11A1, CYP17A1, CYP19, HSD3B1, HSD17B3, SRD5A2) was monitored in the defined epididymal segments applying real-time RT-qPCR. Surprisingly, in addition to a high expression of SRD5A2 in the epididymal head, a substantial expression of HSD3B1 was detected, which increased along the organ.

SULFATION PATHWAYS: Formation and hydrolysis of sulfonated estrogens in the porcine testis and epididymis.

Boars exhibit high concentrations of sulfonated estrogens (SE) mainly originating from the testicular-epididymal compartment. Intriguingly, in porcine Leydig cells, sulfonation of estrogens is colocalized with aromatase and steroid sulfatase (STS), indicating that de novo synthesis of unconjugated estrogens (UE), their sulfonation and hydrolysis of SE occur within the same cell type. So far in boars no plausible concept concerning the role of SE has been put forward. To obtain new information on SE formation and hydrolysis, the porcine testicular-epididymal compartment was screened for the expression of the estrogen-specific sulfotransferase SULT1E1 and STS applying real-time RT-qPCR, Western blot and immunohistochemistry. The epididymal head was identified as the major site of SULT1E1 expression, whereas in the testis, it was virtually undetectable. However, SE tissue concentrations are clearly consistent with the testis as the predominant site of estrogen sulfonation. Results from measurements of estrogen sulfotransferase activity indicate that in the epididymis, SULT1E1 is the relevant enzyme, whereas in the testis, estrogens are sulfonated by a different sulfotransferase with a considerably lower affinity. STS expression and activity was high in the testis (Leydig cells, rete testis epithelium) but also present throughout the epididymis. In the epididymis, SULT1E1 and STS were colocalized in the ductal epithelium, and there was evidence for their apocrine secretion into the ductal lumen. The results suggest that in porcine Leydig cells, SE may be produced as a reservoir to support the levels of bioactive UE via the sulfatase pathway during periods of low activity of the pulsatile testicular steroidogenesis.