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Sulfo-conjugated steroid hormones, such as dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate or estrone-3-sulfate are abundant in the body, but are biologically inactive at classical androgen and estrogen steroid receptors. However, after carrier-mediated import and de-conjugation by the steroid sulfatase, these compounds participate in the overall steroid regulation of reproductive organs. The sodium-dependent organic anion transporter SOAT, coded by the SLC10A6 gene, is specific for the transport of steroid sulfates and is highly expressed in testicular germ cells, including pachytene spermatocytes, secondary spermatocytes, and round spermatids. Therefore, SOAT is supposed to be involved in the regulation of spermatogenesis and male fertility. In the present study, the SLC10A6 gene was analyzed for rare genetic variants, which might affect transport function or membrane expression of SOAT. Among the 31 SOAT variants analyzed, L44P, Q75R, P107L, G109S, S112F, N113K, S133F, G241D, G263E, G294R, and Y308N showed no transport activity for DHEAS at all. In the case of P107L, G241D, G263E, and Y308N, this was most likely due to significantly reduced expression in the plasma membrane. Other variants are located directly at (Q75R, S112F, N113K) or close to (G109S, S133F, and G263E) the supposed SOAT Na + binding sites and thus could disable the sodium-coupled transport cycle. If these loss-of-function SOAT variants are more frequent in men with impaired spermatogenesis or infertility needs further investigation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Sodium-dependent organic anion transporter (SOAT) represents a membrane transporter specific for sulfated steroid hormones, which are supposed to participate in the regulation of reproductive processes. In man, SOAT shows predominant mRNA expression in the testis and here was localized to primary spermatocytes. SOAT mRNA expression is significantly downregulated in different disorders of spermatogenesis, including hypospermatogenesis. The resulting decline of SOAT-mediated transport of sulfated steroids may participate in the impairment of functional spermatogenesis. Apart from downregulation of SOAT mRNA expression, genetic polymorphisms affecting the transport function of SOAT may have the same negative effect on spermatogenesis. Therefore, in the present study we searched for functionally relevant SOAT polymorphisms, aiming to comparatively analyze their occurrence in patients with impaired spermatogenesis vs. patients with intact spermatogenesis. We found that the SOAT polymorphism L204F showed a significantly reduced transport function for DHEAS when expressed in HEK293 cells. Although the K m value was identical with that of the SOAT wildtype, the V max value dramatically declined for the SOAT-L204F variant (942.5 vs. 313.6pmol×mg protein -1 ×min -1 ). Although the same amount of total SOAT-L204F protein was detected in transfected HEK293 cells compared to the SOAT wildtype, plasma membrane expression was significantly reduced, which points to a plasma membrane sorting defect of the SOAT-L204F variant. Groups of 20 subjects with normal spermatogenesis and 26 subjects with hypospermatogenesis were genotyped for this polymorphism. Both groups showed nearly identical distributions of the SOAT-L204F polymorphism (∼10% heterozygous and ∼5% homozygous), indicating that this polymorphism seems not be causative for hypospermatogenesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The sodium-dependent organic anion transporter SOAT (gene name SLC10A6 in man and Slc10a6 in mice) is a plasma membrane transporter for sulfated steroids, which is highly expressed in germ cells of the testis. SOAT can transport biologically inactive sulfated steroids into specific target cells, where they can be reactivated by the steroid sulfatase (STS) to biologically active, unconjugated steroids known to regulate spermatogenesis. Significantly reduced SOAT mRNA expression was previously found in different forms of impaired spermatogenesis in man. It was supposed that SOAT plays a role for the local supply of steroids in the testis and consequently for spermatogenesis and fertility. Thus, an Slc10a6 -/- Soat knockout mouse model was established by recombination-based target deletion of the Slc10a6 gene to elucidate the role of Soat in reproduction. However, the Slc10a6 -/- knockout mice were fertile, produced normal litter sizes, and had normal spermatogenesis and sperm vitality. This phenotype suggests that the loss of Soat can be compensated in the knockout mice or that Soat function is not essential for reproduction. In addition to reproductive phenotyping, a comprehensive targeted steroid analysis including a set of 9 un-conjugated and 12 sulfo-conjugated steroids was performed in serum of Slc10a6 -/- knockout and Slc10a6 +/+ wildtype mice. Only cholesterol sulfate, corticosterone, and testosterone (only in the males) could be detected in considerable amounts. Interestingly, male Slc10a6 -/- knockout mice showed significantly higher serum levels for cholesterol sulfate compared to their wildtype controls. As cholesterol sulfate has a broader impact apart from the testis, further analysis of this phenotype will include other organs such as skin and lung, which also show high Soat expression in the mouse., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The sodium-dependent organic anion transporter SOAT/Soat shows highly specific transport activity for sulfated steroids. SOAT substrates identified so far include dehydroepiandrosterone sulfate, 16α-hydroxydehydroepiandrosterone sulfate, estrone-3-sulfate, pregnenolone sulfate, 17β-estradiol-3-sulfate, and androstenediol sulfate. Apart from these compounds, many other sulfated steroids occur in mammals. Therefore, we aimed to expand the substrate spectrum of SOAT and analyzed the SOAT-mediated transport of eight different sulfated steroids by combining in vitro transport experiments in SOAT-transfected HEK293 cells with LC-MS/MS analytics of cell lysates. In addition, we aimed to better understand the structural requirements for SOAT substrates and so selected structural pairs varying only at specific positions: 3α/3β-sulfate, 17α/17β-sulfate, mono-sulfate/di-sulfate, and 17α-hydroxylation. We found significant and sodium-dependent SOAT-mediated transport of 17α-hydroxypregnenolone sulfate, 17β-estradiol-17-sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and 5α-dihydrotestosterone sulfate. However, 17β-estradiol-3,17-disulfate was not transported by SOAT., In Conclusion: SOAT substrates from the group of sulfated steroids are characterized by a planar and lipophilic steroid backbone in trans-trans-trans conformation of the rings and a negatively charged mono-sulfate group at positions 3' or 17' with flexibility for α- or β- orientation. Furthermore, 5α-reduction, 16α-hydroxylation, and 17α-hydroxylation are acceptable for SOAT substrate recognition, whereas addition of a second negatively charged sulfate group seems to abolish substrate binding to SOAT, and so 17β-estradiol-3,17-disulfate is not transported by SOAT., (Copyright © 2017 Elsevier Ltd. All rights reserved.)