The polymorphism L204F affects transport and membrane expression of the sodium-dependent organic anion transporter SOAT (SLC10A6).

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 Km value was identical with that of the SOAT wildtype, the Vmax 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.

Transport of steroid 3-sulfates and steroid 17-sulfates by the sodium-dependent organic anion transporter SOAT (SLC10A6).

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, 17ß-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/3ß-sulfate, 17α/17ß-sulfate, mono-sulfate/di-sulfate, and 17α-hydroxylation. We found significant and sodium-dependent SOAT-mediated transport of 17α-hydroxypregnenolone sulfate, 17ß-estradiol-17-sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and 5α-dihydrotestosterone sulfate. However, 17ß-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 ß- 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 17ß-estradiol-3,17-disulfate is not transported by SOAT.

Sodium-dependent organic anion transporter (Slc10a6-/-) knockout mice show normal spermatogenesis and reproduction, but elevated serum levels for cholesterol sulfate.

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.

Membrane transporters for sulfated steroids in the human testis--cellular localization, expression pattern and functional analysis.

Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. However, a prerequisite for this mode of action would be a carrier-mediated import of the hydrophilic steroid sulfate molecules into specific target cells in reproductive tissues such as the testis. In the present study we detected predominant expression of the Sodium-dependent Organic Anion Transporter (SOAT), the Organic Anion Transporting Polypeptide 6A1, and the Organic Solute Carrier Partner 1 in human testis biopsies. All of these showed significantly lower or even absent mRNA expression in severe disorders of spermatogenesis (arrest at the level of spermatocytes or spermatogonia, Sertoli cell only syndrome). Only SOAT was significantly lower expressed in biopsies showing hypospermatogenesis. By use of immunohistochemistry SOAT was localized to germ cells at various stages in human testis biopsies showing normal spermatogenesis. SOAT immunoreactivity was detected in zygotene primary spermatocytes of stage V, pachytene spermatocytes of all stages (I-V), secondary spermatocytes of stage VI, and round spermatids (step 1 and step 2) in stages I and II. Furthermore, SOAT transport function for steroid sulfates was analyzed with a novel liquid chromatography tandem mass spectrometry procedure capable of profiling steroid sulfate molecules from cell lysates. With this technique, the cellular inward-directed SOAT transport was verified for the established substrates dehydroepiandrosterone sulfate and estrone-3-sulfate. Additionally, ß-estradiol-3-sulfate and androstenediol-3-sulfate were identified as novel SOAT substrates.

Cloning and functional characterization of the mouse sodium-dependent organic anion transporter Soat (Slc10a6).

The sodium-dependent organic anion transporter SOAT is a member of the Solute Carrier Family SLC10. In man, this carrier is predominantly expressed in the testis and has transport activity for sulfoconjugated steroid hormones. Here, we report on cloning, expression analysis and functional characterization of the mouse Soat (mSoat) and compare its characteristics with the human SOAT carrier. Quantitative mRNA expression analysis for mSoat in male mice revealed very high expression in lung and further high expression in testis and skin. Immunohistochemical studies showed expression of the mSoat protein in bronchial epithelial cells of the lung, in primary and secondary spermatocytes as well as round spermatids within the seminiferous tubules of the testis, in the epidermis of the skin, and in the urinary epithelium of the bladder. Stably transfected mSoat-HEK293 cells revealed sodium-dependent transport for dehydroepiandrosterone sulfate (DHEAS), estrone-3-sulfate, and pregnenolone sulfate (PREGS) with apparent Km values of 60.3µM, 2.1µM, and 2.5µM, respectively. In contrast to human SOAT, which has a preference for DHEAS as a substrate, mSoat exhibits the highest transport rate for PREGS, likely reflecting differences in the steroid pattern between both species. In conclusion, although certain differences between human SOAT and mSoat exist regarding quantitative gene expression in endocrine and non-endocrine tissues, as well as in the transport kinetics for steroid sulfates, in general, both can be regarded as homologous carriers.