Implications of Species Differences in Function and Localization of Transporters at the Blood–Testis Barrier

The testis is the principal site of male reproductive development and regulation of the chemical environment that supports spermatogenesis and is tightly controlled by a selectively permeable barrier. The blood–testis barrier is composed of adjacent Sertoli epithelial cells that form intercellular junctional complexes to limit the movement of chemicals from the blood into Sertoli cells or further into the adluminal compartment of seminiferous tubules (Dym and Fawcett, 1970). Sertoli cells directly interact with and support developing germ cells by supplying them with nutrients. Nutrients bypass the blood–testis barrier through several mechanisms including passive diffusion, receptor-mediated endocytosis, and transporter-mediated uptake. Many chemicals do not readily diffuse across cell membranes and receptors interact with a limited number of ligands. Consequently, membrane transporters facilitate the flux of many hydrophilic chemicals across Sertoli cell membranes. The transepithelial flux of these chemicals requires basal uptake from the blood into Sertoli cells and subsequent apical efflux into the adluminal compartment. However, these transporters also inadvertently allow toxicants to cross and elicit male reproductive toxicity. Some progress has been made in identifying the physiological and pharmacological roles of transporters at the blood–testis barrier in the past decade, although there are still many questions to be answered.

Two fundamental, unanswered questions include: (1) are there differences in blood–testis barrier transporter localization or function between species, and (2) how do these differences impact physiological or pharmacological outcomes in the testis? The lack of human transporter orthologs in model species and differences in substrate selectivity and kinetics poses serious implications when translating findings from pre-clinical models into human subjects (Chu et al., 2013). Although several rodent orthologs exist for various transporters, some transporters such as organic anion transporter 4 are only expressed in humans and do not have a known rodent ortholog. But perhaps the most important factor to consider is the localization and overall function of these transporters between species.

The localization and function of several transporters at the human and rodent blood–testis barrier have been reported, including the equilibrative nucleoside transporters (ENTs). Nucleoside and nucleoside analog uptake into Sertoli cells occur through ENT1 at the basal membrane and efflux into the adluminal compartment occurs through ENT2 at the apical membrane (Klein et al., 2013) in rodents and humans. Therefore, chemicals that are ENT substrates are likely to show similar transport profiles in rodents and humans due to their localization. Typical ENT substrates include many cancer chemotherapeutics and antivirals that may circumvent the blood–testis barrier to effectively treat cancers and viral infections. A targeted therapy approach using drugs that are ENT substrates may lead to a reduced likelihood of cancer relapse in the testis, where the current standard of care is orchiectomy or radiation therapy. These radical approaches leave patients with severely reduced or complete loss of fertility. In addition to treating cancers in the testis, sexually transmitted viruses such as the human immunodeficiency viruses, herpes simplex viruses, or Zika viruses are major targets for drug development. Due to the protective effects of the blood–testis barrier, an unintended consequence is that viral loads can persist in the testis. Antivirals that cross the blood–testis barrier may eliminate a viral sanctuary site, thereby diminishing the possibility of viral transmission from semen. Most importantly, the development of chemotherapeutics and antivirals that effectively penetrate the blood–testis barrier can be conveniently evaluated in rodents and translated into the clinic.

In addition to the ENTs, many of the ATP-binding cassette efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and the multidrug resistance-associated proteins (MRPs) at the blood–testis barrier have been studied in rodents and humans (Bart et al., 2004; Klein et al., 2014). However, there have been conflicting reports on the localization of MRP4 between species, which may impact the use of animal models and the interpretation of the data collected from reproductive toxicity studies. In humans, MRP4 was observed to be localized to the Leydig cells and basal membrane of human and macaque Sertoli cells in three separate studies (Huang et al., 2016; Klein et al., 2014; Morgan et al., 2012). The basal membrane expression is consistent with its barrier function to protect germ cell development. However, MRP4 was observed to be localized to the apical membranes of mouse and rat Sertoli cells (Klein et al., 2014). Because MRP4 is localized to the apical membrane in rodents, the translational application of most data obtained from pre-clinical reproductive toxicity studies using anion drug substrates is misleading. Toxicants that are MRP4 substrates may gain access to the adluminal compartment in rodents to cause greater reproductive toxicity when compared to humans because apical efflux facilitates accumulation in rodent testis, whereas human MRP4 function would restrict access. As a result, drugs may prematurely fail in the pre-clinical phase. Conversely, drugs being developed in rodent models to treat disorders within the male reproductive tract may fail when applied to humans because human MRP4 acts in a barrier function.

These findings are in contrast to other studies which reported Sertoli cell basal membrane and Leydig cell localization in rats and mice (Koraichi et al., 2013; Morgan et al., 2012). Interestingly, one of these studies noted that MRP4 redistributes from the basal membrane to the apical membrane of rat Sertoli cells upon neonatal treatment with an estrogenic mycotoxin, zearalenone (Koraichi et al., 2013). The localization of P-gp, BCRP, MRP1, and MRP5 were unaffected by zearalenone exposure, although MRP4 redistribution suggests that it is plausible that other factors may influence the localization of MRP4 or other transporters in the testis. These data may give credence to the observations made by Klein and colleagues; however, it does not completely explain the inconsistencies between the studies described here. Due to these inconsistencies, the interpretation of reproductive toxicity data must be analyzed cautiously when using rodent models to guide future pre-clinical and clinical studies.

In summary, additional studies are required to correctly identify the localization and function of MRP4 and other transporters in different species. It is imperative that these data are collected to guide pre-clinical and clinical drug development to save drugs from unwarranted failure. Ongoing and future studies will look to answer these questions and new models will be employed to guide translational sciences.

FUNDING

This work was supported by funding from the National Institutes of General Medical Sciences R01 GM123643 and National Institute of Environmental Health Sciences P30 ES006694 and T32 ES007091.

DECLARATION OF CONFLICTS OF INTEREST

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.