Testis and blood-testis barrier in Covid-19 infestation: role of angiotensin-converting enzyme 2 in male infertility.

Severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) that causes COVID-19 infections penetrates body cells by binding to angiotensin-converting enzyme-2 (ACE2) receptors. Evidence shows that SARS-CoV-2 can also affect the urogenital tract. Hence, it should be given serious attention when treating COVID-19-infected male patients of reproductive age group. Other viruses like HIV, mumps, papilloma and Epstein-Barr can induce viral orchitis, germ cell apoptosis, inflammation and germ cell destruction with attending infertility and tumors. The blood-testis barrier (BTB) and blood-epididymis barrier (BEB) are essential physical barricades in the male reproductive tract located between the blood vessel and seminiferous tubules in the testes. Despite the significant role of these barriers in male reproductive function, studies have shown that a wide range of viruses can still penetrate the barriers and induce testicular dysfunctions. Therefore, this mini-review highlights the role of ACE2 receptors in promoting SARS-CoV-2-induced blood-testis/epididymal barrier infiltration and testicular dysfunction.

Matrix metalloproteinase 9 facilitates Zika virus invasion of the testis by modulating the integrity of the blood-testis barrier.

Zika virus (ZIKV) is a unique flavivirus with high tropism to the testes. ZIKV can persist in human semen for months and can cause testicular damage in male mice. However, the mechanisms through which ZIKV enters the testes remain unclear. In this study, we revealed that matrix metalloproteinase 9 (MMP9) was upregulated by ZIKV infection in cell culture and in A129 mice. Furthermore, using an in vitro Sertoli cell barrier model and MMP9-/- mice, we found that ZIKV infection directly affected the permeability of the blood-testis barrier (BTB), and knockout or inhibition of MMP9 reduced the effects of ZIKV on the Sertoli cell BTB, highlighting its role in ZIKV-induced disruption of the BTB. Interestingly, the protein levels of MMP9 were elevated by ZIKV nonstructural protein 1 (NS1) in primary mouse Sertoli cells (mSCs) and other cell lines. Moreover, the interaction between NS1 and MMP9 induced the K63-linked polyubiquitination of MMP9, which enhanced the stability of MMP9. The upregulated MMP9 level led to the degradation of essential proteins involved in the maintenance of the BTB, such as tight junction proteins (TJPs) and type Ⅳ collagens. Collectively, we concluded that ZIKV infection promoted the expression of MMP9 which was further stabilized by NS1 induced K63-linked polyubiquitination to affect the TJPs/ type Ⅳ collagen network, thereby disrupting the BTB and facilitating ZIKV entry into the testes.

Localization of nucleoside transporters in rat epididymis.

The epididymis relies on transporters for the secretion of nucleosides and influence the disposition of nucleoside analogs (NSA). Since these compounds can cross the blood-testis barrier (BTB), it is important to understand if the epididymis reabsorbs NSA drugs. The purpose of this study is to determine the localization of nucleoside transporters expressed within rat epididymis to demonstrate the potential of epididymal reabsorption. Using immunohistochemistry, we determined that equilibrative nucleoside transporter 1 (ENT1) is localized to the basolateral membrane of epithelial cells, ENT2 is expressed in the nucleus of the epithelium and CNT2 is expressed by basal cells. The expression pattern for these transporters suggests that nucleosides are able to access the epithelial cells of the epididymal duct via the blood, but not from the lumen. We did not find any evidence for a transepithelial reabsorption pathway indicating the NSA drugs that cross the BTB remain within the epididymis.

Focal adhesion kinase-Tyr407 and -Tyr397 exhibit antagonistic effects on blood-testis barrier dynamics in the rat.

Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, displays phosphorylation-dependent localization in the seminiferous epithelium of adult rat testes. FAK is an integrated component of the blood-testis barrier (BTB) involved in regulating Sertoli cell adhesion via its effects on the occludin-zonula occludens-1 complex. Herein, we report that p-FAK-Tyr(407) and p-FAK-Tyr(397) display restricted spatiotemporal and almost mutually exclusive localization in the epithelium, affecting BTB dynamics antagonistically, with the former promoting and the latter disrupting the Sertoli cell tight junction-permeability barrier function. Using primary cultured Sertoli cells as an in vitro model that mimics the BTB in vivo both functionally and ultrastructurally, effects of FAK phosphorylation on BTB function were studied by expressing nonphosphorylatable and phosphomimetic mutants, with tyrosine replaced by phenylalanine (F) and glutamate (E), respectively. Compared with WT FAK, Y407E and Y397F mutations each promoted barrier function, and the promoting effect of the Y407E mutant was abolished in the Y397E-Y407E double mutant, demonstrating antagonism between Tyr(407) and Tyr(397). Furthermore, Y407E mutation induced the recruitment of actin-related protein 3 to the Sertoli cell-cell interface, where it became more tightly associated with neuronal Wiskott-Aldrich syndrome protein, promoting actin-related protein 2/3 complex activity. Conversely, Y407F mutation reduced the rate of actin polymerization at the Sertoli cell BTB. In summary, FAK-Tyr(407) phosphorylation promotes BTB integrity by strengthening the actin filament-based cytoskeleton. FAK serves as a bifunctional molecular "switch" to direct the cyclical disassembly and reassembly of the BTB during the epithelial cycle of spermatogenesis, depending on its phosphorylation status, to facilitate the transit of preleptotene spermatocytes across the BTB.

C-Src and c-Yes are two unlikely partners of spermatogenesis and their roles in blood-testis barrier dynamics.

Src family kinases (SFKs), in particular c-Src and c-Yes, are nonreceptor protein tyrosine kinases that mediate integrin signaling at focal adhesion complex at the cell-extracellular matrix interface to regulate cell adhesion, cell cycle progression, cell survival, proliferation and differentiation, most notably in cancer cells during tumorigenesis and metastasis. Interestingly, recent studies have shown that these two proto-oncogenes are integrated components of the stem cell niche and the cell-cell actin-based anchoring junction known as ectoplasmic specialization (ES) at the: (1) Sertoli cell-spermatid interface known as apical ES and (2) Sertoli-Sertoli cell interface known as basal ES which together with tight junctions (TJ), gap junctions and desmosomes constitute the blood-testis barrier (BTB). At the stem cell niche, these SFKs regulate spermatogonial stem cell (SSC) renewal to maintain the proper population of SSC/spermatogonia for spermatogenesis. At the apical ES and the BTB, c-Src and c-Yes confer cell adhesion either by maintaining the proper phosphorylation status of integral membrane proteins at the site which in turn regulates protein-protein interactions between integral membrane proteins and their adaptors, or by facilitating androgen action on spermatogenesis via a nongenomic pathway which also modulates cell adhesion in the seminiferous epithelium. Herein, we critically evaluate recent findings in the field regarding the roles of these two unlikely partners of spermatogenesis. We also propose a hypothetical model on the mechanistic functions of c-Src and c-Yes in spermatogenesis so that functional experiments can be designed in future studies.

Regulation of blood-testis barrier dynamics by focal adhesion kinase (FAK): an unexpected turn of events.

The blood-testis barrier (BTB) is conferred by co-existing tight junctions (TJs), basal ectoplasmic specializations (basal ES), desmosome-like junctions and gap junctions (GJs) between adjacent Sertoli cells near the basement membrane in the seminiferous epithelium. While the concept of the BTB has been known for more than a century and its significance to spermatogenesis discerned for more than five decades, its regulation has remained largely unknown. Recent studies, however, have demonstrated that focal adhesion kinase (FAK), a modulator of the integrin-based signaling that plays a crucial role in cell movement, apoptosis, cell survival and gene expression at the focal adhesion complex (FAC, also known as focal contact, a cell-matrix anchoring junction type), is an integrated component of the BTB, associated with the TJ-integral membrane protein occludin and its adaptor zonula occludens-1 (ZO-1). Herein, we summarize recent findings in the field regarding the significance of FAK in conferring BTB integrity based on some unexpected observations. We also critically discuss the role of FAK in regulating the timely "opening" and "closing" of the BTB to facilitate the transit of primary preleptotene spermatocytes across the BTB at stage VIII of the seminiferous epithelial cycle of spermatogenesis. Lastly, we describe a working model, which can be used to design future functional experiments to explore the involvement of FAK in BTB dynamics by investigators in the field.

Disruption of blood-testis barrier dynamics in ether-lipid-deficient mice.

One of the major roles of Sertoli cells is to establish the blood-testis (Sertoli cell) barrier (BTB), which is permanently assembled and disassembled to accommodate the translocation of leptotene spermatocytes from the basal into the adluminal compartment of the seminiferous epithelium and to guarantee completion of meiosis and spermiogenesis. Recently, we have demonstrated spermatogenesis to be arrested before spermatid elongation in Gnpat-null mice with selective deficiency of ether lipids (ELs) whose functions are poorly understood. In this study, we have focused on the spatio-temporal expression of several BTB tight-junctional proteins in the first wave of spermatogenesis to obtain insights into the physiological role of ELs during BTB establishment and dynamics. Our data confirm the transient existence of Russell's intermediate or translocation compartment delineated by two separate claudin-3-positive luminal and basal tight junctions and reveal that EL deficiency blocks BTB remodeling. This block is associated with (1) downregulation and mistargeting of claudin-3 and (2) impaired BTB disassembly resulting in deficient sealing of the intermediate compartment as shown by increased BTB permeability to biotin. These results suggest that ELs are essential for cyclic BTB dynamics ensuring the sluice mechanism for leptotene translocation into the adluminal compartment.