Src family kinases (SFKs) and cell polarity in the testis

Abstract

Non-receptor Src family kinases (SFKs), most notably c-Src and c-Yes, are recently shown to be expressed by Sertoli and/or germ cells in adult rat testes. Studies have shown that SFKs are involved in modulating the cell cytoskeletal function, and involved in endocytic vesicle-mediated protein endocytosis, transcytosis and/or recycling as well as intracellular protein degradation events. Furthermore, a knockdown to SFKs, in particular c-Yes, has shown to induce defects in spermatid polarity. These findings, coupled with emerging evidence in the field, thus prompt us to critically evaluate them to put forth a developing concept regarding the role of SFKs and cell polarity, which will become a basis to design experiments for future investigations.

Keywords: Cell polarity; Sertoli cells; Spermatids; Spermatogenesis; Src family kinases; Testis; c-Src; c-Yes.

Figure 1. A schematic drawing that illustrates the general morphological features of Sertoli and germ cells in the epithelium of the seminiferous tubule

The blood-testis barrier (BTB) physically divides the seminiferous epithelium into the adluminal (apical) compartment and the basal compartment, which in turn lays on the basement membrane of the tunica propria. Undifferentiated and differentiated spermatogonia are found in the basal compartment. Type B spermatogonia differentiate into preleptotene spermatocytes, which are the germ cells that are being transported across BTB while transforming to leptotene spermatocytes to enter the adluminal compartment to form zygotene and pachytene spermatocytes to prepare for meiosis I/II. The most notable anchoring junction in the testis is the actin-rich adherens junction (AJ) type called ectoplasmic specialization (ES), which is typified by the presence of an array of actin filament bundles found in the Sertoli cell near the plasma membrane and these bundles are sandwiched between the cisternae of endoplasmic reticulum and the apposing Sertoli cell-cell or Sertoli-spermatid plasma membranes. ES is either found at the Sertoli cell-cell interface called basal ES. The basal ES together with the tight junction (TJ) and gap junction all utilize F-actin for attachment. These actin-based junctions together with the intermediate filament-based desmosome constitute the BTB. At the Sertoli cell-spermatid (step 8–19 spermatids in the rat testis) interface, the ES is designated apical ES. The ES is an important structure to support Sertoli cell adhesion at the BTB and also spermatid adhesion. However, the ES is also an important cellular structure to support and Sertoli cell and spermatid polarity, and also spermatid PCP (planar cell polarity) during spermatogenesis.

Figure 2. A schematic drawing that illustrates the various function domains that are conserved among members of the SFK, and the conversion between the active and inactive state of SFKs

Members of SFK (e.g., c-Src, c-Yes) consist of 4 SH (src-homology) domains: SH4 at the N-terminal region, followed by SH3 and SH2, and then a SH1 domain containing the catalytic kinase domain which is linked to the SH2 domain via a short polyproline type II helix called SH2-SH1 linker. In the SH4 domain of c-Src and c-Yes, there is a penultimate glycine (Gly2) residue co-translationally myristoylated (M) to be used for membrane targeting. Also, a cysteine residue (Cys3) near the N-terminus of c-Yes is post-translationally palmitoylated (P) to assist subcellular trafficking of a SFK protein (e.g., facilitating membrane association of c-Yes following palmitoylation). (B) Under normal physiological conditions, SFK is auto-inhibited through intramolecular interactions wherein the SH2 domain binds to the inhibitory phosphotyrosine at the C-terminal tail, while the SH3 domain interacts with the SH2-SH1 polyproline linker so that these two protein interacting motifs block the kinase domain and stabilize SFK in an inactive conformation (right). SFKs are activated through binding of a ligand to the SH2 and SH3 domain, or dephosphorylation of the inhibitory phosphotyrosine by protein tyrosine phosphatases. This leads to a conformational change which allows phosphorylation of the stimulatory tyrosine in the activation loop and to confer intrinsic kinase activity in kinase domain (SH1) (left).

Figure 3. Defects in spermatid polarity following c-Yes knockdown by RNAi

Testes from adult rats received a transfection mixture containing 100 siRNA duplexes specific to c-Yes on day 1 and day 3, i.e., 2 transfections for RNAi to knockdown c-Yes by >70% as described [27]. Control testes received the same amount of non-targeting negative control siRNA duplexes. On day 5, rats were euthanized and testes were removed to freeze in liquid nitrogen. Frozen sections were obtained and cell nuclei stained for DAPI (4′,6-diamidino-2-phenylindole). As noted herein, a knockdown of c-Yes, but not in control testes received the negative non-targeting control siRNA duplexes, caused extensive defects in spermatid polarity in which the head of spermatids no longer pointed towards the basement membrane, but deviating by 90° to 180° from the intended orientation (annotated by white arrowheads). Scale bar, 80 μm and 40 μm in the left and right panel.

Figure 4. A model illustrating the role of c-Src and c-Yes to support germ cell transport and polarity

In this hypothetical model, the BTB integrity is maintained by SFKs such as c-Yes and c-Src such as at stage VII of the epithelial cycle (left panel). For instance, it was shown that c-Src associates with CAR [48], whereas c-Yes associates with occludin and N-cadherin [26]. However, at stage VIII of the epithelial cycle, these two SFKs promote protein endocytosis in which c-Yes targets the endocytosed proteins (e.g., occludin, N-cadherin, CAR) to be recycled to the base of the preleptotene spermatocyte to support the reassembly of a “new” BTB before the “old” BTB is disassembled. At the same time, c-Src promotes the endocytosed proteins to undergo endosome-mediated protein degradation as recently reported [47] (middle panel). As such, the BTB integrity is maintained as noted in the right panel. As noted in Figure 3, SFKs (e.g., c-Yes) also promotes germ cell polarity which may also facilitate the directional transport of preleptotene spermatocytes across the BTB as noted herein.