14-3-3 and its binding partners are regulators of protein-protein interactions during spermatogenesis

Abstract

During spermatogenesis, spermiation takes place at the adluminal edge of the seminiferous epithelium at stage VIII of the epithelial cycle during which fully developed spermatids (i.e. spermatozoa) detach from the epithelium in adult rat testes. This event coincides with the migration of preleptotene/leptotene spermatocytes across the blood-testis barrier from the basal to the apical (or adluminal) compartment. At stage XIV of the epithelial cycle, Pachytene spermatocytes (diploid, 2n) differentiate into diplotene spermatocytes (tetraploid, 4n) in the apical compartment of the epithelium, which begin meiosis I to be followed by meiosis II to form spermatids (haploid, 1n) at stage XIV of the epithelial cycle. These spermatids, in turn, undergo extensive morphological changes and traverse the seminiferous epithelium until they differentiate into elongated spermatids. Thus, there are extensive changes at the Sertoli-Sertoli and Sertoli-germ cell interface via protein 'coupling' and 'uncoupling' between cell adhesion protein complexes, as well as changes in interactions between integral membrane proteins and their peripheral adaptors, regulatory protein kinases and phosphatases, and the cytoskeletal proteins. These precisely coordinated protein-protein interactions affect cell adhesion and cell movement. In this review, we focus on the 14-3-3 protein family, whose members have different binding partners in the seminiferous epithelium. Recent studies have illustrated that 14-3-3 affects protein-protein interactions in the seminiferous epithelium, and regulates cell adhesion possibly via its effects on intracellular protein trafficking and cell-polarity proteins. This review provides a summary on the latest findings regarding the role of 14-3-3 family of proteins and their potential implications on spermatogenesis. We also highlight research areas that deserve attentions by investigators.

Figure 1

A study to assess the cellular localization of 14-3-3 in the seminiferous epithelium of adult rat testes. (A–E) Immunohisto-chemical localization of 14-3-3θ in normal adult rat testes, in which frozen cross sections (~7 μm thick) were immunostained using an anti-14-3-3θ IgG (Santa Cruz Biotechnology, Cat. Sc-732; Lot. A2908) (A and C–E) or normal rabbit IgG (B). Low magnification of testes stained with anti-14-3-3θ polyclonal antibody (A) or normal rabbit IgG (B) illustrating the presence of 14-3-3θ in the seminiferous epithelium. In higher magnification, intense staining is obvious surrounding the heads of elongating spermatids at the apical ES site (C and E) as well as round spermatids (D) and at the basal compartment consistent with its presence at the BTB. Bar in (A)=100 μm, which applies to (B), bar in (C)=30 μm, which applies to (D and E), bar in inset in (C)=15 μm, which applies to insets in (D and E) and (F). A study by immunofluorescence microscopy further illustrates the localization of 14-3-3θ in seminiferous epithelium of normal adult rat testes, by using frozen testis cross sections (~7 μm thick) stained with anti-14-3-3θ IgG. CY3-conjugated donkey anti-rabbit secondary antibody (red fluorescence) was used for visualizing 14-3-3θ, confirming the existence of 14-3-3θ at the apical ES in the seminiferous epithelium in a stage VII tubule. Cell nuclei were stained by DAPI. Bar in (F)=12 μm. The Roman numeral after C, D, E and F represents the seminiferous epithelial stage of the tubule.

Figure 2

The three possible regulatory functions of 14-3-3 in the testis. (A) Effects on intracellular trafficking. 14-3-3 binding modulates endoplasmic reticulum (ER) transportation by masking the COPII (or COPI) binding sites on cargo proteins. (B) Effects on cell-junction dynamics. 14-3-3 participates in the integrin-activated signaling pathways via p130^Cas. (C) Effects on cell polarity. Par1 catalyzes the phosphorylation of Par3, leading to 14-3-3 binding that inhibits the formation of Par3/Par6/aPKC complex. In turn, Par1 can be phosphorylated by aPKC (atypical protein kinase C) and bound with 14-3-3 as inactive form.

Figure 3

A hypothetical model illustrating the possible regulatory role of 14-3-3 during BTB restructuring at stage VIII of the seminiferous epithelial cycle of spermatogenesis. The direct contact between the Par3/Par6/aPKC complex and JAM-A stabilizes the BTB, conferring an intact barrier. At stage VIII of the epithelial cycle, the degradation or endocytosis of JAM-A (dashed line) induces the opening of BTB. This diminishing level of JAM-A at the BTB may be contributed by the disassembly of the Par3/Par6/aPKC protein complex, because of the masking of Par3 and/or Par6 by 14-3-3 protein.