Rabbit Anti-Human S6 monoclonal antibody, clone 0I9M3 [Alexa Fluor 488]

During spermatogenesis, the blood-testis barrier (BTB) undergoes cyclic remodeling that is crucial to support the transport of preleptotene spermatocytes across the immunological barrier at stage VIII to IX of the epithelial cycle. Studies have shown that this timely remodeling of the BTB is supported by several endogenously produced barrier modifiers across the seminiferous epithelium, which include the F5-peptide and the ribosomal protein S6 [rpS6; a downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1)] signaling protein. Herein, F5-peptide and a quadruple phosphomimetic (and constitutively active) mutant of rpS6 [i.e., phosphorylated (p-)rpS6-MT] that are capable of inducing reversible immunological barrier remodeling, by making the barrier "leaky" transiently, were used for their overexpression in the testis to induce BTB opening. We sought to examine whether this facilitated the crossing of the nonhormonal male contraceptive adjudin at the BTB when administered by oral gavage, thereby effectively improving its BTB transport to induce germ cell adhesion and aspermatogenesis. Indeed, it was shown that combined overexpression of F5-peptide and p-rpS6-MT and a low dose of adjudin, which by itself had no noticeable effects on spermatogenesis, was capable of perturbing the organization of actin- and microtubule (MT)-based cytoskeletons through changes in the spatial expression of actin- and MT-binding/regulatory proteins to the corresponding cytoskeleton. These findings thus illustrate the possibility of delivering drugs to any target organ behind a blood-tissue barrier by modifying the tight junction permeability barrier using endogenously produced barrier modifiers based on findings from this adjudin animal model.

During early pregnancy, a well-coordinated communication network between the conceptus and maternal uterus is especially crucial in pigs in which there is a protracted pre-attachment phase prior to implantation. This network is regulated by an astonishing number of molecules such as growth factors. Fibroblast growth factor 4 (FGF4) is a multipotent growth factor that elicits diverse biological actions on various types of cells and tissues. In pigs, FGF4 and its receptors are expressed in the uterine endometrium and conceptus during early pregnancy, but less is known about the FGF4-mediated regulation of conceptus growth during peri-implantation period of pregnancy. Therefore, the aims of the present study were to investigate: 1) expression of endometrial FGF4 mRNA during early pregnancy; 2) up-regulation of FGF receptor expression in porcine trophectoderm (pTr) cells in response to FGF4; and 3) FGF-induced intracellular signaling and cellular activities in pTr cells. In vitro cultured pTr cells incubated with different concentrations of recombinant FGF4 (0-50 ng/ml) responded with a dose-dependent increase in AKT phosphorylation of 2.9-fold at 20 ng/ml FGF4. Within 30 min after treatment with 20 ng/ml FGF4, the abundances of p-AKT, p-P90RSK and p-RPS6 proteins increased 2.1-, 5.2- and 3.2-fold, respectively, and then returned to basal levels by 120 min. To ensure that the stimulatory effect of FGF4 on AKT signaling was p-AKT-dependent, pTr cells were pre-incubated with an AKT inhibitor (LY294002) for 1 h prior to FGF4 treatment. 20 mu M of LY294002 decreased FGF4-induced p-AKT, p-P9ORSK and p-RPS6 proteins. Immunofluorescence analyses revealed that p-RPS6 proteins were abundant within the cytoplasm of FGF4-treated cells, but present at basal levels in the presence of LY294002. Furthermore, FGF4 increased migration of pTr cells and LY294002 significantly reduced this effect. Results of the present study suggest that activation of the FGF receptor(s) on trophectoderm cells by FGF4 secreted by conceptus/endometrium transduces its signal through the phosphatidylinositol 3-kinase (PI3K)/AKT pathway which is linked to migration of trophectoderm cells that is critical to development of the porcine conceptus. (C) 2015 Elsevier Ireland Ltd. All rights reserved.