Unraveling the molecular targets pertinent to junction restructuring events during spermatogenesis using the Adjudin-induced germ cell depletion model

Abstract

During spermatogenesis, extensive restructuring takes place at the Sertoli-Sertoli and Sertoli-germ cell interface, which is regulated via intriguing interactions among cytokines, proteases, protease inhibitors, kinases, phosphatases, and transcription factors. This in turn determines the steady-state levels of integral membrane proteins at the cell junctions. We sought to further expand these observations using the Adjudin model. Adjudin is a potential male contraceptive that targets Sertoli-germ cell adhesion, causing exfoliation of spermatids and spermatocytes, but not spermatogonia, from the seminiferous epithelium. This model thus provides the means to identify crucial regulatory molecules and signaling pathways pertinent to junction restructuring events during spermatogenesis. In this study, genome-wide expression profiling of rat testes after treatment with Adjudin at the time of extensive junction restructuring was performed. Differentially regulated genes, such as cytokines, proteases, protease inhibitors, cell junction-associated proteins, and transcription factors pertinent to junction restructuring were identified. These data were consistent with earlier findings; however, much new information was obtained which has been deposited at the Gene Expression Omnibus data repository website: http://www.ncbi.nih.gov/geo/ with Accession number: GSE5131. The primary signaling events pertinent to junction restructuring in the testis induced by Adjudin were also delineated using bioinformatics. These findings were also consistent with recently published reports. The identified molecular signatures or targets pertinent to junction dynamics in the testis as reported herein, many of which have not been investigated, thus offer a framework upon which the regulation of junction restructuring events at the Sertoli-Sertoli and Sertoli-germ cell interface pertinent to spermatogenesis can be further studied.

Testin and actin are key molecular targets of adjudin, an anti-spermatogenic agent, in the testis

“…Because testin is an ES protein, these findings explain the restricted action of adjudin in the testis. Adjudin apparently exerts its effects by binding to testin since adjudin 28,29 or its analog, gamendazole, 53 are known to upregulate testin expression in the testis, in turn perturbing actin, affecting adhesion and resulting in germ cell loss. Nevertheless, additional research is needed to better understand adjudin's mechanism of action in the testis.…”

“…[22][23][24][25][26][27] Interestingly, testin expression was shown to be transiently induced several-fold whenever apical ES function was disrupted, leading to aspermatogenesis and whenever spermatogenesis was programmed to return to normal. 28 Studies by gene profiling using testes from rats treated with adjudin versus methylcellulose (control) have also identified testin as one of the most induced genes; 29 the only other gene to be induced more than testin was wingless-type MMTV integration site family, member 4 (Wnt4) (Mruk DD, Cheng CY, unpublished observation). Similarly, gamendazole, an analog of adjudin, was also shown to stimulate testin expression in the adult testis by 3.2-, 17.6-and 52.4-fold versus control rats by 4, 12, and 24 hours, respectively, in a gene profiling study.…”

VGF: An inducible gene product, precursor of a diverse array of neuro-endocrine peptides and tissue-specific disease biomarkers

“…VGF mRNA was induced in the brain by erythropoietin treatment (Girgenti et al, 2009), in gastric epithelial cells upon Helicobacter pylori infection (Liu et al, 2006), and in the testis upon germ cell depletion, compared to normally very low levels in the latter organ (Xia et al, 2007). Conversely, a reduced expression of VGF mRNA was found in the carotid body upon chronic hyperoxia (Dmitrieff et al, 2011), and in breast cancer cell lines (Herná ndez-Vargas et al, 2006;Mitra et al, 2008), possibly via cancer-specific gene methylation (Ostrow et al, 2009), with an associated decrease of VGF products in the secreted proteome (Mitra et al, 2008).…”

Regulation of cell junction dynamics by cytokines in the testis—A molecular and biochemical perspective

“…Recent studies using genome wide gene profiling techniques and an in vivo animal model in which rats were treated with Adjudin to induce junction restructuring mimicking similar events that occur during spermatogenesis have identified several transcription factors, namely Egr-1 & -2, Bhlhb2, Jun, Nupr1, Pawr, c-fos, Atf3, Irf1, Myc, and Stat3, that are pertinent to these events [84]. The information obtained from such gene profiling and bioinformatics approach should be helpful to expand this area of research, unraveling the cytokine-mediated biochemical and molecular regulation of junction dynamics.…”