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.

Figure 1

Morphological analysis of testes after Adjudin treatment and a summary of the gene expression profiles in testes at the time of extensive junction restructuring at the Sertoli–germ cell interface. (A; a–c). Paraffin sections of testes stained with hematoxylin in normal (Ctrl) rats (a) versus 8 h (b) and day 4 (c) after Adjudin treatment, spermatids were still loosely attached to Sertoli cells in the seminiferous epithelium by 8 h (b) post-treatment even though elongating spermatids and spermatocytes were found in lumen of most tubules examined versus day 4 (c), when virtually all spermatids were depleted from the epithelium,and control (Ctrl, normal) testes. Bar in a=40 μm which applies to (b and c); bar in inset in a=150 μm, which applies to insets in (b and c). (d) Changes in testis weight (per organ pair) after Adjudin treatment as a result of germ cell loss (n=5). Percentage of testis weight in comparison with control (Ctrl, normal rats) testes was also annotated. ns, Not significantly different from control (normal) rats by ANOVA; *P<0.01. (e) Each of the total of 31 099 probe sets on the Affymetrix GeneChip Rat Genome 230 2.0 array, was assigned a Flag to indicate its presence (P), marginal (M), or absence (A) based on the signal detected. The solid bar summarized the mean percentage of genes in each of the three groups from a total of eight samples (n=3 for control testes, n=2 for 8 h and n=3 for 4 days after Adjudin treatment). All the raw microarray data have been deposited at the Gene Expression Omnibus (GEO) data repository website: http://www.ncbi.nih.gov/geo/ with the accession number: GSE5131. (f and g) Normalized expression level of each gene at 8 h or at day 4 was plotted against its raw expression level (signal intensity) in control (Ctrl) testes. Each dot is a mean of data from two (8 h) or three (Ctrl and day 4) gene chips using different samples for hybridization. The two green lines above and below the green line marked at 1 represent a twofold change, namely induction or suppression, in gene expression level versus control. The color scheme (red signifies an increased expression and blue decreased expression) shown herein is the same as in Fig. 4A. (B) Venn diagram summarizing the number of genes with at least twofold changes in expression levels at 8 h and at day 4 from a total of 1466 transcripts that were found to be differentially regulated by one-way ANOVA.

Figure 2

Real-time RT-PCR quantification of the steady-state mRNA levels of selected target genes after Adjudin treatment by 8 h and day 4 versus control (Ctrl, normal testes). Note that the relative mRNA levels (fold changes against control) determined by real-time PCR for these nine genes were consistent with results of microarray experiments (see Tables 2–4). The activation of selected target genes during Adjudin-induced germ cell loss from the seminiferous epithelium as described herein have not previously been reported. ns, Not significantly different by ANOVA, *P<0.05; ^†P<0.01.

Figure 3

A study by immunoblotting and immunohistochemistry to assess changes in selected target proteins in rat testes after Adjudin treatment to validate microarray results. (A; a) Immunoblot analysis of cathepsin L in control (Ctrl), 8-h and 4-day rat testes. (b–d) Immunohistochemistry localization of cathepsin L in control, 8-h and 4-day testis respectively. Bar in b=70 μm, also applies to (c–d), as well as (b–d) in (B), (C) and (D). (B–D) Corresponding immunoblot analysis and immunohistochemistry results for α₂-macroglobulin (α₂-MG), testin, and claudin 3 respectively, similar to those shown in (A). The induction of testin during Adjudin-induced germ cell loss from the testis is consistent with earlier reports (Grima et al. 1997, Cheng et al. 2001), but changes of α₂-MG and cathepsin or the unresponsiveness of claudin 3 have not been reported.

Figure 4

Hierarchical clustering of 1466 differentially regulated transcripts in rat testes from eight samples (n=3 for control (Ctrl) testes, n=2 for 8 h, n=3 for day 4 following Adjudin treatment). (A) Expression fold changes are depicted by the color map in the left panel. The dendrogram in the right panel illustrates that these 1466 probe sets displayed consistent patterns of up- or down-regulation by 8 h or 4 days after Adjudin treatment versus controls. Note that these patterns are consistent across different chips using different batches of samples within the same group. Five domains can be identified (a–e) that comprise the entire spectrum, which are also the five subtrees in the dengrogram (branches were removed without distracting the main diagram). These domains can be further grouped into two categories of i and ii, the subtrees at a higher level, which show a more pronounced mRNA induction at day 4 than 8 h (i) and vice versa (ii). Two boxed areas shown in (A) were selected and examined in detail and shown in (B) (top panel) and (C) (bottom panel). (B) Selected genes with significantly increased expression at day 4 but not at 8 h. (C) Selected genes with significant reduced expression at 8 h but not at day 4. The gene/Affymetrix IDs are listed on the right side of each row.