Figure 1.
A Pearson correlation analysis was performed on fold-change values of 123 differentially expressed genes that were retrieved after comparing ours and Beissbarth et al datasets [8]. A correlation coefficient of r = 0.885 shows a high correlation between both datasets.
Table 1.
qPCR data validation.
Figure 2.
Differentially expressed (DE) genes were compared to the Crem ChIP-seq dataset from Martianov et al [9] from 2010 (dataset of genes that are bound by Crem in testis), the Affymetrix MG U 74A dataset from Beissbarth et al [8] from 2003 (dataset of DE genes between WT and KO mice testis) and the TFCat dataset from Fulton et al [38] (a hand curated database of transcriptional factors). A – DE genes that are bound by Crem; B – DE genes that are transcriptional factors; C – DE genes that are transcriptional factors bound by Crem; D, E and F are genes common to both analysis using Affymetrix microarrays. Numbers represent the number of genes in each dataset or the number of common genes between comparisons.
Figure 3.
Euler diagrams representing genes from different datasets.
Euler diagrams were drawn to visualize the comparisons of genes from different datasets. The size of the circles corresponds to the number of genes present in each dataset. Three comparisons were made in order to retrieve the data for further functional analysis of DE genes.
Figure 4.
Functional characterization of genes deregulated in Crem KO mice.
Biological processes deregulated in Crem KO mice together with the total number of genes deregulated in each process (A) and the number and relative level of DE genes that are bound by CREM (B).
Table 2.
Differentially expressed genes essential for spermatogenesis, epididymal maturation and fertilization.
Figure 5.
Apoptosis induction pathways in round spermatids.
Synthesis of TNFα by germ cells induces the expression of Fas ligand on their surface which in turn activates the Fas receptor and caspase 8 or Ask1 mediated apoptosis in germ cells. The production of TNFα is mediated by the up-regulation of metalloproteinase's such as Timp1. Similar to Ask1, BAD also represses the action of the anti-apoptotic protein Bcl-2. BAD's activity is inhibited by phosphorylation with PKA which is down regulated in KO testis. Other apoptosis induction pathways through calpain and fodrin are also possible.
Figure 6.
Steroidogenesis, melatonin and the circadian clock.
Our data showed that several genes involved in steroid hormone synthesis (Cyp11a1, Hsd17b3, Hsd3b6, Srd5a1) and cholesterol transport (Star, Tsop, Scp2) are up-regulated in testes of Crem KO mice. On the other hand genes involved in the production of estrogens (Cyp19a1) and up-take of cholesterol (Scarb1 and Lipe) are down-regulated. Many of these genes are under the control of both circadian factors (Bmal1 and Per1) and the hormone melatonin. Melatonin can exert its effect either through the membrane receptor Mtnr1a or inside the cells through yet unresolved ways. Figure 6 also depicts the regulation of melatonin synthesis in the pineal gland. Here the main regulatory enzyme of melatonin synthesis Aanat is activated by phosphorylated CREB and inhibited by ICER, which in absent Crem KO animals.
Table 3.
Genes involved in cholesterol and androgen transport, synthesis and signal transduction.
Figure 7.
Two cell-cell junctions present between Sertoli and germ cells are presented: A - desmosome-like junctions and B - Ectoplasmic specialization. In DJ the down regulation of desmoglein and plakoglobin could lead to destabilization and separation of germ cells from Sertoli cells as seen by [16]. B - Apical EC are present between elongating or elongated spermatids and Sertoli cells. Down regulation of both germ and Sertoli specific nectin as well as its adaptor proteins that connect it to F-actin shows destabilization of the ES junction complex.
Table 4.
Proteins affecting actin and microtubule dynamics in testis.