Table 1.
Phenotypical and histological description of the testicular samples included in the study.(a).
Figure 1.
DNA methylation microarray analysis determined disease-associated profiles.
(A) Unsupervised hierarchical clustering separated testis with a complete absence of germ cells (SCO) from those with the presence of germ cell lineage, and testis with conserved spermatogenesis (CS) from those with spermatogenic failure (SpF) human samples. (B) Hierarchical clustering of CS, SpF and SCO samples, displaying the 633 CpG sites differentially methylated between CS and SpF samples. (C) Hierarchical clustering of PIWIL1/2 and TDRD1/9 involved in the piRNA processing machinery. Sample number corresponding to that in Table 1 is also indicated.
Figure 2.
PIWIL2 and TDRD1 become more methylated in human infertility syndromes.
Bisulfite sequencing of the piRNA processing genes PIWIL1 (A), PIWIL2 (B), TDRD1 (C) and TDRD9 (D). Black and white squares indicate CpG methylation and unmethylated sites, respectively. One representative sample of a large difference between testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF) or at the round spermatid (rsMF) stage, and in Sertoli cell-only syndrome (SCO) are displayed. Sample number corresponding to that in Table 1 is also indicated. (E) Methylation level of gene promoters of PIWIL1, PIWIL2, TDRD1 and TDRD9 in testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF) or at the round spermatid (rsMF) stage, and in Sertoli cell-only syndrome (SCO) samples. Independent data are also shown in Figure S1 and S2. Significant differences compared to CS samples are indicated (*).
Figure 3.
SpF samples show a PIWIL2 and TDRD1 hypermethylation pattern.
Methylation levels of PIWIL2 (A) and TDRD1 (B) in mature spermatozoa (sperm), testis with a conserved spermatogenic pattern (CS), maturation failure at the round spermatid (rsMF), the spermatocyte (scMF) and the spermatogonia (sgMF) stages, Sertoli cell-only syndrome (SCO) and somatic tissue measured by pyrosequencing. The black bar indicates the mean methylation level. Methylation per cell profiling of PIWIL2 and TDRD1, displayed as methylation per somatic cell (x100) (C, D) and methylation per germ cell (x100) (E, F) in testis with conserved spermatogenesis (CS), maturation failure at the round spermatid (rsMF), the spermatocyte (scMF) and the spermatogonia (sgMF) stages and Sertoli cell-only syndrome (SCO). The horizontal bar displays the mean cellular expression level. Significant differences from the control are indicated: *p<0.05; **p<0.01.
Figure 4.
PIWIL2 and TDRD1 hypermethylation is negatively associated with PIWIL2 and TDRD1 transcript levels in SpF samples.
Tissular expression profiling of PIWIL2 (A) and TDRD1 (B) by quantitative real-time qPCR in testis with conserved spermatogenesis (CS), maturation failure at the round spermatid (rsMF), the spermatocyte (scMF) and the spermatogonia (sgMF) stages and Sertoli cell-only syndrome (SCO). Expression levels relative to PGM1 are shown. Expression per cell profiling of PIWIL2, displayed as expression ratio per spermatogonia/spermatocyte (x1000) (C) and expression per cell of TDRD1, displayed as expression ratio per spermatocyte (X1000) (D) in testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF), the round spermatid (rsMF), and the spermatogonia (sgMF) stages. The horizontal bar displays the mean cellular expression level. Significant differences from the control are indicated: *p<0.05; **p<0.01.
Table 2.
Pearson correlation coefficients and adjusted p values (r;p) between the molecular and the histological parameters for all the samples analysed.
Figure 5.
Downregulation of PIWIL2 and TDRD1 is associated with piRNA reduction and LINE-1 hypomethylation.
(A–E) Expression levels of selected piRNAs in testis with conserved spermatogenesis (CS), spermatogenic failure (SpF), Sertoli cell-only syndrome (SCO) phenotypes, measured by qPCR. Expression levels relative to RNU48, RNU19 and RNU6B are shown. (F) Methylation profiling of LINE-1 in mature spermatozoa (sperm), testis with conserved spermatogenesis (CS), spermatogenic failure (SpF), Sertoli cell-only syndrome (SCO) phenotypes and somatic tissue, measured by pyrosequencing. Significant differences from the control are indicated: *p<0.05; **p<0.01. Mean expression levels are depicted by horizontal bars.