Germ cell-specific targeting of DICER or DGCR8 reveals a novel role for endo-siRNAs in the progression of mammalian spermatogenesis and male fertility

Abstract

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process.

Figure 1. Germ cell-specific depletion of miRNAs in GC-Dcr1 and GC-Dgcr8 mutant testes.

(A) Schematic representation of Dicer1 and Dgcr8 loci. Exon 24 of the Dicer1 gene is flanked with loxP sites (grey triangles) and excision occurs upon Ddx4-Cre recombinase expression leading to loss of the RNaseIII domain 2. Excision of exon 3 of the Dgcr8 locus leads to a sequence frameshift and introduces multiple premature STOP codons. (B) Histograms showing the absence of expression of spermatocyte-specific miRNAs in GC-Dgcr8 and GC-Dcr1 mutant total testes. In contrast, expression of Sertoli cell-specific miRNAs in both mutants was not affected (C). Results are mean ± SEM, * = p<0.05, ** = p<0.005, *** = p<0.0001 GC-Dcr1 mutant vs. control, GC-Dgcr8 mutant vs. control or GC-Dcr1 vs. GC-Dgcr8.

Figure 2. Reduction in testis size and near complete absence of mature spermatozoa in GC-Dcr1 and GC-Dgcr8 mutant mice.

(A–C, M) At P60, testis weight showed a 55% and 50% reduction in GC-Dcr1 (n = 16) and GC-Dgcr8 mutants (n = 5) compared to control testes (n = 17). H&E staining of testis sections (D–F) revealed several defects in the architecture of the seminiferous epithelium, near complete absence of mature spermatozoa, and reduced tubular diameter (N) (Control: 170.9 µm²±1.7, GC-Dcr1 mut: 122.5 µm²±2.2, GC-Dgcr8 mut: 142.6 µm²±1.8). (G–I) Anti-H3K9me3 stained sex-body in round spermatids (RS). We observed a 60% reduction in the number of RS per tubule in GC-Dcr1 mutants and 55% reduction in GC-Dgcr8 mutants compared to control testes (P). (J–L) Anti-protamine revealed a 75% reduction in the number of elongated spermatids (ES) per tubule in GC-Dcr1 mutants and a 62% reduction in GC-Dgcr8 mutants compared to control testes (Q). DAPI (blue) was used for nuclear staining. (O) Epididymal sperm count analysis showed a 99% decrease in GC-Dcr1 mutants and 96% in GC-Dgcr8 mutants. TW: Testis Weight, BW: Body Weight, RS: Round Spermatids, ES: Elongated Spermatids. Results are mean ± SEM, * = p<0.05, ** = p<0.005, *** = p<0.0001 GC-Dcr1 mutant vs. control, GC-Dgcr8 mutant vs. control or GC-Dcr1 vs. GC-Dgcr8. Scale bars: 20 µm (D–L).

Figure 3. Tubular defects in GC-Dcr1 and GC-Dgcr8 mutant testes appear from P15.

H&E staining of control (A,D,G), GC-Dcr1 mutant (B,E,H) and GC-Dgcr8 mutant (C,F,J) testes at P12 (A–C), P15 (D–F) and P21 (G–J). The anatomical defects include germ cells sloughing, Sertoli cell cytoplasmic extensions (arrowheads), reduction in the number of round spermatids (asterisks), and apoptosis and disorganization of the seminiferous epithelium. The increase of apoptotic rate (M) correlates with the reduced testis weight ratio (N) during the first wave of spermatogenesis. (J–L) Apoptotic cells revealed by TUNEL assay on testis sections at P60. Results are mean ± SEM (n = 3/genotype), a = p<0.05 GC-Dcr1 mut vs. control, b = p<0.05 GC-Dgcr8 mut vs. control and c = p<0.05 GC-Dcr1 mut vs. GC-Dgcr8 mut. Scale bars: 20 µm.

Figure 4. Meiotic defects in GC-Dcr1 and GC-Dgcr8 mutant testes.

(A–D) Anti-γH2AX (green), present in the entire nucleus in early meiotic stages and located to the XY body during the pachytene stage, indicates a decreased number of tubules containing XY body positive cells in GC-Dcr1 and GC-Dgcr8 mutant testes compared to controls at P12. (E–H) Anti-pH3 (green), present in metaphasic cells, reveals a reduction in the number of metaphasic tubules within GC-Dcr1 and GC-Dgcr8 mutants in P60 testes compared to controls. DAPI (blue) was used for nuclear staining. Results are mean ± SEM (n = 3/genotype), * = p<0.05, ** = p<0.005, *** = p<0.0001 GC-Dcr1 mutant vs. control, GC-Dgcr8 mutant vs. control or GC-Dcr1 vs. GC-Dgcr8. Scale bars: 20 µm.

Figure 5. Impaired spermiogenesis leads to altered morphology of spermatozoa in both GC-Dcr1 and GC-Dgcr8 mutants at P60.

Representative transmission electron micrographs from P60 control (A, D), GC-Dcr1 mutants (B,E) and GC-Dgcr8 mutants (C,F). In round spermatids (A–C), the acrosome is fragmented or asymmetric in both mutants (arrowheads). In elongated spermatids (D–F), nuclear shape (red arrows), chromatin condensation and the acrosome (arrowheads) are abnormal in both GC-Dcr1 and GC-Dgcr8 mutants. Note the presence of vacuoles within nuclei (blue arrows) in both mutants. Scale bar: 2 µm. H&E staining of epididymal sperm spreads of control (G) and mutant (H–L) adult mice. In contrast to control mice, spermatozoa of both mutant mice exhibited multiple defects of morphology, such as falciform head (H), intermediate head (I), round head (J), elongated head (K), pinhead (L) and abnormal midpiece (J). Scale bars: 10 µm. The histogram shows the percentage of spermatozoa in each category (M). Results are mean ± SEM (minimum n = 3/genotype), a = p<0.005 GC-Dcr1 mut vs. control, b = p<0.005 GC-Dgcr8 mut vs. control and c = p<0.005 GC-Dcr1 mut vs. GC-Dgcr8 mut.

Figure 6. Altered mRNA transcriptome in GC-Dcr1 spermatocytes.

(A) Hierarchical clustering of the six sequenced samples. We calculated the distance between each pair of samples as 1 - rho, where rho was the Spearman correlation coefficient for the gene expression levels in the two samples. Clustering was performed using the hclust function in R and Ward's method. (B) Overview of the normalized gene expression data. Genes were grouped into 10 equally sized bins based on their combined expression in GC-Dcr1 and control spermatocytes. Highly expressed genes are shown on the right. Expression change was calculated as the difference between the log₂-transformed normalized expression values in GC-Dcr1 and control spermatocytes. (C) Comparison of expression change in genes targeted by highly and lowly expressed miRNAs (see text for details). Expression change was calculated as in (B).