Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis

Abstract

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L-/- germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L-/- meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.

Fig. 1.

Disruption of Dnmt3L and its expression in gonocytes. (A) The Dnmt3L protein, plant homeodomain (PHD)-like domain, nuclear localization signal (*), and C-terminal motifs (I, IV, VI, and IX) relative to the insertion site of the LacZ gene (arrow) (3). (B) Targeted disruption of Dnmt3L.E, EcoRI; B, BamHI; PA, polyadenylation signal; PGK-Neo, phosphoglycerate kinase neomycin. (C) Southern blot analysis of DraI-digested ES cell DNA (3′ probe) gave products of 8.8 kb (lane 2), 14.0 kb (lane 1), and 12.2 kb (lane 3) for the WT, targeted, and excised alleles, respectively. (D) LacZ reporter gene expression in gonocytes (G) of newborn testis. ST, seminiferous tubule.

Fig. 2.

Dnmt3L^–/– germ cells are lost by sloughing and apoptosis after failure of homologous chromosome alignment and synapsis. WT (A, C, E, and G) and Dnmt3L^–/– (KO) (B, D, F, H, and J) testes are shown. (A) WT d 14 testes. (B) KO d 14 testes containing cells with pyknotic nuclei (Pk) consistent with apoptosis and cells with characteristics of early pachytene spermatocytes (PS). (C) TUNEL staining of d 41 WT. (D) TUNEL staining of d 41 KO reveals an increase in apoptotic cells (A) (brown staining). Germ cell sloughing (Sl) was frequently observed. (E) EM of d 22 WT testes. (F) EM of d 22 KO testes shows many degenerating cells and vacuolation (*) indicative of recent germ cell loss. (G) The tripartite appearance of homologous chromosomes synapsed by means of the SC (Inset) in WT pachytene spermatocytes. (H and J) KO testes contained single unpaired chromosomes and partially paired chromosomes (arrows). Abnormal ribosomal clumps were frequently found (open arrow). (I) Graphical representation of the number of TUNEL-positive cells per section of WT, heterozygous, and KO testes. In, intermediate spermatogonia; E, elongating spermatid; S, Sertoli cell. Spermatocytes: Pl, preleptotene; L, leptotene; Z, zygotene; D, diplotene; 2S, secondary.

Fig. 3.

Abnormal DNA methylation in Dnmt3L^–/– germ cells. (A and B) Bisulfite genomic sequencing of H19 and Rasgrf1 paternally (P) imprinted loci from WT and KO germ cells. Each line corresponds to a single strand of DNA, and each circle represents an individual CpG dinucleotide. Filled circles, methylated CpG; open circles, unmethylated CpG; gray circles, incomplete sequence. (C) Portion of a gel showing an AMP-detected DNA methylation polymorphism in KO spermatogonia. Absence of the locus (arrow) only in digested KO DNA indicates loss of methylation. (D) Portion of the gel showing the AMP locus (arrow) detected in KO spermatogonia but not in WT spermatogonia or KO somatic cells. (E) Southern blot confirmation of the AMP locus shown in D. M, size marker.

Fig. 4.

Differential detection of histone modifications in Dnmt3L^–/– germ cells. (A and B) Acetylated H4 was detected in leptotene (L), zygotene (Z), and pachytene (PS) spermatocytes in KO (B) but not in WT (A) testes at all ages (d 21 shown). (C and D) Acetylated H3K9 was detected in leptotene and zygotene spermatocytes in KO (D) but not in WT (C) adult testes (d 42). (E and F) Dimethylated H3K9 was detected in leptotene and zygotene spermatocytes in WT (E) but not in KO (F) testes at all ages (d 21 shown). (G and H) Unmodified histone H3 stained strongly in pachytene spermatocytes of KO (H) but not WT (G) testes. Spermatogonia: In, intermediate; A, type A; B, type B; Pl, preleptotene spermatocyte; R, round spermatid; E, elongating spermatid. Roman numerals indicate seminiferous tubule stage.