Figure 1.
Dnmt3L haploinsufficiency resulted in XY body abnormalities.
(A) A typical pachytene spermatocyte from a Dnmt3L wildtype male. (B) A pachytene spermatocyte from a Dnmt3L heterozygous male. The XY body appeared to be twisted and longer when compared to the XY body from wildtype males. Green = SCP3, red = γH2AX. Scale bars = 10 µm. (C) Lengths of XY bodies from Dnmt3L heterozygous (white bars, n = 712 cells, n = 8 mice) and wildtype males (black bars, n = 650 cells, n = 6 mice).
Figure 2.
Dnmt3L haploinsufficiency resulted in an increased risk of XY sperm.
FISH on sperm from a Dnmt3L heterozygous male with X and Y chromosome probes. Green (FITC) = X chromosome, Red (CY3) = Y chromosome. Arrow indicates an XY spermatozoon. Scale bar = 10 µm.
Figure 3.
QPCR on selected genes differentially expressed in Dnmt3L wildtype versus Dnmt3L heterozygous germ cells by microarray.
(A) X-linked genes from spermatocytes. (B) Autosomal genes from spermatocytes. (C) Autosomal genes in spermatids. Expression was normalized to β-actin and the wildtype values were set to 1. Black bars = wildtype, white bars = heterozygous, n = 3 biological replicates (10 mice/replicate). Results are presented as means ± SEM. * Indicates a statistically significant increase or decrease in gene expression with a p≤0.05.
Figure 4.
Dnmt3L expression in Dnmt3L haploinsufficient males.
(A) A box plot representing the total Dnmt3L expression from microarray on Dnmt3L heterozygous and wildtype spermatocytes and spermatids. The expression of Dnmt3L was normalised to the whole genome, n = 3 biological replicates (10 mice per replicate). (B) The Dnmt3L wildtype allele contains 13 exons. Disruption of Dnmt3L was achieved through homologous recombination and resulted in the insertion of a LacZ gene within exon 5. Three stop codons and a polyadenylation signal were included after the LacZ gene. (C) The Dnmt3L-5 primer sets were located in exons 3–4, 5′ to the insertion of the LacZ gene. The Dnmt3L-mid primer sets were located in exons 7–8, 3′ of the insertion of the LacZ gene. The Dnmt3L-3 primer sets were located in exons 12–13, which is common to all four Dnmt3L isoforms. A second promoter can be activated in intron 9 of the Dnmt3L gene to produce a novel exon, 9b, from which transcription of the three alternative transcripts starts. Form 1 includes exon 9b and large 9c (9cL). Form 2 includes exon 9b and short 9c (9cS). Form 3 only includes exon 9b. Forms 1, 2 and 3 all contain exons 10–13. Primers for Form 1 were located in exons 9b and 9cL. Primers for Form 2 were located in exons 9b and 9cS. Primers for Form 3 were located in exon 9b and exon 11. All primer sets are represented by arrows. (A, B) Black boxes represent exons. Thin lines between exons represent introns.
Figure 5.
Dnmt3L haploinsufficiency resulted in altered expression of the Dnmt3L splice variants.
The level of Dnmt3L mRNA transcripts in spermatocytes (A) and spermatids (B) from Dnmt3L heterozygous and wildtype males as defined by QPCR. Each gene was normalized to β-actin and the wildtype values were set to 1. Black bars = wildtype, white bars = heterozygous, n = 3 biological replicates (10 mice/replicate). Results are presented as means ± SEM. * Indicates a statistically significant increase or decrease in gene expression with a p≤0.05.
Figure 6.
DNMT3L was found within the adult testis and mature sperm.
(A) DNMT3L was localized to the nuclei of round spermatids (R), elongating spermatids (E) and peritubular cells (Pt). The omission of primary antibody was used as a negative control (inset). S = spermatogonia, P = pachytene spermatocyte. Scale bar = 50 µm. (B) Western Blot analysis of DNMT3L (50 kDa) within Dnmt3L wildtype and heterozygous sperm. (C) Relative content of DNMT3L within sperm. Each sample was normalized to Actin and the wildtype value was set to 1. Black bars = wildtype, white bars = heterozygous, n = 3 biological replicates (2 mice/replicate). Results are presented as means ± SEM. * Indicates a statistically significant decrease in relative intensity of DNMT3L with a p≤0.05.