Conditional inactivation of Miwi2 reveals that MIWI2 is only essential for prospermatogonial development in mice

Abstract

The PIWI-piRNA pathway serves as a critical defense mechanism through which the genome of the male germline is protected from invasion by transposable elements (TEs). MIWI2/PIWIL4, a member of the murine PIWI subclade of the Argonaute family, has been shown to be expressed in primordial germ cells (PGCs) and prospermatogonia in fetal and prepubertal testes. Global inactivation of Miwi2 leads to male sterility due to an early meiotic arrest, which correlates with retrotransposon desuppression. However, it remains unclear whether MIWI2 functions beyond the PGC stage and whether MIWI2 has a role beyond TE suppression during male germ line development. Through conditional inactivation of Miwi2, we demonstrate herein that MIWI2 function is restricted to a narrow time window during male PGC reprograming and that Miwi2 is dispensable for postnatal male germline development and testicular function in mice. Moreover, persistent activation of LINE1 and IAP retrotransposons caused by Miwi2 inactivation is compatible with mitotic cell cycle progression of spermatogonia during the first wave of spermatogenesis, but can cause zygotene to pachytene arrest in early meiosis due to multiple defects including enhanced DNA double-strand breaks, aberrant histone modifications and altered mRNA transcriptome. Our data not only validate those from global Miwi2 KO studies, but also suggest that MIWI2 and MIWI2-associated piRNAs have functions beyond TE suppression.

Figure 1

Generation of Miwi2 conditional knockout mice. (a) Strategy for generating a floxed Miwi2 allele in murine embryonic stem (ES) cells. In the targeted ES cells, exons 9–12 are flanked by two loxp sequences, and Cre-mediated recombination leads to the deletion of the four exons, which causes a frame shift and a complete lack of MIWI2 protein production. Arrows depict the locations of PCR genotyping primers (For, forward primer; Rev, reverse primer). The restriction enzyme SpeI was used to digest genomic DNA in Southern blot analyses shown in panel b. (b) A representative Southern blot showing two WT ES cell clones (Lanes 1 and 4) and two targeted ES cell clones (Lanes 2 and 3) carrying one WT allele (13 062 bp) and the other Miwi2 floxed allele (Miwi2^lox) (8726 bp). (c) Representative PCR genotyping results showing tail samples with different genotypes (WT=438 bp, miwi2^lox=315 bp; Ddx4-Cre=240 bp). (d) A schematic diagram showing the timeline of male germ-line development and the onset of Cre expression in three germline-specific Cre deletor lines

Figure 2

Miwi2 is required between embryonic day 15.5 (E15.5) and postnatal day 3 (P3), but dispensable after P3 during murine testicular development. (a) A representative image showing the testes of Ddx4-cre; Miwi2^lox/Δ male mice were smaller than Stra8-Cre; Miwi2^lox/Δ testes, which were similar to WT controls. (b) Testis growth curves in male mice with different genotypes indicated. Stra8-Cre; Miwi2^lox/Δ and Prm-Cre; Miwi2^lox/Δ WT male mice displayed the same testicular growth rate as WT controls, which are omitted here. (c–h) Testicular histology of two control (Miwi2^+/lox as a negative control, whereas Stra8-Cre; Mov10l1^lox/Δ as a positive control), and four male germ cell-specific Miwi2 conditional knockout mouse lines. Note that all testes samples were from postnatal day 21 (P21). Scale bar=200 μm

Figure 3

Inactivation of Miwi2 in primordial germ cells (PGCs)/prospermatogonia at E15.5 does not disrupt mitotic progression of spermatogonia, but leads to delayed entry into the meiotic phase and an eventual block in the zygotene stage of prophase I during the first wave of spermatogenesis. (a) Histology of the testes from control (Miwi2^+/lox) and Miwi2 cKO (Ddx4-Cre; Miwi2^lox/Δ) mice at various developmental time points (P4, P7, P10 and P14). Note that the morphology and total number of germ cells are indistinguishable between control and Miwi2 cKO testes before P10. Scale bar=120 μm; (b) Immunohistochemical staining of germ cell nuclear antigen (GCNA), a marker for germ cells, in developing testes of control and Miwi2 cKO mice. Typical germ cell types were labeled at specific time points during postnatal testicular developmental. spg, spermatogonia; lep, leptotene; pachy, pachytene. Scale bar=100 μm; (c) Delayed meiotic entry in the testes of Ddx4-Cre; Miwi2^lox/Δ mice at P10 as revealed by γH2Ax immunofluorescent staining. A large number of γH2Ax-positive germ cells (that is, leptotene spermatocytes) are present in the control (Miwi2^+/lox) testis, whereas only a few exist in the Ddx4-Cre; Miwi2^lox/Δ testis. Scale bar=100 μm. (d) Testicular histology of a 10-month-old Ddx4-Cre; Miwi2^lox/Δ male mouse. Numerous vacuoles can be observed in the seminiferous tubules, as indicated by arrows. Although some tubules contain only Sertoli cells (Ser), resembling ‘Sertoli-only' histology in humans (*), many tubules still possess meiotic germ cells, for example, zygotene spermatocytes (Zyg). (e) Quantitative analyses of germ cell number based on GCNA staining in developing testes, as shown in Panel B. Only round tubule cross sections were selected for counting germ cells and at least 20 tubules were analyzed for each time point. *P<0.05

Figure 4

Activation of retrotransposons and DNA double-strand breaks (DSBs) in Ddx4-Cre; Miwi2^lox/Δ testes during postnatal development. (a) Quantitative real-time PCR (qPCR) analyses of five types of transposable elements (TEs) in WT and Ddx4-Cre; Miwi2^lox/Δ testes at P12. Transcripts of retrotransposons, such as LINE1 and IAP, were significantly upregulated in Ddx4-Cre; Miwi2^lox/Δ testes, whereas the DNA transposons, for example, Charlier, Mariner and MusD, remained unaffected. *P<0.05; (b) Immunofluorescent staining of ORF1 of LINE1 and IAP in control (Miwi2^+/lox) and Miwi2 cKO (Ddx4-Cre;Miwi2^lox/Δ) testes with Stra8-Cre; Mov10l1^lox/Δ testes as a positive control. Inset represents the magnified view of the dashed area. Scale bar=100 μm. (c) Immunofluorescent staining of ORF1/LINE1 and γH2Ax in control (Miwi2^+/lox) and Miwi2 cKO (Ddx4-Cre; Miwi2^lox/Δ) testes at birth (P0) and P7. Although germ cells are rarely positive for either ORF1 or γH2Ax staining in control testes at P0 and P7, numerous prospermatogonia or spermatogonia are ORF1- (arrows) and γH2Ax-positive (arrowheads) although γH2Ax-positve germ cells are much fewer in Miwi2 cKO testes at the same ages. Scale bar=100 μm

Figure 5

Immunofluorescent staining pattern of H3K4me3 in spermatogonia and spermatocytes isolated from control (Miwi2^+/lox) and Miwi2 cKO (Ddx4-Cre; Miwi2^lox/Δ) testes at postnatal day 21 (P21). SCP3 staining was used to distinguish the various stages of meiotic cells. spg, spermatogonia; eL, preleptotene; L, leptotene; eZ, early zygotene; mP, middle pachytene; IP, late pachytene. Scale bar=5 μm

Figure 6

Immunofluorescent staining pattern of H3K27me3 in spermatogonia and spermatocytes isolated from control (Miwi2^+/lox) and Miwi2 cKO (Ddx4-Cre; Miwi2^lox/Δ) testes at postnatal day 21 (P21). SCP3 staining was used to distinguish the various stages of meiotic cells. spg, spermatogonia; eL, preleptotene; L, leptotene; eZ, early zygotene; mP, middle pachytene; IP, late pachytene. Scale bar=5 μm

Figure 7

Microarray analyses of mRNA transcriptome in spermatocytes purified from WT and Ddx4-Cre; Miwi2^lox/Δ testes at postnatal day 11 (P11). (a) A scatter plot showing a total of 2642 up or downregulated mRNA transcripts (mean >10, P<0.05, fold change (FC) >1.2). Black dots: 1.2<FC<1.5; Green dots: 1.5<FC<2.0; Red dots: FC>2.0; (b) GO term analyses revealing an enrichment of genes involved in transcription-related biological processes among deregulated genes. (c) Bioinformatic analyses of potential complementary annealing between MIWI2-associated piRNAs and deregulated mRNA transcripts based on the microarray analyses