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Meiotic Knockdown and Complementation Reveals Essential Role of RAD51 in Mouse Spermatogenesis - PubMed

  • ️Sun Jan 01 2017

Meiotic Knockdown and Complementation Reveals Essential Role of RAD51 in Mouse Spermatogenesis

Jieqiong Dai et al. Cell Rep. 2017.

Abstract

Meiotic homologous recombination (HR) is important for proper chromosomal segregation during gametogenesis and facilitates evolutionary adaptation via genomic reshuffling. In most eukaryotes, HR is mediated by two recombinases, the ubiquitous RAD51 and the meiosis-specific DMC1. The role of RAD51 in mammalian meiosis is unclear and study of its function is limited due to embryonic lethality of RAD51 knockouts. Here, we developed an in vivo meiotic knockdown and protein complementation system to study RAD51 during mouse spermatogenesis. We show that RAD51 is crucial during meiotic prophase and its loss leads to depletion of late prophase I spermatocytes through a p53-dependent apoptotic pathway. This phenotype is distinct from that observed in the DMC1 knockdown. Our meiotic knockdown and complementation system establishes an experimental platform for mechanistic studies of meiotic proteins with unknown functions or essential genes for which a testis-specific knockout is not possible.

Keywords: RAD51; homologous recombination; in vivo meiotic gene knockdown; knockdown-protein complementation; meiosis.

Published by Elsevier Inc.

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Figures

Figure 1
Figure 1. Analysis of DMC1 Knockdowns Generated by the in vivo siRNA microinjection approach

Testes of 8 dpp pups were injected with control or DMC1 siRNAs and collected 10 days after microinjection. The samples were fixed, paraffin embedded, stained using appropriate techniques, and analyzed by microscopy. Scale bars represent 40 μm. (A) Immunostaining of DMC1 in tubule cross sections. (B) Hematoxylin and eosin (H&E) staining was used for the morphological analysis of the DMC1 knockdown phenotype. Spermatogonia (Sg) and pre-leptotene (PL) and leptotene (L) spermatocytes are indicated with a green arrow, the zygotene (Z) cells are indicated with a blue arrow, and pachytene (P) and diplotene (Di) spermatocytes are indicated by a red arrow. Examples of magnified nuclei morphology of different germ cells are shown. (C) Apoptosis was detected using the TUNEL assay. See also Figure S2 for the quantification of cell populations in DMC1 knockdown tubules, and Figure S3 for the quantification of apoptosis.

Figure 2
Figure 2. Summary of Results from in vivo Knockdown Experiments

Testes of 8 dpp or 20 dpp pups were injected with siRNAs (control: 100 μM, RAD51: 50 μM, p53: 50 μM, DMC1: 100 μM). Samples were collected after 7, 10, or 14 days of injection, fixed and paraffin embedded. The proportion of tubules showing phenotypes was estimated after the evaluation of cell morphology on H&E stained tissue sections. In RAD51 knockdown and RAD51/p53 double-knockdown samples, the phenotype we scored was spermatocyte depletion. In DMC1 knockdown and DMC1/p53 double-knockdown samples, the phenotype we scored was zygotene arrest. Although tubules depleted of both somatic and germ cells were likely the result of tissue damage caused by microinjection, they were included in the quantification of knockdown phenotypes. See also Figure S1 for the quantification of the proportion of tissue damage in each sample. N – Number of testes evaluated. For each testis, at least 6 sections were checked, and more than 100 tubules were assessed for each section. Results are presented as means ± SD of the values. Statistical significance was determined using ANOVA. ****p < 0.001, NS: no significance.

Figure 3
Figure 3. Depletion of Primary Spermatocytes in RAD51 Seminiferous Tubules from in vivo Knockdown Testes

Testes of 8 dpp pups were injected with control or RAD51 siRNA. Collected samples were fixed and paraffin embedded for tissue sectioning (A, C and D), or prepared for chromosome spreads (B). Scale bars represent 40 μm. (A) Samples were collected 10 days after injection. Immunostaining of RAD51 in the tubule cross sections show a reduction of RAD51 staining in the RAD51 siRNA injected tubules. (B) Spermatocyte chromosome spreads were prepared from the whole testis. RAD51 foci were quantified in zygotene spermatocytes from 3 experimental repeats. N – Number of cells evaluated. Results are presented as means ± SD of the values. Statistical significance was determined using t-tests. ****p < 0.001. (C) Samples were collected 10 days (upper two rows) and 14 days (lower two rows) after injection. Morphological analysis of the H&E stained seminiferous tubule cross sections reveals the depletion of primary spermatocytes in the knockdown testes. Spermatogonia and pre-leptotene and leptotene spermatocytes are indicated with green arrows, zygotene cells are indicated with blue arrows, pachytene and diplotene spermatocytes are indicated with red arrows, metaphase I cells are indicated with grey arrows, and round spermatids are indicated with orange arrows. (D) Samples were collected 10 days after injection. Apoptosis was detected using the TUNEL assay. The numbers of apoptotic cells per tubule cross section are plotted. N – Number of tubules evaluated. Results are presented as means ± SD of the values. Statistical significance was determined using t-test. ****p < 0.001. See also Figures S2, S3, and S4.

Figure 4
Figure 4. Normal Autosomal Synapsis but Reduced Crossover Formation in Rad51-Knockdown Spermatocytes

Testes of 8 dpp pups were injected with siRNAs and collected 10 days after injection. Samples were fixed and paraffin embedded for tissue sectioning (A), or prepared for chromosome spreads (B). (A) Autosomal synapsis was determined by SYCP1 (red) and SYCP3 (green) staining in the spermatocytes. Over 600 tubule sections were examined for each sample. Scale bars represent 40 μm. (B) Crossover formation was analyzed by counting MLH1 foci numbers in the H1t-positive pachytene cells. Spermatocyte chromosome spreads were prepared from the whole testes. N – Number of cells evaluated. Results are presented as means ± SD of the values. Statistical significance was determined using ANOVA. ****p < 0.001. See also Figure S5.

Figure 5
Figure 5. p53 Knockdown Prevents Spermatocyte Depletion in RAD51-Knockdown Testes by Suppressing Apoptosis

Testes of 8 dpp pups were injected with siRNAs and collected 10 days after injection. Samples were fixed and paraffin embedded. Scale bars represent 40 μm. (A) Morphological analysis of the H&E stained seminiferous tubule cross sections reveals that p53 depletion rescues the spermatocyte depletion phenotype in RAD51 knockdown testes, but not the zygotene arrest phenotype in DMC1 knockdown testes. Expression of p53 (red) was detected by immunofluorescence. Apoptosis was detected using the TUNEL (green) assay. (B) Immunostaining of RAD51 in the tubule cross sections. (C) Immunostaining of DMC1 in the tubule cross sections. See also Figures S3, S4, and S6.

Figure 6
Figure 6. Rescue of the RAD51-Knockdown Phenotype by Complementation with RAD51 Protein

Testes of 8dpp or 20 dpp pups were injected with siRNAs. Frozen tissue sections were prepared from samples collected 7 days after injection, and paraffin embedded tissue sections were prepared from samples collected 10 days after injection. The knockdown phenotype was determined by morphological analysis based on H&E staining (1st and 2nd rows). The RAD51 transcription level was analyzed using mRNA FISH (3rd row). Injected His-tag–fused proteins were detected using a His-tag antibody with a secondary signal amplification system (4th row). The boxed regions are displayed with magnification separately (5th row). Scale bars represent 40 μm. See also S7.

Figure 7
Figure 7. Summary of the Results from in vivo Protein-Complementation Experiments

Testes of 8 dpp or 20 dpp pups were injected with siRNAs (control: 100 μM, RAD51: 50 μM,) and proteins (6 μM). Samples were collected after 7, 10, or 14 days of injection, fixed and paraffin embedded. (A) The proportion of tubules showing phenotypes was estimated after the evaluation of cell morphology on H&E stained tissue sections. The phenotype of RAD51 knockdown we scored was spermatocyte depletion. Although tubules depleted of both somatic and germ cells were likely the result of tissue damage caused by microinjection, they were included in the quantification of knockdown phenotypes. See also Figure S1 for the quantification of the proportion of tissue damage in each sample. N – Number of testes evaluated. For each testis, at least 6 sections were checked, and more than 100 tubules were assessed for each section. (B) Spermatocyte chromosome spreads were prepared from whole testes. Crossover formation was analyzed by counting MLH1 foci number in H1t-positive pachytene cells. N - Number of cells evaluated. Results are presented as means ± SD of the values. Statistical significance was determined using ANOVA. ***p < 0.005, ****p < 0.001, NS: no significance.

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