Argonaute proteins regulate the timing of the spermatogenic transcriptional program

  1. Maria de las Mercedes Carro
  2. Alexis Dziubek
  3. Amanda Touey
  4. Elizabeth A. Popkowski
  5. Mark Abdelmassih
  6. Leah Simon
  7. Stephanie Tanis
  8. Faraz Ahmed
  9. Jen K. Grenier
  10. Andrew Grimson
  11. Paula E. Cohen

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Abstract

Argonaute proteins are best known for their role in microRNA-mediated post-transcriptional gene silencing. Here, we show that AGO3 and AGO4, but not AGO2, localize to the sex chromatin of pachytene spermatocytes where they are required for transcriptional silencing of XY-linked genes, known as Meiotic Sex Chromosome Inactivation (MSCI). Using an Ago413 -/- mouse, we show that AGO3 and AGO4 are key regulators of spermatogenesis, orchestrating expression of meiosis-related genes during prophase I while maintaining silencing of spermiogenesis genes. Premature overexpression of spermiogenesis genes during prophase I in Ago413 -/- mice results in subfertility, altered sperm morphology and reduced fertilization capability. We also identify BRG1, a BAF complex subunit, as an AGO3 interactor. Loss of AGO3 and AGO4 results in increased BRG1 in spermatocytes, suggesting that AGO3 aids in removing BRG1 from the XY chromatin to achieve MSCI and demonstrating a meiotic role for AGO3 in transcriptional control through the chromatin remodeling machinery.

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    Reviewer #1

    Evidence, reproducibility and clarity

    In their manuscript de las Mercedes Carro et al investigated the role of Ago proteins during spermatogenesis by producing a triple knockout of Ago 1, 3 and 4. They first describe the pattern of expression of each protein and of Ago2 during the differentiation of male germ cells, then they describe the spermatogenesis phenotype of triple knockout males, study gene deregulation by scRNA seq and identify novel interacting proteins by co-IP mass spectrometry, in particular BRG1/SMARCA4, a chromatin remodeling factor and ATF2 a transcription factor. The main message is that Ago3 and 4 are involved in the regulation of XY gene silencing during meiosis, and also in the control of autosomal gene expression during meiosis. Overall the manuscript is well written, the topic, very interesting and the experiments, well-executed. However, there are some parts of the methodology and data interpretation that are unclear (see below).

    Major comments

    1= Please clarify how the triple KO was obtained, and if it is constitutive or specific to the male germline. In the result section a Cre (which cre?) is mentioned but it is not mentioned in the M&M. On Figure S1, a MICER VECTOR is shown instead of a deletion, but nothing is explained in the text nor legend. Could the authors provide more details in the results section as well as in the M&M ? This is essential to fully interpret the results obtained for this KO line, and to compare its phenotype to other lines (such as lines 184-9 Comparison of triple KO phenotype with that of Ago4 KO). Also, if it is a constitutive KO, the authors should mention if they observed other phenotypes in triple KO mice since AGO proteins are not only expressed in the male germline.

    Response: We apologize for omitting this vital information. We have now incorporated a more detailed description of how the Ago413 mutant was created in the results and M&M sections (line 120 and 686 respectively).

    As mentioned in the manuscript, Ago4, Ago1 and Ago3 are widely expressed in mammalian somatic tissues. Mutations or deletions of these genes does not disrupt development; however, there is limited research on the impact of these mutations in mammalian models in vivo. In humans, mutations in Ago1 and Ago3 genes are associated with neurological disorders, autism and intellectual disability (Tokita, M.J.,et al. 2015- doi: 10.1038/ejhg.2014.202., Sakaguchi et al. 2019- doi: 10.1016/j.ejmg.2018.09.004, Schalk et al 2021- doi: 10.1136/jmedgenet-2021-107751). In mouse, global deletion of Ago1 and Ago3 simultaneously was shown to increase mice susceptibility to influenza virus through impaired inflammation responses (Van Stry et al 2012- doi.org/10.1128/jvi.05303-11). Studies performed in female Ago413 mutants (the same mutant line used herein) have shown that knockout mice present postnatal growth retardation with elevated circulating leukocytes (Guidi et al 2023- doi: 10.1016/j.celrep.2023.113515). Other studies of double conditional knockout of Ago1 and Ago3 in the skin associated the loss of these Argonautes with decreased weight of the offspring and severe skin morphogenesis defects (Wang et al 2012- doi: 10.1101/gad.182758.111). In our study, we did not observe major somatic or overt behavioral phenotypes, and we did not observe statistical differences in body weights of null males compared to WT as shown in figure below.

    2= The paragraph corresponding to G2/M analysis is unclear to me. Why was this analysis performed? What does the heatmap show in Figure S4? What is G2/M score? (Fig 2D). Lines 219-220, do the authors mean that Pachytene cells are in a cell phase equivalent to G2/M? All this paragraph and associated figures require more explanation to clarify the method and interpretation.

    __Response: __We have modified the methods to include more information about how the cell cycle scoring used in Figures 2D and S4 were calculated and will add more information regarding the interpretation of these figures.

    3= I have concerns regarding Fig2G: to be convincing the analysis needs to be performed on several replicates, and, it is essential to compare tubules of the same stage - which does not seem to be the case. This does not appear to be the case. Besides, co (immunofluorescent) staining with markers of different cell types should be shown to demonstrate the earlier expression of some markers and their colocalization with markers of the earlier stages.

    __Response: __We agree with the Reviewer. New images with staged tubules will be added to the analysis of Figure 2G.

    4= one important question that I think the authors should discuss regarding their scRNAseq: clusters are defined using well characterized markers. But Ago triple KO appears to alter the timing of expression of genes... could this deregulation affects the interperetation of scRNAseq clusters and results?

    __Response: __We thank the reviewer for this suggestion and agree that including this information is important. We expect that, at most, this dysregulation impacts the edges of these clusters slightly. Given that marker genes that have been used to define cell types in these data are consistently expressed between the knockout and wildtype mice (see Figure S4A), we do not think that the cells in these clusters have different identities, just dysregulated expression programs. We have added the relevant sentence to the discussion, and will include additional supplemental figure panels to document this point more comprehensively.

    5= XY gene deregulation is mentioned throughout the result section but only X chromosome genes seem to have been investigated.... Even the gene content of the Y is highly repetitive, it would be very interesting to show the level of expression of Y single copy and Y multicopy genes in a figure 3 panel.

    __Response: __We agree with the reviewer that including analysis of Y-linked genes is important. We will add a supplemental figure which includes the Y:Autosome ratio and differential expression analysis.

    6= Can the authors elaborate on the observation that X gene upregulation is visible in the KO before MSCI; that is in lept/zygotene clusters (and in spermatogonia, if the difference visible in 3A is significant?)

    Response: We do see that X gene expression is upregulated before pachynema. Previous scRNA-seq studies that have looked at MCSI have seen that silencing of genes on the X and Y chromosomes starts before the cell clusters that are defined as pachynema, though silencing is not fully completed until pachynema. We have clarified this point in the manuscript.

    7 = miRNA analysis: could the authors indicate if X encoded miRNA were identified and found deregulated? Because Ago4 has been shown to lead to a downregulation of miRNA, among which many X encoded. It is therefore puzzling to see that the triple KO does not recapitulate this observation. Were the analyses performed differently in the present study and in Ago4 KO study?

    __Response: __The analysis identifying downregulation of miRNA in the original Ago4 mutant analysis was conducted relative to total small RNA expression. Amongst those altered miRNA families in the Ago4 mutants, we demonstrated both upregulation and downregulation of miRNA. We agree that confirming a similar global downregulation of miRNA counts compared to other small RNAs is important. Therefore, in a revised manuscript, we will add this information to the miRNA analysis section, especially highlighting the X chromosome-associated miRNAs, as well as whether the ratios between other small RNA classes change.

    8 = The last results paragraph would also benefit from some additional information. It is not clear why the authors focused on enhancers and did not investigate promoters (or maybe they were but it's unclear). Which regions (size and location from TSS) were investigated for motif enrichment analyses? To what correspond the "transcriptional regulatory regions previously identified using dREG" mentioned in the M&M? I understand it's based on a previous article, but more info in the present manuscript would be useful.

    Response: We thank the reviewer for this suggestion. The regions that were used for motif enrichment will be included as a supplementary information in the fully revised manuscript. We have also clarified in the methods that these transcriptional regulatory regions were downloaded from GEO and obtained from previous ChRO-seq data (from GEO) analysis. These data are run through the dREG pipeline that identifies regions predicted to contain transcription start sites, which include promoters and enhancers.

    Minor comments

    1. In the introduction: The sentence "Ago1 is not expressed in the germline from the spermatogonia stage onwards allowing us to use this model to study the roles of Ago4 and Ago3 in spermatogenesis." is misleading because Ago1 is expressed at least in spermatogonia; It would be more precise to write "after spermatogonia stage" and rephrase the sentence. Otherwise it is surprising to see AGO1 protein in testis lysate and it is not in line with the scRNA seq shown in figure 2.

    __Response: __We agree with the Reviewers suggestion and have edited the sentence on line 100. This sentence now reads "Ago1 is not expressed in the germline after the spermatogonia stage allowing us to use this model to study the roles of Ago4 and Ago3 in spermatogenesis".

    1. Could the authors precise if AGO proteins are expressed in other tissues? In somatic testicular cells?

    __Response: __Expression patterns of mammalian AGOs have been described in somatic and testicular tissues for the mouse by Gonzales-Gonzales et al (2008) by qPCR. They found that Ago2 is expressed in all the somatic tissues analyzed (brain, spleen, heart, muscle and lung) as well as the testis, with the highest expression in brain and lowest in heart. Ago1 is highly expressed in spleen compared to all the tissues analyzed, while Ago3 and Ago4 showed highest expression in testis and brain. Within somatic tissues of the testis, the four argonautes are expressed in Sertoli cells, however, Ago1,3 and 4 expression is very low compared to Ago2, with the latter showing a 10-fold higher transcript level. We have included a sentence with this information in the introduction in line 89.

    1. Pattern of expression: How do the authors explain that AGO3 disappears at the diplotene stage and reappears in spermatids?

    __Response: __ Single cell RNAseq data in the germline shows reduced transcript for Ago3 from the Pachytene stage onwards, suggesting minimal if any new transcription in round spermatids. We hypothesize that the AGO3 protein present in the round spermatid stage is cytoplasmic, presumably coming from the pool of AGO3 in the chromatoid body, a cytoplasmic structure with functional association with the nucleus in round spermatids (Kotaja et al, 2003 doi: 10.1073/pnas.05093331).

    1. It would be useful to show the timing of expression of AGO 1 to 4 throughout spermatogenesis in the first paragraph of the article. Maybe the authors could present data from fig2B earlier?

    Response: We understand the Reviewers concern, however, given that Ago expression throughout spermatogenesis was obtained from scRNA seq, we consider that this data should be presented after introducing the Ago413 knockout and the scRNA seq experiment. As Ago1-4 expression was also described in an earlier manuscript by Gonzales-Gonzales et al in the mouse male germline, and our data aligns with this report, we included a sentence about these previous findings in the earlier results section.

    1. Line 190: please modify the sentence "reveal no differences in cellular architecture of the seminiferous tubules when compared to wild-type males" to " reveal no gross differences..." since even without quantification of the different cell types it is visible that KO seminiferous tubules are different from WT tubules.

    __Response: __We agree with the reviewer, and we modified line 190 (now 173) as suggested. Grossly, seminiferous tubules from Ago413 null males contain the same cell types as in wild type tubules, including spermatozoa. However, our studies show that the number and quality of germ cells is compromised in knockouts, as shown by sperm counts and TUNEL staining.

    1. TUNEL analysis: please stage the tubules to determine the stage(s) at which apoptosis is the most predominant.

    __Response: __We have complied with the reviewer suggestion. Figure 1G now shows staged seminiferous tubules, and we have replaced the wild type image for one where the staged tubules match the knockout image.

    1. Figure S4B does not show an increase of cells at Pachytene stage but at Lepto/zygotene stage (as well as an increase of spermatogonia). Please comment this discrepancy with results shown in Fig2.

    __Response: __Figures 2 and S4 show distribution of cells in different substages of spermatogenesis and prophase I measured with very different methods: a cytological approach using chromosome spreads cells vs a transcriptomic approach that involves clustering of cells. We attribute the differences in cell type distribution to differences in the sensitivity of the methods to identify each cell type and therefore identify differences between the number of cells for each group. Moreover, our scRNA-seq data groups the leptotene and zygotene stages together, while the cytological approach allows for separation of these two sub-stages. Importantly, both results show that Ago413 spermatocytes are progressing slower from pachynema into diplonema and/or are dying after pachynema, as stated in line 194 in our manuscript.

    1. Fig5H and 5I are not mentioned in the result section. Also, it would be useful to label them with "all chromosomes" and "XY" to differentiate them easily

    __Response: __We apologize for the omission and have now cited Figures 5H and 5I in the manuscript (line 453). We have added the suggested labels.

    1. Line 530 "data provide further evidence for a functional association between AGO-dependent small RNAs and heterochromatin formation, maintenance and/or silencing." Please rephrase, the present article does not really show that AGO nuclear role depends on small RNAs.

    __Response____: __We agree with the reviewer that these data do not directly show a dependence on small RNAs. As our identified localization of AGO proteins to the pericentric heterochromatin coincides with localization of DICER shown previously by Yadav and collaborators (2020, doi: 10.1093/nar/gkaa460), we do believe that our data further implicates small RNAs in the silencing of heterochromatin. Yadav et al shows that DICER localizes to pericentromeric heterochromatin and processes major satellite transcripts into small RNAs in mouse spermatocytes, and cKO germ cells have reduced localization of SUV39H2 and H3K9me3 to the pericentromeric heterochromatin. Given the colocalization of both small RNA producing machinery and AGOs at pericentromeric heterochromatin, the AGOs may bind these small RNAs, and the statement in line 530 refers to how our results provide evidence for the involvement of other RNAi machinery in the silencing of pericentromeric heterochromatin investigated by Yadav et al which likely includes small RNAs.

    To clarify this point, we have modified the text accordingly.

    1. Line 1256: replace "cite here " by appropriate reference

    __Response: __The reference was added to line 1256.

    1. Please use SMARCA4 instead of BRG1 name as it is its official name.

    __Response: __We have replaced BRG1 with SMARCA4 in the text and figures.

    Figures:

    Figure 1: Are the pictures shown for Ago3-tagged and floxed from the same stages ? The leptotene stage in 1A looks like a zygotene, while some pachytene/diplotene stage pictures do not look alike.

    __Response: __New representative images have been added to figure 1 to match the same substages across the figure.

    Figure 1D, please label the Y scale properly (testis weight related to body weight)

    __Response: __We have fixed this.

    FigS1: Please comment the presence of non-specific bands in the figure legend

    __Response: __We have added a sentence in Figure S1 Legend.

    Fig 2E and F, please indicate on the figure (in addition to its legend), what are the X and Y axes respectively to facilitate its reading.

    __Response: __X and Y axes are now labelled in Figure 2E and F.

    2F: please use an easier abbreviation for Spermatocyte than Sp (which could spermatogonia, sperm etc..) such as Scyte I ? (same comment for Fig 3C)

    Response: The abbreviation for spermatocyte was changed from Sp to Scyte I in Figures 2 and 3.

    Overall, for all figures showing GSEA analyses, could the authors explain what a High positive NES and a High negative NES mean in the results section?

    Response: Thank you for this suggestion. We have added this information where the GSEA score of the cell markers is initially introduced.

    Significance

    Ago proteins are known for their roles in post transcriptional gene regulation via small RNA mediated cleavage of mRNA, which takes places in the cytoplasm. Some Ago proteins have been shown to be also located in the nucleus suggesting other non-canonical roles. It is the case of Ago4 which has been shown to localize to the transcriptionally silenced sex chromosomes (called sex body) of the spermatocyte nucleus, where it contributes to regulate their silencing (Modzelewski et al 2012). Interestingly, Ago4 knockout leads to Ago3 upregulation, including on the sex body indicating that Ago3 and Ago4 are involved in the same nuclear process. In their manuscript, de las Mercedes Carro et al., investigate the consequences of loss of both Ago3 and Ago4 in the male germline by the production of a triple knockout of Ago1, 3 and 4 in the mouse. With this model, the authors describe the role of Ago3 and Ago4 during spermatogenesis and show that they are involved in sex chromosome gene repression in spermatocytes and in round spermatids, as well as in the control of autosomal meiotic gene expression. Triple KO males have impaired meiosis and spermiogenesis, with fewer and abnormal spermatozoa resulting in reduced fertility. Since Ago1 male germline expression is restricted to pre-meiotic germ cells, it is not expected to contribute to the meiotic and postmeiotic phenotypes observed in the triple KO. The strengths of the study are i) the thorough analyses of mRNA expression at the single cell level, and in purified spermatocytes and spermatids (bulk RNAseq), ii) the identification of novel nuclear partners of AGO3/4 relevant for their described nuclear role: ATF2, which they show to also co-localize with the sex body, and BRG1/SMARCA4, a SWI/SNF chromatin remodeler. The main limitation of the study is the lack of information in the method regarding the production of the triple KO, as well as some aspects of the transcriptome and motif analyses. It is also surprising to see that the triple KO does not recapitulate the miRNA deregulation observed in Ago4 KO. The characterization of a non-canonical role of AGO3/4 in male germ cells will certainly influence researchers of the field, and also interest a broader audience studying Argonaute proteins and gene regulation at transcriptional and posttranscriptional levels.

    Reviewer #2

    Evidence, reproducibility and clarity

    In the manuscript titled "Argonaute proteins regulate the timing of the spermatogenic transcriptional program" by Carro et al., the authors present their findings on how Argonaute proteins regulate spermatogenic development. They utilize a mouse model featuring a deletion of the gene cluster on chromosome 4 that contains Ago1, Ago3, and Ago4 to investigate the cumulative roles of AGO3 and AGO4 in spermatogenic cells. The authors characterize the distribution of AGO proteins and their effects on key meiotic milestones such as synapsis, recombination, meiotic transcriptional regulation, and meiotic sex chromosome inactivation (MSCI). They analyze stage-specific transcriptomes in spermatogenic cells using single-cell and bulk RNA sequencing and determine the interactome of AGO3 and AGO4 through mass spectrometry to examine how AGO proteins may regulate gene expression in these cells during meiotic and post-meiotic development. The authors conclude that both AGO3 and AGO4 are essential for regulating the overall gene expression program in spermatogenic cells and specifically modulate MSCI to repress sex-linked genes in pachytene spermatocytes, which may be partially mediated by the proper distribution of DNA damage repair factors. Additionally, AGO3 is suggested to interact with the chromatin remodeler SWI/SNF factor BRG1, facilitating its removal from the sex-chromatin to enable the repression of sex-linked genes during MSCI.

    Major Comments:

    1. The study utilized a triple knockout mouse model to determine the effect of AGO3 on spermatogenesis, following up on their previous report about the role of AGO4 in spermatogenesis, which resulted from an upregulation of AGO3 in Ago4-/- spermatocytes. However, the results are more difficult to interpret and ascertain the role of AGO3 in these cells, given the absence of any observable phenotype from Ago3 interruption. AGO4 regulates sex body formation, meiotic sex chromosome inactivation (MSCI), and miRNA production in spermatocytes, all of which were noted in the absence of both AGO3 and AGO4, with only an increased incidence of cells containing abnormal RNAPII at the sex chromosomes. It will be necessary to characterize how AGO3 regulates spermatogenic development, including meiotic progression and the regulation of the meiotic transcriptome, and compare these findings with the current observations to determine if the proposed mechanism involving AGO3, BRG1, and possibly AP2 is relevant in this context.

    __Response: __While we agree with Reviewer that a single Ago3 knockout will help understand distinct roles of AGO3 and AGO4 in spermatogenesis, the time and resources required to generate a new mouse model are substantial. The analysis included in this current manuscript has already taken over seven years, and with the lengthy production of a new single mutant mouse, validation of the new mouse, and then final analysis, we would be looking at another 3-5 years of analysis. In the current funding climate, and with strong concerns over ensuring reduction in utilization of laboratory mice, we consider this request to be far in excess of what is required to move this important story forward.

    The Ago413-/- mouse model has allowed us to associate a nuclear role of Argonaute proteins with a strong reproductive phenotype in the mouse germline. Given the redundancy between *Ago3 *and Ago4, it is likely that a single *Ago3 *knockout would have a mild phenotype just like the *Ago4 *KO. All this said, we agree with the reviewer that analysis of an Ago3 knockout mouse is a valuable next step, just not within this chapter of the story.

    1. Does Ago413-/- mice recapitulate the early meiotic entry phenotype observed in Ago4-/- mice? If not, could it be possible that AGO3 promotes meiotic entry, given its strong mRNA expression in spermatogonia according to the scRNAseq data (Fig. 2B)

    Response: Our scRNA-seq data shows strong expression of Ago3 in spermatogonia, as mentioned by the Reviewer. Analysis of cell cycle marker expression also shows that the transcriptomic profile of spermatogonia is altered, with higher levels of transcripts corresponding to the later G2/M stages (Figure 2D). Moreover, Ago413 knockouts present an increase in the number of spermatogonial stem cells (Supplementary Figure S4B). However, this cluster represents a pool of quiescent and mitotically active cells entering meiosis, therefore interpretation of these data might be challenging. While specific experiments could be conducted to answer this question, this is outside of the scope of our manuscript. The manuscript as it stands is already rather large, and a full analysis of meiotic entry dynamics would dilute the core message relating to chromatin regulation in the sex body.

    1. The authors suggested that the removal of BRG1 by AGO3 is necessary during sex body formation and the eventual establishment of MSCI. However, the BAF complex subunit ARID1A has been shown to facilitate MSCI by regulating promoter accessibility. It will be interesting to determine how BRG1 distribution changes across the genome in the absence of AGO proteins and how that correlates with alterations in sex-linked gene expression.

    __Response: __We agree that changes in BRG1 distribution across the genome would be very interesting to identify. However, in this work we show that BRG1/SMARCA4 protein changes its localization in the sex body very rapidly between early to late pachynema. These two substages are only discernable by immunofluorescence using synaptonemal complex markers, as there are currently no available techniques to enrich for these subfractions. Therefore, study of genome occupancy of BRG1 in these specific substages by techniques such as CUT&Tag are not currently possible. However, we are currently working on new methods to distinguish these cell populations and hope eventually to use these purification strategies to perform the studies suggested by this reviewer. Alternatively, the hope is that single cell CUT&Tag methods will become more reliable, and will enable us to address these questions. Both of these options are not currently available to us. The studies by Menon et al (2024-doi:10.7554/eLife.88024.5) provide strong evidence to support that ARID1A is needed to reduce promoter accessibility of XY silenced genes in prophase I through modulation of H3.3 distribution. However, this mechanism and our identification of the removal of BRG1 between early and late pachytema are not inconsistent with one another, as either SMARCA4 or SMARCA2 can associate with ARID1A as part of the cBAF complex, and ARID1A is also not in all forms of the BAF complex which BRG1 are in. The difference between our results and those seen in Menon et al likely indicate that there are multiple forms of the BAF complex which are differentially regulated during MSCI and play different roles in silencing transcription. Further studies of specific BAF subunits are needed to elucidate how different flavors of the BAF complex act at specific genomic locations and meiotic time points.

    1. The observations presented in this manuscript (Fig. 1D, 2C, 3D, and 4) suggest a haploinsufficiency of the deleted locus in spermatogenic development. How does this compare with the ablation of either Ago3 or Ago4? Please explain.

    Response: Our previous studies in single Ago4 knockouts did not present a heterozygous phenotype (Modzelewski et al 2012, doi: 10.1016/j.devcel.2012.07.003, data not shown). Triple Ago413 knockouts show a much stronger fertility phenotype than single Ago4 knockout. Testis weight of *Ago413 *homozygous null present a 30% reduction while heterozygous mice show a 15% reduction (Figure 1D), comparable to the 13% reduction previously observed in Ago4-/- males. Sperm counts of Ago413 null and heterozygous males are reduced by 60% and 39% compared to wild type (Figure 1E), respectively, whereas Ago4 null mice have a milder phenotype, with only a 22% reduction in sperm counts. At the MSCI level, both homozygous and heterozygous Ago413 mutant spermatocytes show a similar increase in pachytene spermatocytes with increased RNA pol II ingression into the sex body with respect to wild-type of 35% and 30%, respectively. Ago4 single knockouts show an almost 18% increase in Pol II ingression when compared to wild type. These comparisons are now included in our manuscript in lines 170, 172 and 288. A milder phenotype of the Ago4 knockout and haploinsufficiency in triple Ago413 knockouts but not in Ago4 single knockouts is likely a consequence of the overlapping functions of Ago3 and Ago4 in mammals (and/or overexpression of Ago3 in Ago4 knockouts). In the context of their role in RISC, Wang et al (doi: 10.1101/gad.182758.111) studied the effects of single and double conditional knockouts for Ago1 and Ago2 in miRNA-mediated silencing. They discovered that the interaction between miRNAs and AGOs is highly correlated with the abundance of each AGO protein, and only double knockouts presented an observable phenotype.

    Minor Comments: Based on the interactome analysis, it was argued that AGO3 and AGO4 may function separately. Please discuss how AGO3 might compensate for AGO4 (Line 109).

    Response: We hypothesize that the combined function of AGO3 and AGO4 is needed for proper sex chromosome inactivation during meiosis. We base this hypothesis on the facts that (i) both proteins localize to the sex body in pachytene spermatocytes, (ii) loss of Ago4 leads to upregulation of Ago3, and (iii) the MSCI phenotype of Ago413 knockout mice is much stronger than the single Ago4 knockout (see above). However, AGO3 and AGO4 might not induce silencing through the same mechanism or pathway. In this work, we observed that their temporal expression in prophase I is different; while AGO3 protein seems to disappear by the diplotene stage, AGO4 is present in the sex body of these cells. Moreover, the proteomic analysis revealed a very low number of common interactors, an observation which could support the idea of AGO3 and AGO4 acting by different (albeit perhaps related) mechanisms to achieve MSCI. It is also possible that common interactors were not identified in our proteomic analysis due to the low abundance of AGO3 and AGO4 in the germ cells, limiting the resolution of the proteomics analysis (note that in order to visualize AGO proteins in WB experiments, at least 60 μg of enriched germ cell lysate must be loaded per lane). Moreover, given the difficulty in obtaining enough isolated pachytene and diplotene spermatocytes to perform immunoprecipitation experiments, we performed IP experiments in whole germ cell lysates, which limits the interpretation of our analysis. If AGO3 and AGO4 protein interactors overlap, then AGO3 would directly substitute for AGO4 leading to silencing in single Ago4 knockouts. However, if AGO3 and AGO4 work together through different, complementary mechanisms, then Ago4 mutant mice likely compensates loss of Ago4 by upregulation of Ago3along with specific interactors of the given pathway. We have added a sentence addressing this matter in line 411 of the results section and lines 506 and 513 of the discussion in the revised manuscript.

    In Line 221, it is unclear what is meant by 'cell cycle transcripts'. Does this refer to meiotic transcripts? It is also important to discuss the relevance of the G2/M cell cycle marker genes at later stages of meiotic prophase.

    Response: Thank you for this suggestion. We have changed the relevant text to remove redundancies and include more information. We agree that considering the importance of these genes across meiotic prophase is needed, as cells which are in the dividing stage will already have produced the proteins necessary for division. These cells likely correspond to the diplotene/M cluster cells that have a lower G2/M score, potentially causing the bimodal distribution seen in Figure 2D. We have added a sentence addressing this to the manuscript.

    While identified as a common interactor of both AGO3 and AGO4 in lines 440-445, HNRNPD is not listed among AGO4 interactors in Table S6. Please correct or explain this discrepancy.

    Response: HNRPD was originally identified as an AGO4 interactor using a less strict criteria than the one used in our manuscript: we required consistent enrichment in at least two rounds of IP MS experiments. This reference to HNRNPD was a mistake, given that HNRPD was only enriched in one of our three replicates. Thus, we apologize and have removed the sentence in lines 440-445.

    It is unclear whether wild-type cell lysate or lysate containing FLAG-tagged AGO3 was used for BRG1 immunoprecipitation, and which antibody was used to detect AGO3 in the BRG1 IP sample. A co-IP experiment demonstrating interaction between BRG1 and wild-type AGO3 would be ideal in this context. Furthermore, co-localization by IF would be beneficial to determine the subcellular localization and the cell stages the interaction may be occurring. Additionally, co-IP and Western blot methodologies should be included in the methods section.

    __Response: __MYC-FLAG tagged AGO3 protein lysates were used for BRG1 Co-Immunoprecipitation, along with an anti MYC antibody to detect AGO3. This is now detailed in the Methods section of our revised manuscript (line 1133).

    Regarding BRG1 and AGO3 colocalization by IF, we can confidently show that both AGO3 and BRG1 localize to the sex chromosomes in early pachynema by comparing BRG1/SYCP3 and FLAG-AGO3/SYCP3 stained spreads. We were not able to show colocalization simultaneously on the same cells, given the lack of appropriate antibodies. Our anti FLAG antibody is raised in mouse, while anti BRG1 is raised in rabbit, therefore a non-rabbit, non-mouse anti SYCP3 would be needed to identify prophase I substages, and our lab does not possess such a validated antibody. However, we now have access to a multiplexing kit that allows to use same-species antibodies for immunofluorescence and we can perform these experiments for a revised manuscript.

    __Response: __The methods section now includes description of co-IP methodologies (line 1132). Western Blot methodologies are explained in lane 718, under the "Immunoblotting" title.

    In line 599, it is unclear what is meant by 'persistence of sex chromosome de-repression'. Please correct or clarify this.

    Response: This sentence has been changed and reads: "The persistence of sex chromosome gene expression".

    If possible, please add an illustration to summarize the findings together.

    Response: We thank the reviewer for this suggestion, and have now added this in Figure 6

    Significance

    Overall, this study enhances the understanding of gene expression regulation by AGO proteins during spermatogenesis. Several approaches, including functional, histological, and molecular characterization of the triple knockout phenotype, were instrumental in elucidating the role of AGO proteins in MSCI and meiotic as well as postmeiotic gene regulation. The main limitation of the study is that it is challenging to appreciate the role of AGO3 in addition to the previously published role of AGO4 without the inclusion of necessary control groups. Furthermore, the mechanism of action for AGO proteins in meiotic gene regulation was left relatively unexplored. This study presents new findings that will be significant for the research community interested in gene regulation, chromatin biology, and reproductive biology with the above suggestions considered.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    The authors characterize a CRISPR-Cas9 mouse mutant that targets 3 genes that encode AGO family proteins, 2 of which are expressed during spermatogenesis (AGO3 and AGO4) and one that is said is not expressed, AGO1. This mouse mutant showed that AGO3 and AGO4 both contribute to spermatogenesis success as the "Ago413" mutation gave rise to an additive reduction in testis weight, due to spermatocyte apoptosis, and reduction in sperm count. Furthermore, they use insertion mouse mutants for Ago3 and Ago2 that express tagged versions of their corresponding proteins, which they use in combination with pan-AGO antibodies and Ago mutants to show differential expression and localization properties of AGO2, AGO3, and AGO4 (and the absence of AGO1) during spermatogenesis with a particular focus on meiotic prophase. They perform single-cell RNAseq and intricate analyses to demonstrate a change in distribution of meiotic stages in Ago413 mutants, and the overall cell cycle in spermatogonia and spermatocytes is altered. This analysis shows that the mutation leads to an inability to downregulate prior spermatogonia/spermatocyte stage transcripts in a timely manner. On the other hand, later-stage spermatocytes are abnormally expressing spermiogenesis genes. Similar to the Ago4 mutant previously characterized MSCI is disrupted. The authors also show that AGO3 has different interaction partners compared to AGO4 and focus their final assessment on a novel interaction partner of AGO3, BRG1. They show that this factor, which is involved in chromatin remodeling, is aberrantly localized to the sex body during meiotic prophase and diplonema. As BRG1 is involved in open chromatin, it is proposed that AGO3 restricts BRG1 (and related proteins) from the XY chromosome to ensure MSCI. Overall, this paper is very well constructed with mechanistic insights that make this a very impactful contribution to the research community. Major Comments:

    1. The abstract contains "Ago413-/- mouse" without any explanation of what that is. The abstract needs to be a stand-alone document that does not require any referencing for context.

    Response: We have included a sentence describing Ago413 in line 27

    Figure 2C. - The significance bars are confusing as they appear to overlap strangely.

    Response: We have modified this figure and now present the significance bars are on top of the data points.

    On line 235, the authors state that "we first identified the top non-overlapping upregulated genes for Ago413+/+ germ cells in each cluster. Why did the authors not also select down-regulated genes in each cluster to perform a similar analysis?

    __Response: __Thank you for this question. As our goal was to identify genes that are markers of the transcriptional program in each cell type, we used only uniquely upregulated genes for each cluster. Genes that are downregulated for a cluster may be indicative of the transcription in several other cell types, which is not easily interpretable. For a revised manuscript, we will perform this analysis to determine if there is any specific alterations in these downregulated genes.

    Their Ago413 mutant characterization does a good job of assessing meiotic prophase and spermatozoa. However, their assessment of the stages in between these is lacking (meiotic divisions and spermiogenesis).

    Response: We understand the reviewer's concern, however, it is not usual to study stages between the first meiotic division and spermiogenesis because meiosis II is so rapid and thus we lack tools to dissect it. In general, any defect that impacts meiosis I (and particularly prophase I) leads to cell death during prophase I or at metaphase I due to strictly adhered checkpoints that eradicate defective cells. Thus, the increased TUNEL staining in prophase I indicates to us that defective cells are cleared before exit from meiosis I, and those cells progressing to the spermatid stage are "normal" for meiosis II progression. For these cells that did complete meiosis I and progressed normally through meiosis II, we analyzed their spermiogenic outcome extensively (see section entitled "Post-meiotic spermatids from Ago413-/- males exhibit defective spermiogenesis and poor spermatozoa function"). This section included extensive sperm morphology, sperm motility and sperm fertility through in vitro fertilization assays. That said, we have added a sentence on line 268 to explain the transit through meiosis II.

    The discovery of the interaction between BRG1 and AGO3 is exciting. They should assess BRG1 localization in later sub-stages, including late diplonema and diakinesis.

    __Response: __BRG1(SMARCA4) was analyzed throughout prophase I, as shown in image 5G, including quantification of fluorescence intensity included the analysis of diplonema (5H-I). However, diakinesis was not included here since there was no observable signal of BRG1 in these cells. We have explained this in lines 459.

    ATF2 should have been assessed in more detail, as was done for BRG1 in Figure 5.

    __Response: __We agree with the Reviewer, however, staining of chromosome spreads with the anti ATF2 antibody was not possible in our hands after several attempts and changes in staining conditions. However, as staining of sections was successful, we showed localization of ATF2 on spermatocytes by co staining sections with SYCP3 and ATF2.

    Reviewer #3 (Significance (Required)): Overall, this paper is very well constructed with mechanistic insights, as described in my reviewer comments, that make this a very impactful contribution to the research community.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors characterize a CRISPR-Cas9 mouse mutant that targets 3 genes that encode AGO family proteins, 2 of which are expressed during spermatogenesis (AGO3 and AGO4) and one that is said is not expressed, AGO1. This mouse mutant showed that AGO3 and AGO4 both contribute to spermatogenesis success as the "Ago413" mutation gave rise to an additive reduction in testis weight, due to spermatocyte apoptosis, and reduction in sperm count. Furthermore, they use insertion mouse mutants for Ago3 and Ago2 that express tagged versions of their corresponding proteins, which they use in combination with pan-AGO antibodies and Ago mutants to show differential expression and localization properties of AGO2, AGO3, and AGO4 (and the absence of AGO1) during spermatogenesis with a particular focus on meiotic prophase. They perform single-cell RNAseq and intricate analyses to demonstrate a change in distribution of meiotic stages in Ago413 mutants, and the overall cell cycle in spermatogonia and spermatocytes is altered. This analysis shows that the mutation leads to an inability to downregulate prior spermatogonia/spermatocyte stage transcripts in a timely manner. On the other hand, later-stage spermatocytes are abnormally expressing spermiogenesis genes. Similar to the Ago4 mutant previously characterized MSCI is disrupted. The authors also show that AGO3 has different interaction partners compared to AGO4 and focus their final assessment on a novel interaction partner of AGO3, BRG1. They show that this factor, which is involved in chromatin remodeling, is aberrantly localized to the sex body during meiotic prophase and diplonema. As BRG1 is involved in open chromatin, it is proposed that AGO3 restricts BRG1 (and related proteins) from the XY chromosome to ensure MSCI. Overall, this paper is very well constructed with mechanistic insights that make this a very impactful contribution to the research community.

    Major Comments:

    1. The abstract contains "Ago413-/- mouse" without any explanation of what that is. The abstract needs to be a stand-alone document that does not require any referencing for context.
    2. Figure 2C. - The significance bars are confusing as they appear to overlap strangely.
    3. On line 235, the authors state that "we first identified the top non-overlapping upregulated genes for Ago413+/+ germ cells in each cluster. Why did the authors not also select down-regulated genes in each cluster to perform a similar analysis?
    4. Their Ago413 mutant characterization does a good job of assessing meiotic prophase and spermatozoa. However, their assessment of the stages in between these is lacking (meiotic divisions and spermiogenesis).
    5. The discovery of the interaction between BRG1 and AGO3 is exciting. They should assess BRG1 localization in later sub-stages, including late diplonema and diakinesis.
    6. ATF2 should have been assessed in more detail, as was done for BRG1 in Figure 5.

    Significance

    Overall, this paper is very well constructed with mechanistic insights, as described in my reviewer comments, that make this a very impactful contribution to the research community.

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    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript titled "Argonaute proteins regulate the timing of the spermatogenic transcriptional program" by Carro et al., the authors present their findings on how Argonaute proteins regulate spermatogenic development. They utilize a mouse model featuring a deletion of the gene cluster on chromosome 4 that contains Ago1, Ago3, and Ago4 to investigate the cumulative roles of AGO3 and AGO4 in spermatogenic cells. The authors characterize the distribution of AGO proteins and their effects on key meiotic milestones such as synapsis, recombination, meiotic transcriptional regulation, and meiotic sex chromosome inactivation (MSCI). They analyze stage-specific transcriptomes in spermatogenic cells using single-cell and bulk RNA sequencing and determine the interactome of AGO3 and AGO4 through mass spectrometry to examine how AGO proteins may regulate gene expression in these cells during meiotic and post-meiotic development. The authors conclude that both AGO3 and AGO4 are essential for regulating the overall gene expression program in spermatogenic cells and specifically modulate MSCI to repress sex-linked genes in pachytene spermatocytes, which may be partially mediated by the proper distribution of DNA damage repair factors. Additionally, AGO3 is suggested to interact with the chromatin remodeler SWI/SNF factor BRG1, facilitating its removal from the sex-chromatin to enable the repression of sex-linked genes during MSCI.

    Major Comments:

    The study utilized a triple knockout mouse model to determine the effect of AGO3 on spermatogenesis, following up on their previous report about the role of AGO4 in spermatogenesis, which resulted from an upregulation of AGO3 in Ago4-/- spermatocytes. However, the results are more difficult to interpret and ascertain the role of AGO3 in these cells, given the absence of any observable phenotype from Ago3 interruption. AGO4 regulates sex body formation, meiotic sex chromosome inactivation (MSCI), and miRNA production in spermatocytes, all of which were noted in the absence of both AGO3 and AGO4, with only an increased incidence of cells containing abnormal RNAPII at the sex chromosomes. It will be necessary to characterize how AGO3 regulates spermatogenic development, including meiotic progression and the regulation of the meiotic transcriptome, and compare these findings with the current observations to determine if the proposed mechanism involving AGO3, BRG1, and possibly AP2 is relevant in this context.

    Does Ago413-/- mice recapitulate the early meiotic entry phenotype observed in Ago4-/- mice? If not, could it be possible that AGO3 promotes meiotic entry, given its strong mRNA expression in spermatogonia according to the scRNAseq data (Fig. 2B) The authors suggested that the removal of BRG1 by AGO3 is necessary during sex body formation and the eventual establishment of MSCI. However, the BAF complex subunit ARID1A has been shown to facilitate MSCI by regulating promoter accessibility. It will be interesting to determine how BRG1 distribution changes across the genome in the absence of AGO proteins and how that correlates with alterations in sex-linked gene expression. The observations presented in this manuscript (Fig. 1D, 2C, 3D, and 4) suggest a haploinsufficiency of the deleted locus in spermatogenic development. How does this compare with the ablation of either Ago3 or Ago4? Please explain.

    Minor Comments:

    Based on the interactome analysis, it was argued that AGO3 and AGO4 may function separately. Please discuss how AGO3 might compensate for AGO4 (Line 109).
    In Line 221, it is unclear what is meant by 'cell cycle transcripts'. Does this refer to meiotic transcripts? It is also important to discuss the relevance of the G2/M cell cycle marker genes at later stages of meiotic prophase.
    While identified as a common interactor of both AGO3 and AGO4 in lines 440-445, HNRNPD is not listed among AGO4 interactors in Table S6. Please correct or explain this discrepancy. It is unclear whether wild-type cell lysate or lysate containing FLAG-tagged AGO3 was used for BRG1 immunoprecipitation, and which antibody was used to detect AGO3 in the BRG1 IP sample. A co-IP experiment demonstrating interaction between BRG1 and wild-type AGO3 would be ideal in this context. Furthermore, co-localization by IF would be beneficial to determine the subcellular localization and the cell stages the interaction may be occurring. Additionally, co-IP and Western blot methodologies should be included in the methods section. In line 599, it is unclear what is meant by 'persistence of sex chromosome de-repression'. Please correct or clarify this. If possible, please add an illustration to summarize the findings together.

    Significance

    Overall, this study enhances the understanding of gene expression regulation by AGO proteins during spermatogenesis. Several approaches, including functional, histological, and molecular characterization of the triple knockout phenotype, were instrumental in elucidating the role of AGO proteins in MSCI and meiotic as well as postmeiotic gene regulation. The main limitation of the study is that it is challenging to appreciate the role of AGO3 in addition to the previously published role of AGO4 without the inclusion of necessary control groups. Furthermore, the mechanism of action for AGO proteins in meiotic gene regulation was left relatively unexplored. This study presents new findings that will be significant for the research community interested in gene regulation, chromatin biology, and reproductive biology with the above suggestions considered.

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    Referee #1

    Evidence, reproducibility and clarity

    In their manuscript de las Mercedes Carro et al investigated the role of Ago proteins during spermatogenesis by producing a triple knockout of Ago 1, 3 and 4. They first describe the pattern of expression of each protein and of Ago2 during the differentiation of male germ cells, then they describe the spermatogenesis phenotype of triple knockout males, study gene deregulation by scRNA seq and identify novel interacting proteins by co-IP mass spectrometry, in particular BRG1/SMARCA4, a chromatin remodeling factor and ATF2 a transcription factor. The main message is that Ago3 and 4 are involved in the regulation of XY gene silencing during meiosis, and also in the control of autosomal gene expression during meiosis. Overall the manuscript is well written, the topic, very interesting and the experiments, well-executed. However, there are some parts of the methodology and data interpretation that are unclear (see below).

    Major comments

    1. Please clarify how the triple KO was obtained, and if it is constitutive or specific to the male germline. In the result section a Cre (which cre?) is mentioned but it is not mentioned in the M&M. On Figure S1, a MICER VECTOR is shown instead of a deletion, but nothing is explained in the text nor legend. Could the authors provide more details in the results section as well as in the M&M ? This is essential to fully interpret the results obtained for this KO line, and to compare its phenotype to other lines (such as lines 184-9 Comparison of triple KO phenotype with that of Ago4 KO). Also, if it is a constitutive KO, the authors should mention if they observed other phenotypes in triple KO mice since AGO proteins are not only expressed in the male germline.
    2. The paragraph corresponding to G2/M analysis is unclear to me. Why was this analysis performed? What does the heatmap show in Figure S4? What is G2/M score? (Fig 2D). Lines 219-220, do the authors mean that Pachytene cells are in a cell phase equivalent to G2/M? All this paragraph and associated figures require more explanation to clarify the method and interpretation.
    3. I have concerns regarding Fig2G: to be convincing the analysis needs to be performed on several replicates, and, it is essential to compare tubules of the same stage - which does not seem to be the case. This does not appear to be the case. Besides, co (immunofluorescent) staining with markers of different cell types should be shown to demonstrate the earlier expression of some markers and their colocalization with markers of the earlier stages.
    4. one important question that I think the authors should discuss regarding their scRNAseq: clusters are defined using well characterized markers. But Ago triple KO appears to alter the timing of expression of genes... could this deregulation affects the interperetation of scRNAseq clusters and results?
    5. XY gene deregulation is mentioned throughout the result section but only X chromosome genes seem to have been investigated.... Even the gene content of the Y is highly repetitive, it would be very interesting to show the level of expression of Y single copy and Y multicopy genes in a figure 3 panel.
    6. Can the authors elaborate on the observation that X gene upregulation is visible in the KO before MSCI; that is in lept/zygotene clusters (and in spermatogonia, if the difference visible in 3A is significant?)
    7. miRNA analysis: could the authors indicate if X encoded miRNA were identified and found deregulated? Because Ago4 has been shown to lead to a downregulation of miRNA, among which many X encoded. It is therefore puzzling to see that the triple KO does not recapitulate this observation. Were the analyses performed differently in the present study and in Ago4 KO study?
    8. The last results paragraph would also benefit from some additional information. It is not clear why the authors focused on enhancers and did not investigate promoters (or maybe they were but it's unclear). Which regions (size and location from TSS) were investigated for motif enrichment analyses? To what correspond the "transcriptional regulatory regions previously identified using dREG" mentioned in the M&M? I understand it's based on a previous article, but more info in the present manuscript would be useful.

    Minor comments

    1. In the introduction: The sentence "Ago1 is not expressed in the germline from the spermatogonia stage onwards allowing us to use this model to study the roles of Ago4 and Ago3 in spermatogenesis." is misleading because Ago1 is expressed at least in spermatogonia; It would be more precise to write "after spermatogonia stage" and rephrase the sentence. Otherwise it is surprising to see AGO1 protein in testis lysate and it is not in line with the scRNA seq shown in figure 2.
    2. Could the authors precise if AGO proteins are expressed in other tissues? In somatic testicular cells?
    3. Pattern of expression: How do the authors explain that AGO3 disappears at the diplotene stage and reappears in spermatids?
    4. It would be useful to show the timing of expression of AGO 1 to 4 throughout spermatogenesis in the first paragraph of the article. Maybe the authors could present data from fig2B earlier?
    5. Line 190: please modify the sentence "reveal no differences in cellular architecture of the seminiferous tubules when compared to wild-type males" to " reveal no gross differences..." since even without quantification of the different cell types it is visible that KO seminiferous tubules are different from WT tubules.
    6. TUNEL analysis: please stage the tubules to determine the stage(s) at which apoptosis is the most predominant.
    7. Figure S4B does not show an increase of cells at Pachytene stage but at Lepto/zygotene stage (as well as an increase of spermatogonia). Please comment this discrepancy with results shown in Fig2.
    8. Fig5H and 5I are not mentioned in the result section. Also, it would be useful to label them with "all chromosomes" and "XY" to differentiate them easily
    9. Line 530 "data provide further evidence for a functional association between AGO-dependent small RNAs and heterochromatin formation, maintenance and/or silencing." Please rephrase, the present article does not really show that AGO nuclear role depends on small RNAs.
    10. Line 1256: replace "cite here " by appropriate reference
    11. Please use SMARCA4 instead of BRG1 name as it is its official name.

    Figures:

    Figure 1: Are the pictures shown for Ago3-tagged and floxed from the same stages ? The leptotene stage in 1A looks like a zygotene, while some pachytene/diplotene stage pictures do not look alike.

    Figure 1D, please label the Y scale properly (testis weight related to body weight)

    FigS1: Please comment the presence of non-specific bands in the figure legend

    Fig 2E and F, please indicate on the figure (in addition to its legend), what are the X and Y axes respectively to facilitate its reading.

    2F: please use an easier abbreviation for Spermatocyte than Sp (which could spermatogonia, sperm etc..) such as Scyte I ? (same comment for Fig 3C)

    Overall, for all figures showing GSEA analyses, could the authors explain what a High positive NES and a High negative NES mean in the results section?

    Significance

    Ago proteins are known for their roles in post transcriptional gene regulation via small RNA mediated cleavage of mRNA, which takes places in the cytoplasm. Some Ago proteins have been shown to be also located in the nucleus suggesting other non-canonical roles. It is the case of Ago4 which has been shown to localize to the transcriptionally silenced sex chromosomes (called sex body) of the spermatocyte nucleus, where it contributes to regulate their silencing (Modzelewski et al 2012). Interestingly, Ago4 knockout leads to Ago3 upregulation, including on the sex body indicating that Ago3 and Ago4 are involved in the same nuclear process. In their manuscript, de las Mercedes Carro et al., investigate the consequences of loss of both Ago3 and Ago4 in the male germline by the production of a triple knockout of Ago1, 3 and 4 in the mouse. With this model, the authors describe the role of Ago3 and Ago4 during spermatogenesis and show that they are involved in sex chromosome gene repression in spermatocytes and in round spermatids, as well as in the control of autosomal meiotic gene expression. Triple KO males have impaired meiosis and spermiogenesis, with fewer and abnormal spermatozoa resulting in reduced fertility. Since Ago1 male germline expression is restricted to pre-meiotic germ cells, it is not expected to contribute to the meiotic and postmeiotic phenotypes observed in the triple KO. The strengths of the study are i) the thorough analyses of mRNA expression at the single cell level, and in purified spermatocytes and spermatids (bulk RNAseq), ii) the identification of novel nuclear partners of AGO3/4 relevant for their described nuclear role: ATF2, which they show to also co-localize with the sex body, and BRG1/SMARCA4, a SWI/SNF chromatin remodeler. The main limitation of the study is the lack of information in the method regarding the production of the triple KO, as well as some aspects of the transcriptome and motif analyses. It is also surprising to see that the triple KO does not recapitulate the miRNA deregulation observed in Ago4 KO. The characterization of a non-canonical role of AGO3/4 in male germ cells will certainly influence researchers of the field, and also interest a broader audience studying Argonaute proteins and gene regulation at transcriptional and posttranscriptional levels.

  5. Version published to 10.1101/2024.12.31.630913v1 on bioRxiv