KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.


2) Chimeric (XX/XY) mice were generated by injection of Zfp541 KO ES cells into blastocysts
to study the requirement of Zfp541 function. This approach lacks an explanation/rationale.
Why not injecting Zfp541+/-(Het) ES cells to generate chimera mice for germline transmission? Is it because Zfp541+/-(Het) ES cells were never obtained? If this were the case, a few sentences of explanation/description would be very useful.
 We obtained Zfp541+/-(Het) ES cells without any trouble. However, we injected KO ES cells to examine the phenotype quickly. By taking advantage of the chimeric analysis that has neighboring WT cells as control, we clearly showed that the interaction contributes to the translocation of KCTD19 from the cytosol to the nucleus. We mentioned these points in the manuscript (L.235-237 and L.250-252) L.235-237: "To reveal the function of ZFP541 and its relationship with KCTD19, we analyzed Zfp541 KO phenotype with chimeric mice (chimeric analysis), which enables rapid gene function analysis (4-5)" L. 250-252: "The KCTD19 intensity became weaker, although not lost, in the nuclei of Zfp541 deficient spermatocytes than that of adjacent WT spermatocytes ( Fig 7A)." Minor: 1) Line 35, change "that consisting…" to "that consists…" 2) Line 202, change "anit-KCT19" to "anti-KCTD19".
 Thank you for pointing these points. We revised them. KO mouse line, demonstrated the loss of KCTD19 expression using polyclonal and monoclonal antibodies, and show that these mice are unable to produce litters with wildtype females ( fig. 1). Histological analysis of the Kctd19 null male testes also revealed significantly smaller testis-to-body weight ratios, a loss of normal testicular architecture, and an increase in the number of cells undergoing apoptosis, implicating Kctd19 in sperm production in male mice ( fig. 2). These spermatocytes also could not complete meiosis and accumulated in the seminiferous tubules ( fig. 2). Immunostaining of prophase spermatocytes with antibodies against the synaptonemal complex and spindle apparatus show that Kctd19 null spermatocytes experience a significant increase in chromosome misalignment and the formation of SYCP3 polycomplexes, indicating that KCTD19 may be important for metaphase I organization ( fig. 3). In fig. 4, the authors were able to rescue the Kctd19 null phenotype with an epitope-tagged Kctd19 transgene. In fig. 5, Oura et al. show that ZFP541 and HDAC1 are putative interactors with KCTD19 via IP-MS. Overall, the authors claim that KCTD19 associates with ZFP541 after the late pachytene stage, in addition to HDAC1, though the interaction timing of both complexes remains unknown.
One major strength of this paper is the interesting finding that KCTD19 likely associates with ZFP541 after the late pachytene stage and that KCTD19 is essential for the metaphaseanaphase transition. However, the following suggestions may improve the manuscript: MAJOR CONCERNS 1. One major concern of this paper is that Oura et al. recognize that Choi et al. already demonstrated that KCTD19 complexes with ZFP541, which was the main conclusion of this paper by Oura et al. This suggests that the findings in this paper have already been reported and more follow-up experiments are needed to offer novel results.
 As the reviewer pointed out, Choi et al. previously demonstrated that KCTD19 complexes with ZFP541. In the present study, as mentioned above, we clearly showed that the interaction contributes to the translocation of KCTD19 from the cytosol to the nucleus using the chimeric analysis. More importantly, we knocked out both genes and revealed their necessity during male meiosis. To emphasize this point and avoid misleading, we changed the title of this paper. Fig. 1h, 4c, 4e, 5a, and 5b do not appear to be specific. This is particularly concerning since these antibodies were used for the IP-MS experiment.

The antibodies used in
 Thank you for pointing this out. We deleted the word "specifically" in L.116. As the reviewer mentioned, antibodies always have issues in specificity. To make our data concrete, we used two different antibodies for both IHC (mAB #22-15 for endogenous KCTD19 and anti-HA for transgenically expressed KCTD19-HA) and IP (pAB and mAB #19-3).

MINOR CONCERNS
1. The labeling system of the KO lines, transgenic lines, and the antibodies used was not clear or easy to follow.
 We clarified gene names for KO line labeling and epitope tags for transgenic lines. For monoclonal antibodies, we used their original clone number to clearly show they were produced in different clones of hybridomas.
2. The chromosome spreads presented in fig. 3 are not spread enough to allow the reader to look at SC morphology. It would also be helpful to have the yH2AX signal overlayed with the SYCP3 channel to investigate sex body formation.
 We revised the Figure 3 with well-spread spermatocytes and overlaid images. To do so, we moved PND20 spread pictures to the supplemental figure (Fig. S3).
3. There are grammatical and syntax errors throughout this manuscript which made it hard to understand some of the descriptions. The title states that KCTD19 is required for meiotic exit. And the abstract states that KCTD19 and ZFP541 are essential for meiotic exit. The phenotype observed by the authors in mice lacking KCTD19 is metaphase arrest and lack of round spermatids. The phenotype observed in mice lacking ZFP541 is depletion of diplotene cells, presence of gamma-H2AX during late prophase and absence of round spermatids. Although these phenotypes occur late during prophase, they do not necessarily suggest a role for KCTD19 and ZFP541 in "meiotic exit".
And there is no data provided in the manuscript to suggest that. I suggest modifying the text and title to better represent what is shown, or providing additional background and discussion detailing why the authors conclude this role for KCTD19 and ZFP541.
 We sincerely appreciate this comment. We realized "meiotic exit" was an exaggeration and incorrect. We revised the title and changed "meiotic exit" to "meiosis" throughout the manuscript.
There is another instance where the language used is too strong and not supported by the data: in the abstract the authors state that KCTD19 interacts with HDAC1 and that this indicated that KCTD19 is involved in chromatin modification. While this may be the case, this statement should be modified or moved to the discussion section given there is no additional data presented to support a role for KCTD19 in chromatin modification.  We showed high-magnified images as well and quantified them (Fig.2D, E).
In Figure 6(F), cell types should be labeled to help interpretation of the data.  We agree that it is unreasonable to conclude whether Zfp541 KO spermatocytes reached metaphase or not by images only. Since the same conclusion can be supported by Fig.7E (Fig. 6J in the original manuscript), we revised the text as below: L. 248-249: "Zfp541 deficient spermatocytes were mainly eliminated by apoptosis in stage X -I seminiferous tubules (Fig 6I and 6J)" Minor comments: Since the antibodies used to detect KCTD19 are C-terminally located, can the authors exclude the possibility that the N-terminal region containing the BTB domain is expressed and stable in the del allele? This can be easily tested if there is an available antibody against the N-terminus.
 Thank you for the comment. Actually, we gave up producing antibodies against Nterminus because we could not obtain enough amount of recombinant protein in the E.coli expression system.
In figures plotting testis weights, the same units (either mg or g) should be used for both the testis and body weights.
 We used g throughout the manuscript.
In Figure 2(G) it would be useful to have cell types annotated (e.g. pachytene, round spermatid etc.).
 We labeled the cell type.
In figure 5(F) the DNA and HDAC1 images don't match for stage VII-VIII. Please correct.
 We showed low-magnified images for comparison of WT and KO, and showed highmagnified images only in WT for mentioning the similar immunostaining pattern with KCTD19.
A figure showing protein domain structure of ZFP541 within Figure 6 would be nice.
 We prepared the figure (Fig.6C) The absence of nucleus-wide gamma-H2AX in early pachytene but presence in late pachytene in cells lacking ZFP541 is interesting. Does this imply that new damage is being introduced at late pachytene? This phenotype is not discussed. How do the authors interpret this?
 We interpret the result in the same way as the reviewer. In addition to regained γH2AX foci, we observed SYCP1 spreading outside of the synaptonemal complex axis.
Considering DSBs persist on chromosomes into the pachytene stage, we assumed that precocious desynapsis or delays in other processes reactivated DSB protein activity. We mentioned this point in the discussion section (L.308-312).
L.308-312: "It is known that Spo11 and other DSBs persist on chromosomes into the pachytene stage, and engagement of homologous chromosomes is one mechanism for restraining DSB proteins activity (42). Therefore, precocious SYCP1 dissociation (desynapsis) or delays in other processes might cause the reactivation of DSB proteins remaining on chromosomes." There are several typos in figures: e.g. the words metaphase (3D) and pachytene (6J) are misspelled. And typos are present in figure legends: e.g. legend of Figure 5 (D) is incorrect, there is a typo in legend of Figure 5 (E) and S2(E), and S2 refers delta-POZ mice whereas figure labels state delta-BTB mice. The title of the first results section also has a typo.
 Thank you for pointing out these mistakes. We revised all of them.
Have all data underlying the figures and results presented in the manuscript been provided?
Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.
 We showed whole semi-quantitative data for MS analysis in supplemental table S3. Also, we prepared supplemental table S4 for numerical data of the pups number ( Fig. 1I and J), testis and body weight ( Fig. 2B and 4K), TUNEL staninig ( Fig. 2E and 6J), and meiotic prophase I ratio (Fig. 3B, Fig. S3B, Fig.7E).