Loss of circSRY reduces γH2AX level in germ cells and impairs mouse spermatogenesis

The study proved that the loss of Sry circular RNA in testis causes impaired spermatogenesis, and this damage may be related to the decline in the γH2AX level during the meiosis I stage.

Referee #1 Review Report for Author: I raised two major concerns in the initial review: 1) co-localization of circSRY with miR-138-5p in spermatocytes; 2)a lack of rescue experiments to validate the proposed sponge function. The first concern was addressed adequately, but the second remains because the experiments performed were highly questionable. The antagomir strategy is not reliable at all. They injected 10 microliters of solution into the testis, which for sure would cause damage to the spermatogenic cells due to increased intratubular pressure.
Referee #3 Review Report for Author: In this manuscript, Song and colleagues examine the role of the circular form of the Sry RNA (circSry), which is a master regulator of mammalian sex (testis) determination. They find that, while it is not required for somatic sex determination, circSry is required in meiotic germ cells, as circSry KO mice exhibit reduced sperm count, likely due to increased apoptosis and loss of spermatocytes. They propose a mechanism in which circSry acts as a sponge for miR-138-5p to regulate H2ax expression. Overall, the studies are rigorously performed and the data is clearly presented in detail. The strength of the study is in its novel examination of the circular version of Sry and finding a germ-cell-specific role for this version of the RNA. These findings are of interest to both the general molecular biology, for its study of circular RNA and gene regulation, and to the reproductive biology field, for its examination of a new role for Sry. However, there are some shortcomings in the manuscript, the main one being that the link between circSry, miR-138-5p, and yH2Ax is not strongly demonstrated, which weakens support for the model proposed by the authors. Fig. EV5 are rather limited, especially when compared to similar analyses in Figs. 4 and 5. In particular, it is unclear what the source of the increased yH2AX levels is. Is it due to more spermatocytes? More expression within the same number of spermatocytes? More DNA damage in general (since yH2AX also can be due to general DNA damage and is not specific to meiosis)? Furthermore, it is unclear why only a subset of testes were chosen in Fig. EV5H  Thank you for submitting your manuscript entitled "Loss of circSRY impairs mouse spermatogenesis via reducing H2AX level in germ cells" to Life Science Alliance. We invite you to re-submit the manuscript, revised to address the following points:

The antagomir experiments in
-Due to the concerns with the antagomir assays, the claims made regarding links between H2AX and miR-138-5p should be toned down. -Address Reviewer 3's comments.
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Thank you for this interesting contribution to Life Science Alliance. We are looking forward to receiving your revised manuscript.
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Editor:
I am happy to inform you that Dr. Sawey is interested in your findings, and would like to publish this manuscript at LSA pending the following revisions: Due to the concerns with the antagomir assays, the claims made regarding links between H2AX and miR-138-5p should be toned down.
Response: We thank you for your valuable comment and will address the reviewers' concerns the best way possible.
We did not observe a phenotypic recovery in antagomir assays, our current model only partially explained the relationship between circSRY, miR-138-5p, and γH2AX, thus we have revised the summary in the last paragraph to "In summary, our results suggest that circSRY acts as a sponge to competitively bind miR-138-5p in vivo, and the loss of circSRY leads to reduced γH2AX level and defected meiosis, which further impairs spermatogenesis in adult mice." We also replaced the manuscript title with "Loss of circSRY reduces γH2AX level in germ cells and impairs mouse spermatogenesis" for a more accurate statement.

Referee #3:
In this manuscript, Song and colleagues examine the role of the circular form of the Sry RNA (circSry), which is a master regulator of mammalian sex (testis) determination. They find that, while it is not required for somatic sex determination, circSry is required in meiotic germ cells, as circSry KO mice exhibit reduced sperm count, likely due to increased apoptosis and loss of spermatocytes. They propose a mechanism in which circSry acts as a sponge for miR-138-5p to regulate H2ax expression. Overall, the studies are rigorously performed and the data is clearly presented in detail. The strength of the study is in its novel examination of the circular version of Sry and finding a germ-cell-specific role for this version of the RNA.
These findings are of interest to both the general molecular biology, for its study of circular RNA and gene regulation, and to the reproductive biology field, for its examination of a new role for Sry. However, there are some shortcomings in the manuscript, the main one being that the link between circSry, miR-138-5p, and yH2Ax is not strongly demonstrated, which weakens support for the model proposed by the authors.
Response: Thanks for your time reviewing our manuscript. Your encouraging comment is greatly appreciated. Due to the unsuccessful phenotypic recovery in antagomir assays, our current model only partially explained the relationship between circSRY, miR-138-5p, and γH2AX. Thus, we agree with you about the shortcomings in the manuscript which is also mentioned by the editor and toned down the claims regarding links between H2AX and miR-138-5p. We have revised the summary in the last paragraph to "In summary, our results suggest that circSRY acts as a sponge to competitively bind miR-138-5p in vivo, and the loss of circSRY leads to reduced γH2AX level and defected meiosis, which further impairs spermatogenesis in adult mice." We also changed the manuscript title into "Loss of circSRY reduces γH2AX level in germ cells and impairs mouse spermatogenesis" for a more accurate statement. Fig. EV5 are rather limited, especially when compared to similar analyses in Figs. 4 and 5. In particular, it is unclear what the source of the increased yH2AX levels is. Is it due to more spermatocytes? More expression within the same number of spermatocytes? More DNA damage in general (since yH2AX also can be due to general DNA damage and is not specific to meiosis)?

The antagomir experiments in
Response: We thank the reviewer for this comment. We performed co-immunostaining of DDX4 with γH2AX or SOX9 in miR-138-5p antagomir-injected testes of circSRY KO mice to verify the source of increased γH2AX. The ratio of DDX4 positive cells to SOX9 positive cells in antagomir-injected testes was comparable to that of the control group. However, the γH2AX level was increased in antagomir-injected testes, implying the increased γH2AX came from the elevated expression of γH2AX within germ cells. The data were shown below. Elevated expression of γH2AX was detected in antagomir-injected testis (A) Representative image of immunofluorescence staining of SOX9 (red), MVH (green) and DAPI (blue) in antagomir-injected or control circSRY KO mice testes (n=3 biologically independent experiments). Scale bar indicates 25 μm.
Furthermore, it is unclear why only a subset of testes were chosen in Fig. EV5H (6 testes), when the authors had many samples from which to choose. If there was truly a significant increase in yH2AX expression, a larger sample size would have strengthened this analysis; conversely, if they were to use all the samples and would find a large variability that results in a non-significant difference, then the current limited analysis appears to be misleading.
Response: Indeed, a larger sample size would offer stronger proof for an increased γH2AX expression. However, due to insufficient experimental materials for performing all the related assays using one testes sample, we randomly selected 6 testes for γH2AX expression detection, the remaining was used for paraffin embedding and staining experiment of MVH、SOX9 and γH2AX.
Because the correlation between miR-138-5p and γH2AX was not strongly demonstrated, we have changed our conclusion in the last paragraph into "In summary, our results suggest that circSRY acts as a sponge to competitively bind miR-138-5p in vivo, and the loss of circSRY leads to reduced γH2AX level and defected meiosis, which further impairs spermatogenesis in adult mice." We also changed the title with "Loss of circSRY reduces γH2AX level in germ cells and impairs mouse spermatogenesis".
Finally, are there any positive controls available to verify that the antagomir actually worked to reduce miR-138-5p activity? Overall, there are several major concerns with the antagomir assays.
Response: Antagomir is a modified inhibitor of miR-138-5p, of which activity on reducing miR-138-5p has been shown in vitro (Fig. 6H). Furthermore, antagomir is a validated commercial product, which has been reported in related studies to inhibit miR-138-5p activity in vivo (Morton et al., 2008;Nama et al., 2019).
At a minimum, any claims about links between H2ax and miR-138-5p should be softened.
Response: We agree with your comments. Due to the weak correlation between miR-138-5p and γH2AX, we have toned down our conclusion in the last paragraph into "In summary, our results suggest that circSRY acts as a sponge to competitively bind miR-138-5p in vivo, and the loss of circSRY leads to reduced γH2AX level and defected meiosis, which further impairs spermatogenesis in adult mice." We also changed the title with "Loss of circSRY reduces γH2AX level in germ cells and impairs mouse spermatogenesis". Figure 2F, the authors need to quantify cell death (TUNEL+ cells).

In
Response: Thank the reviewer for the helpful suggestion. We performed immunostaining of DDX4 and TUNEL assay to quantify the death cells within seminiferous tubules. The number of TUNEL-positive cells were significantly higher than control group. We also calculated the ratio of apoptotic cells to DDX4-positive cells. The ratio of TUNEL-positive cells to DDX4-positive cells was significantly higher than control group. The data were shown in Figure 2F. DDX4 and TUNEL co-staining in circSRY KO and control mice testis (A) Germ cells were labeled with the antibody against DDX4 (red) and demonstrated TUNEL assay (green) in circSRY KO and control 9-week-old mouse testes. Scale bar indicates 50 μm. (B and C) Number of TUNEL-positive cells or ratio of TUNEL-positive cells to DDX4-positive cells within seminiferous tubules in circSRY KO and control 9-week-old mouse testes. The total tubule number reached 100 from more than 3 biologically independent experiments (****P <0.0001; unpaired, Student's t-test).
3. In Fig. 2I, 3B, and EV3B (and elsewhere), the y-axis label units are misleading for the sperm counts since it jumps from 0 to 5x10^6. This scale is somewhat misleading in terms of the magnitude of change.
Response: We thank the reviewer for pointing out this issue. We have changed the scale of Y axis of Figure 2I, 3B, 3F and Figure   6. Statistics should be performed and significant differences should be denoted in Fig.   6B.
Response: We thank the reviewer for pointing out this issue. We have performed the statistical analysis in Figure 6B. The data were shown below.

section.
Response: Thank the reviewer for the suggestion. We have added information on mouse strains used in our manuscript, please see the Materials and Methods section.
8. Minor point: GAPDH should be labeled as Gapdh in qRT-PCR graphs, since it refers to RNA and not protein.
Response: Thank the reviewer for pointing out this error. We have replaced GAPDH with Gapdh in Figure 2C and Figure 5H. 9. Minor point: There are still many remaining minor typographical and grammatical errors in the manuscript. These likely can be addressed during copyediting.
Response: Thank the reviewer for the helpful suggestion. We have carefully revised our manuscript and corrected typographical and grammatical errors. Thank you for submitting your revised manuscript entitled "Loss of circSRY reduces yH2AX level in germ cells and impairs mouse spermatogenesis". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. Thank you for submitting your Research Article entitled "Loss of circSRY reduces yH2AX level in germ cells and impairs mouse spermatogenesis". It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance. Congratulations on this interesting work.
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