The oocyte zinc transporter Slc39a10/Zip10 is a regulator of zinc sparks during fertilization in mice

Reviewer #1 (Public review):

The revised manuscript addresses several reviewer concerns, and the study continues to provide useful insights into how ZIP10 regulates zinc homeostasis and zinc sparks during fertilization in mice. The authors have improved the clarity of the figures, shifted emphasis in the abstract more clearly to ZIP10, and added brief discussion of ZIP6/ZIP10 interactions and ZIP10's role in zinc spark-calcium oscillation decoupling. However, some critical issues remain only partially addressed.

(1) Oocyte health confound: The use of Gdf9-Cre deletes ZIP10 during oocyte growth, meaning observed defects could result from earlier disruptions in zinc signaling rather than solely from the absence of zinc sparks at fertilization. The authors acknowledge this and propose transcriptome profiling as a future direction. However, since mRNA levels often do not accurately reflect protein levels and activity in oocytes, transcriptomics may not be particularly informative in this context. Proteomic approaches that directly assess the molecular effects of ZIP10 loss seem more promising. Although current sensitivity limitations make proteomics from small oocyte samples challenging, ongoing improvements in this area may soon allow for more detailed mechanistic insights.

(2) ZIP6 context and focus: The authors clarified the abstract to emphasize ZIP10, enhancing narrative clarity. This revision is appropriate and appreciated.

(3) Follicular development effects: The biological consequences of ZIP6 and ZIP10 knockout during folliculogenesis are still unknown. The authors now say these effects will be studied in the future, but this still leaves a major mechanistic gap unaddressed in the current version.

(4) Zinc spark imaging and probe limitations: The addition of calcium imaging enhances the clarity of Figure 3. However, zinc fluorescence remains inadequate, and the authors depend solely on FluoZin-3AM, a dye known for artifacts and limited ability to detect subcellular labile zinc. The suggestion that C57BL/6J mice may differ from CD1 in vesicle appearance is plausible but does not fully address concerns about probe specificity and resolution. As the authors acknowledge, future studies with more selective probes would increase confidence in both the spatial and quantitative analysis of zinc dynamics.

(5) Mechanistic insight remains limited: The revised discussion now recognizes the lack of detailed mechanistic understanding but does not significantly expand on potential signaling pathways or downstream targets of ZIP10. The descriptive data are useful, but the inability to pinpoint how ZIP10 mediates zinc spark regulation remains a key limitation. Again, proteomic profiling would probably be more informative than transcriptomic analysis for identifying ZIP10-dependent pathways once technical barriers to low-input proteomics are overcome.

Overall, the authors have reasonably revised and clarified key points raised by reviewers, and the manuscript now reads more clearly. However, the main limitation, lack of mechanistic insight and the inability to distinguish between developmental and fertilization-stage roles of ZIP10, remains unresolved. These should be explicitly acknowledged when framing the conclusions.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

The authors investigated the role of the zinc transporter ZIP10 in regulating zinc sparks during fertilization in mice. By utilizing oocyte-specific Zip6 and Zip10 conditional knockout mice, the authors effectively demonstrate the importance of ZIP10 in zinc homeostasis, zinc spark generation, and early embryonic development. The study is overall useful as it identifies ZIP10 as an important component of oocyte processes that support embryo development, thus opening the door for further investigations. While the study provides solid evidence for the requirement of ZIP10 in the regulation of zinc sparks and zinc homeostasis, it falls short of revealing the underlying mechanism of how ZIP10 exerts this important function.

This report is the first to clarify the role of the zinc transporters ZIP10 expressed in oocytes, which was previously unknown, and does not focus on the detailed mechanism. As you pointed out, we believe that the mechanism will also be important information in the field of fertilization and embryogenesis research, and we believe that it is necessary to consider this issue in the future.

(1) The zinc transporters the authors are knocking out are expressed in mouse oocytes through follicular development, and the Gdf9-cre driver used means these oocytes were grown in the absence of appropriate Zinc signaling. Thus, it would be difficult to assert that the lack of fertilization associated with zinc sparks is solely responsible for the failure of embryo development. Spindle morphology and other meiotic parameters do not necessarily report oocyte health, so normalcy of these features may not be a strong argument when it comes to metabolic issues.

As you rightly observe, the results of this study do not entirely exclude the possibility of oocyte health in the absence of adequate zinc homeostasis during oocyte growth. However, evidence has been presented demonstrating that spindle formation does occur in Zip10^d/d mouse oocytes (Fig.2 C), that fertilization occurs despite the absence of zinc spark (Fig.3 and Fig. 4A), and that some embryos develop to blastocysts (Fig. 4 B). We believe that future studies should evaluate the transcriptome profile of Zip10^d/ mouse oocytes.

(2) While comparing ZIP6 and ZIP10 in the abstract provides context, focusing more on ZIP10 would improve reader comprehension, as ZIP10 is the primary focus of the study. Emphasizing the specific role of ZIP10 will help the reader grasp the core findings more clearly.

Thank you for your valuable input. We have revised the summary to focus more on ZIP10 by removing the section in the summary that mentions ZIP6 (P.1-2 Line 34-52).

(3) Zinc transporters ZIP6 and ZIP10 are expressed during follicular development, but the biological significance of the observation is not clearly addressed. The authors should investigate whether the ZIP6 and ZIP10 knockout affects follicular development and discuss the potential implications.

Thank you for your valuable input. As you mentioned, we have not been able to clarify the effects of ZIP6 and ZIP10 knockout on follicle formation. However, this report clarifies the role of ZIP-mediated zinc ions in their inclusion. The effect of ZIP knockout on follicle formation will be discussed in the future.

(4) In Figure 3, the zinc fluorescence images are unclear, making it difficult for readers to interpret the data. Including snapshot images of calcium and zinc spikes as part of the main figure would improve clarity. Moreover, adding more comparative statements and a deeper explanation of why Zip10 KO mice exhibit normal calcium oscillations but lack zinc sparks would strengthen the manuscript.

Thank you for your suggestion. We have also added images of calcium elevation after fertilization to Fig. 3 and Fig. S3. In addition, the figure legends have been changed (P.29 Line 937-939, P.34 Line 1104-1106). As to why Zip10 KO mice show normal calcium oscillations but lack zinc spikes, as mentioned in Discussion (P. 10 Line 299-300), we speculate that zinc ions existed in Zip10d/d mouse oocytes induce Ca2+ release without compromising IP3R1 sensitivity. We also assume that the lack of zinc spark is due to low accumulation of zinc ion levels in the oocytes via ZIP10, as described in Discussion (P.10 Line 300-302).

(5) While the study identifies the role of ZIP10 in zinc spark generation, it lacks a clear mechanistic insight. The topic itself is interesting, but without providing a more detailed explanation of the underlying mechanisms, the study leaves an important gap. Further discussion on the signaling pathways potentially involved in zinc spark regulation would add depth to the findings.

Thank you for your input. This report is the first to clarify the role of the zinc transporters ZIP6 and ZIP10 expressed in oocytes, which was previously unknown, and does not focus on the detailed mechanism. As you pointed out, we believe that the mechanism and signaling pathways will also be important information, and we believe that it is necessary to research this issue in the future.

Reviewer #2 (Public review):

Summary:

In this important study, the authors examine the role of two zinc uptake transporters, Zip6 and Zip10, which are important during the maturation of oocytes, and are critical for both successful fertilization and early embryogenesis.

Strengths:

The authors report that oocytes from Zip10 knockout mice exhibit lower labile zinc content during oocyte maturation, decreased amounts of zinc exocytosis during fertilization, and affect the rate of blastocyst generation in fertilized eggs relative to a control strain. They do not observe these changes in their Zip6 knockout animals. The authors present clear and well-documented results from a broad range of experimental modalities in support of their conclusions.

Thank you for your positive comments.

Weaknesses:

(1) The authors' statement that Zip10 is not expressed in the oocyte nuclei (line 252). Furthermore, in that study, ZIP10 was detected in the nuclear/nucleolar positions of oocytes of all follicular stages (Chen et al., 2023), which we did not observe. This is not supported by Figure 1, where some Zip10 signal is apparent in the primordial, primary, and secondary follicle oocytes. This statement should be corrected.

Thank you for pointing this out. Our results of ISH staining (Fig. 1A) and immunofluorescence staining (Fig. 1B) showed that it was not detected at the nucleus/nucleolus location. In other words, they could not be detected at the mRNA and protein levels. Based on the results of ISH staining and immunofluorescence staining, we conclude that it is expressed in the plasma membrane.

(2) Based on the FluoZin-3AM data, there appears to be less labile zinc in the Zip10d/d oocyte, eggs, and embryos; however, FluoZin-3AM has a number of well-known artifacts and does not accurately capture the localization of labile zinc pools. The patterns do not correspond to the well-documented zinc-containing cortical vesicles. Another zinc probe, such as ZinPyr-4 or ZincBY-1 should be used to visualize the zinc vesicles and confirm that there is less labile zinc in these locations as well.

Thank you for your suggestion. Previous studies (Lisle et al., 2013, Reproduction) and our report (Kageyama et al., 2022, Animal Science Journal) have shown that it is possible to examine the presence of labile zinc ions in oocytes and embryos. In addition, mouse oocytes (embryos) reported in previous studies are from CD1 (ICR) mice, whereas our study was conducted using C57BL/6J mice. In our report (Kageyama et al., 2024, Journal of Reproduction and Development), we reported that the appearance of zinc vesicles in the oocytes observed by Fluozin-3AM staining in CD1 and C57BL/6J mice is different, and we believe that this appearance of cortical vesicles in C57BL/6J mice is not a problem. As you say, we have not used other zinc probes and will consider this in the future.

(3) Line 268 The results indicate that ZIP10 is mostly responsible for the uptake of zinc ions in mouse oocytes. The situation seems a bit more complicated given that the differences in labile zinc content between oocytes from the WT and Zip10d/d animals are small (only 20-30 %) and that the zinc spark is diminished but still apparent at a low level in the Zip10d/d oocytes. Clearly, other factors are involved in zinc uptake at these stages. A variety of studies have suggested that Zip6 and Zip10 work together, perhaps even functioning as a heterodimer in some systems. The double KO would address this more clearly, but if it is not available, it might be more prudent to state that Zip10 plays some role in uptake of zinc in mouse oocytes while the role of Zip6 remains uncertain.

We would like to express our gratitude for the comments received. The phenotype of double knockout mice for ZIP6 and ZIP10 will be discussed at a future date. We have also added to the text that the role of ZIP6 remains uncertain (P. 11 Line 353-354).

(4) Zip6d/d oocytes did not have changes in labile zinc, nor did the lack of Zip6 have an impact on the zinc spark. However, Figure S1 does show a small amount of detectable Zip6 in the western blot. It is possible that this small amount could compensate for the complete lack of Zip6. Can ZIP6 be found in immunofluorescence of GV oocytes or MII eggs from the Zip6d/d animals? Additionally, it is possible that Zip6's role is only supplementary to that of Zip10. The authors should discuss this possibility. It would also be interesting to see if the Zip6/Zip10 double knockout displays greater defects compared to the Zip10 knockout when considering previous studies.

Thank you for your input. The mice are deficient in the gene so that ZIP6 is not functional. It is our notion that the results of WB analysis are not indicative of protein structural functionality, even in cases where the ZIP6 antibody detects a small amount of protein. Since the role of ZIP6 was not elucidated in this study, we added a statement to that effect in the text (P. 11 Line 353-354). In addition, studies using ZIP6/Zip10 double knockout mice will be discussed in the future.

Recommendations for the authors: please note that you control which revisions to undertake from the public reviews and recommendations for the authors.

We have revised the text based on the reviewerʼs suggestions.

Reviewer #1 (Recommendations for the authors)：

(1) In lines 133-136, it seems that the authors would like to aim to emphasize the lack of research on oocytes compared to other tissues and cells. However, the inclusion of unrelated contexts, such as the role of ZIP10 in cancer and skin, appears unnecessary and detracts from the focus on oocyte-specific mechanisms. Removing these unrelated sentences would help maintain clarity and relevance in the introduction.

*As you indicated, we removed the sentence that is not related to oocytes (P.4 Line 120-125). Further, they reported that targeted disruption using Zip6- and Zip10- specific morpholino injection or antibody incubation induced alteration of the intracellular labile zinc content, spontaneous resumption of meiosis from the PI arrest and premature arrest at a telophase I-like state (Kong et al., 2014). It is clear from these reports that ZIP6 and ZIP10 are involved in zinc transport in oocytes, but the function is not elucidated.”

(2) Ensure that all video files are properly labeled to enhance understanding.

Improved video labels for clarity (Movie 1-8, Movie S1-S4)

(3) Correct mislabeling issues, such as the one in line 209.

Corrected as follows: Zip10^d/d mouse oocytes can be fertilized but were unlikely to develop to blastocysts (P. 6-7 Line 196-197).

(4) In Figure 4D, the amount of ZIP2 appears to increase relative to actin. Including quantification would make the data more robust. Similarly, in Figure 4F, JUNO levels appear increased in Zip10 KO. Please provide quantification.

The WB band images in Fig. 4D were quantified and their graphs were added to lower part of Fig. 4D. Furthermore, the Juno of Immunofluorescent images in Figure 4F were quantified and their graphs were added to Fig. S4. Figure legends and text were corrected and added.P. 30 Line 975-979: Expression level of β-actin serves as a protein loading control and quantified the expression level of ZP2. Molecular mass is indicated at the left. Statistical differences were calculated according to the one-way ANOVA. Different letters represent significant differences (p < 0.05).

P. 35 Line: Fig. S4 Comparison of JUNO expression in Zip10^f/f and Zip10^d/d mouse MII oocytes. To measure JUNO-immunofluorescence intensity, oocytes images were selected as regions of interest (ROIs) and measured using ImageJ. Statistical differences were calculated according to student’s t-test (p > 0.05; no significant difference).P.7 Line 206-209: As for the expression of JUNO, it had the same expression than between null and control oocytes (Fig. S4) and the temporal dynamics of its disappearance from the cortex after fertilization was similar for both Zip10^f/f and Zip10^d/d groups (Fig. 4F).

(5) Some of the sentences lack proper references.

The entire text was reviewed and references inserted where necessary.

P.7 Line 221, P.7 Line222-223, P.8 Line 253-254, P.12 Line 358-360 and P.24 Line 698-699.

Reviewer #2 (Recommendations for the authors)：

Revisions are warranted in order to address the issues noted in the Weaknesses section of the Public Review.

Thank you for your comments, we have individually addressed the areas you pointed out in the Weaknesses section. The following text has also been corrected and edited.

(1) Line 247 "In primordial follicles, the ooplasmic staining of ZIP10 we anticipate corresponds to ooplasmic vesicular sites.

The text of P. 8 Line 230-232 was revised as follows.

"In primordial follicles, the ooplasm staining of ZIP10 we anticipate corresponds to ooplasmic vesicular sites.

(2) Line 926 "ZP2 was not stained in primordial follicle, but primary, secondary, and antral follicles stained. FOXL2 was observed in granulosa cells in 928 of all stage follicles. The scale bar represents 20 μm of primordial-secondary follicle and 150 μm of antral follicle." All three sentences have grammar issues that should be fixed.

The text of p.28 Line 908-911 was revised as follows.

It was observed that ZP2 was not present in the primordial follicle; however, it was present in the primary, secondary and antral follicles. Furthermore, FOXL2 was observed at granulosa cells of all stage follicles. Scale bar: 20 µm (primordial, primary and secondary follicle); 150 µm (antral follicle).