The oocyte zinc transporter Slc39a10/Zip10 is a regulator of zinc sparks during fertilization in mice
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eLife Assessment
This study presents significant and novel insights into the roles of zinc in mammalian meiosis/fertilization events. These findings are useful to our understanding of these processes. The evidence presented is solid, with experiments being well-designed, carefully described, and interpreted with appropriate rigor.
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Abstract
Abstract
In all vertebrates studied to date, a rise(s) in intracellular calcium is indispensable for successful fertilization and further embryonic development. Recent studies demonstrated that zinc is ejected to the extracellular milieu, the ‘zinc spark’, and follows the first few calcium rises of fertilization. However, the role of the zinc sparks in fertilization and development, and the supporting influx mechanism(s) are unknown. In this study, we focused on zinc transporters Zip6/Slc39a6 and Zip10/Slc39a10 both of which are expressed in mouse oocytes through follicular development, and investigated the oocyte-specific deficient mice for Zip6 (Zip6d/d: Zip6flox/flox Gdf9Cre/+) and Zip10 (Zip10d/d: Zip10flox/flox Gdf9Cre/+). Zip10 mRNA or ZIP10 protein was expressed throughout folliculogenesis in the oocyte or plasma membrane, respectively. ZIP6 protein was also expressed in the nuclear localization in the oocytes and granulosa cells throughout folliculogenesis. The number of ovulated oocytes was examined in Zip6d/d and Zip10d/d mice, and no change from the number of oocytes was observed for either strain. Zip10d/d oocytes decreased zinc level in the oocytes, but did not affect maturation and metaphase II spindles formation. The levels of zinc fluorescence intensity in the Zip6d/d oocytes were not different from the Zip6f/f oocytes. Fertilization-induced calcium oscillations were present in both Zip6d/d and Zip10d/d oocytes, but zinc sparks were not observed in Zip10d/d oocytes. Despite other events of egg activation proceeding normally in Zip10d/d oocytes, embryo development into 4-cells and beyond was compromised. We show here for the first time that the zinc transporter ZIP10 contributes to zinc homeostasis in oocytes and embryos, highlighting the role of labile zinc ions in early development.
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eLife Assessment
This study presents significant and novel insights into the roles of zinc in mammalian meiosis/fertilization events. These findings are useful to our understanding of these processes. The evidence presented is solid, with experiments being well-designed, carefully described, and interpreted with appropriate rigor.
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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.
(1) The zinc transporters the authors are knocking out are expressed in mouse oocytes …
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.
(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.
(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.
(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.
(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.
(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.
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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.
Weaknesses:
(1) The authors' statement that Zip10 is not expressed in the oocyte nuclei (line …
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.
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.
(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.
(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.
(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.
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