Tmc7 deficiency causes acrosome biogenesis defects and male infertility in mice

Reviewer #1 (Public Review):

Summary:
TMC7 knockout mice were generated by the authors and the phenotype was analyzed. They found that Tmc7 is localized to Golgi and is needed for acrosome biogenesis.

Strengths:
The phenotype of infertility is clear, and the results of TMC7 localization and the failed acrosome formation are highly reliable. In this respect, they made a significant discovery regarding spermatogenesis.

Weaknesses:
There are also some concerns, which are mainly related to the molecular function of TMC7 and Figure 5. It is understandable that TMC7 exhibits some channel activity in the Golgi and somehow affects luminal pH or Ca2+, leading to the failure of acrosome formation. On the other hand, since they are conducting the pH and calcium imaging from the cytoplasm, I do not think that the effect of TMC7 channel function in Golgi is detectable with their methods. Rather, it is more likely that they are detecting apoptotic cells that have no longer normal ion homeostasis. Another concern is that n is only 3 for these imaging experiments.

Reviewer #2 (Public Review):

Summary:

This study presents a significant finding that enhances our understanding of spermatogenesis. TMC7 belongs to a family of transmembrane channel-like proteins (TMC1-8), primarily known for their role in the ear. Mutations to TMC1/2 are linked to deafness in humans and mice and were originally characterized as auditory mechanosensitive ion channels. However, the function of the other TMC family members remains poorly characterized. In this study, the authors begin to elucidate the function of TMC7 in acrosome biogenesis during spermatogenesis. Through analysis of transcriptomics datasets, they identify TMC7 as a transmembrane channel-like protein with elevated transcript levels in round spermatids in both mouse and human testis. They then generate Tmc7-/- mice and find that male mice exhibit smaller testes and complete infertility. Examination of different developmental stages reveals spermatogenesis defects, including reduced sperm count, elongated spermatids, and large vacuoles. Additionally, abnormal acrosome morphology is observed beginning at the early-stage Golgi phase, indicating TMC7's involvement in proacrosomal vesicle trafficking and fusion. They observed localization of TMC7 in the cis-Golgi and suggest that its presence is required for maintaining Golgi integrity, with Tmc7-/- leading to reduced intracellular Ca2+, elevated pH, and increased ROS levels, likely resulting in spermatid apoptosis. Overall, the work delineates a new function of TMC7 in spermatogenesis and the authors suggest that its ion channel activity is likely important for Golgi homeostasis. This work is of significant interest to the community and is of high quality.

Strengths:

The biggest strength of the paper is the phenotypic characterization of the TMC7-/- mouse model, which has clear acrosome biogenesis/spermatogenesis defects. This is the main claim of the paper and it is supported by the data that are presented.

Weaknesses:

The claim is that TMC7 functions as an ion channel. It is reasonable to assume this given what has been previously published on the more well-characterized TMCs (TMC1/2), but the data supporting this is preliminary here, and more needs to be done to solidify this hypothesis. The authors are careful in their interpretation and present this merely as a hypothesis supporting this idea.

Reviewer #3 (Public Review):

Summary:

In this study, Wang et al. have demonstrated that TMC7, a testis-enriched multipass transmembrane protein, is essential for male reproduction in mice. Tmc7 KO male mice are sterile due to reduced sperm count and abnormal sperm morphology. TMC7 co-localizes with GM130, a cis-Golgi marker, in round spermatids. The absence of TMC7 results in reduced levels of Golgi proteins, elevated abundance of ER stress markers, as well as changes of Ca2+ and pH levels in the KO testis. However, further confirmation is required because the analyses were performed with whole testis samples in spite of the differences in the germ cell composition in WT and KO testis. In addition, the causal relationships between the reported anomalies await thorough interrogation.

Strengths:
The microscopic images are of great quality, all figures are properly arranged, and the entire manuscript is very easy to follow.

Weaknesses:
Tmc7 KO male mice show multiple anomalies in sperm production and morphogenesis, such as reduced sperm count, abnormal sperm head, and deformed midpiece. Thus, it is confusing that the authors focused solely on impaired acrosome biogenesis. Further investigations are warranted to determine whether the abnormalities reported in this manuscript (e.g., changes in protein, Ca2+, and pH levels) are directly associated with the molecular function of TMC7 or are the byproducts of partially arrested spermiogenesis. Please find additional comments in "Recommendations for the authors".