LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility

Reviewer #1 (Public Review):

Hwang et al., report that LRRC23 is required for RS3 head assembly and sperm motility, and the truncating LRRC23 is associated with asthenozoospermia in humans. They identified an LRRC23 variant in a consanguineous Pakistani family with infertile males diagnosed as asthenozoospermia and found this variant leads to early termination of LRRC23 translation with loss of 136 amino acids at the C-terminus. They generated Lrrc23 mutant mice that mimic the predicted outcome in human patients and found the truncated LRRC23 specifically disorganizes RS3 and the junctional structure between RS2 and RS3 in the sperm axoneme, which causes sperm motility defects and male infertility. These dates try to elucidate the pathogenicity of LRRC23 in asthenozoospermia. The conclusions of this paper are mostly well supported by data, but many aspects of data analyses and data interpretations need to be improved.

1. The pathogenesis of truncating LRRC23 in asthenozoospermia needs to be further considered. The molecular mechanism of LRRC23 demonstrated in mice should be tested in patients with the LRRC23 variant. As it may be difficult to determine the structures of RS3 in the infertile male sperm, the LRRC23 localization should be observed in the sperm from patients with the LRRC23 variant.
2. The absence of the RS3 head in LRRC23Δ/Δ mouse sperm is not sufficient to support the specific localization of LRRC23 in RS3 head. Although LRRC23 might bind to RS head protein RSPH9, the authors state that "RSPH9 is a head component of RS1 and RS2 like in C. reinhardtii (Gui et al, 2021), but not of RS3" as the protein level and the localization of RSPH9 is not altered in LRRC23Δ/Δ sperm. Thus, the specific localization of LRRC23 in RS3 head should be further confirmed.
3. The interaction between LRRC23 and RSPH9 needs to be defined. AlphaFold models could help determine the likelihood of a direct interaction. In addition, the structure of the 96-nm modular repeats of axonemes from the flagella of human respiratory cilia has been determined (PMID: 37258679), and the localization of LRRC23 in RS could be further predicted.
4. The ortholog of the RSP15 may also be predicted or confirmed by using the reported structure in human respiratory cilia (PMID: 37258679). Whether the LRCC34 in RS2 is LRRC34?

Reviewer #2 (Public Review):

Summary:
The present study explores the molecular function of LRRC23 in male fertility, specifically in the context of the regulation of spermiogenesis. The author initiates the investigation by identifying LRRC23 mutations as a potential cause of male sterility based on observations made in closely related individuals affected by asthenozoospermia (ASZ). To further investigate the function of LRRC23 in spermatogenesis, mutant mice expressing truncated LRRC23 proteins are created, aligning with the identified mutation site. Consequently, the findings confirm the deleterious effects of LRRC23 mutations on sperm motility in these mice while concurrently observing no significant abnormalities in the overall flagella structure. Furthermore, the study reveals LRRC23's interaction with the RS head protein RSPH9 and its active involvement in the assembly of the axonemal RS. Notably, LRRC23 mutations result in the loss of the RS3 head structure and disruption of the RS2-RS3 junction structure. Therefore, the author claimed that LRRC23 is an indispensable component of the RS3 head structure and suggests that mutations in LRRC23 underlie sterility in mice.

Strengths:
The key contribution of this article lies in confirming LRRC23's involvement in assembling the RS3 head structure in sperm flagella. This finding represents a significant advancement in understanding the complex architecture of the RS3 structural complex, building upon previous studies. Moreover, the article's topic is interesting and originates from clinical research, which holds significant implications for potential clinical applications.

Weaknesses:
1. While the author generated mutant mice expressing truncated LRRC23 proteins, the expression of these truncated proteins was not detected in sperm. This implies that, in terms of sperm structure, the mutant LRRC23 protein behaves similarly to the complete knockout of the LRRC23 protein, which has been previously reported and characterized (Zhang et al., 2021).

2. This reviewer questions the proposal that LRRC23 is an integral component of RS3, as the results indicate not only the loss of the RS3 head structure but also an incomplete RS2-RS3 junction structure. In addition, the interaction of LRRC23 with RSPH9 alone does not fully explain its involvement solely in RS3 assembly. Additional evidence is required to examine the influence of LRRC23 on the RS2-RS3 junction.

3. The article does not explore how these mutations affect the flagella structure in human sperm, which needs further study. Expanding the study to include human sperm structure would undoubtedly enhance the quality of the article.