FBXO24 modulates mRNA alternative splicing and MIWI degradation and is required for normal sperm formation and piRNA production

Reviewer #1 (Public Review):

In this study, Li et al., report that FBXO24 contributes to sperm development by modulating alternative mRNA splicing and MIWI degradation during spermiogenesis. The authors demonstrated that FBXO24 deficiency impairs sperm head formation, midpiece compartmentalization, and axonemal/peri-axonemal organization in mature sperm, which causes sperm motility defects and male infertility. In addition, FBXO24 interacts with various mRNA splicing factors, which causes altered splicing events in Fbxo24-null round spermatids. Interestingly, FBXO24 also modulates MIWI levels via its polyubiquitination in round spermatids. Thus, the authors address that FBXO24 modulates global mRNA levels by regulating piRNA-mediated MIWI function and splicing events in testicular haploid germ cells.

This study is performed with various experimental approaches to explore and elucidate underlying molecular mechanisms for the FBXO24-mediated sperm defects during germ cell development. Overall, the experiments were designed properly and performed well to support the authors' observation in each part. In addition, the finding in this study is useful for understanding the physiological and developmental significance of the FBXO24 in the male germ line, which can provide insight into impaired sperm development and male infertility. However, there are several concerns to be explained more in this study. In addition, some results should be revised and updated.

Reviewer #2 (Public Review):

Spermatogenesis is a process of cell differentiation necessary to produce fertile spermatozoa. It consists of three parts, the last of which is called spermiogenesis, in which the size, shape, and organelle composition of the spermatids undergo significant changes that result in the formation of fully elongated spermatozoa. Defects in spermatogenesis or spermiogenesis can lead to male infertility. In this study, Li et al. identified FBXO24 as a highly expressed protein in human and mouse testis that is required for modulating alternative gene splicing in round spermatids through interaction with various splicing factors. They also found that deletion of FBXO24 in mice results in disorganized mitochondrial packing along the midpiece of the tail and chromatoid body architecture, which may account for the observed male sterility. The authors discovered that FBXO24 interacts with the subunits of MIWI and SCF and is required for normal piRNA biogenesis.

The major strengths of the study are the rigorous phenotypic and molecular analysis by using two complementary animal models (knock-out mouse model but also HA-tagged transgenic mouse model) to pinpoint the protein levels and localization in time and space during normal spermatogenesis and when the protein is absent.

The minor weakness of the study is inconsistent use of terminology throughout the manuscript, occasional logic-jump in their flow, and missing detailed description in methodologies used either in the text or Materials and Methods section, which can be easily rectified.

Overall, this study highlights the relevance and importance of FBXO24 in male fertility and provides a better understanding of the MIWI/piRNA pathway, mitochondrial organization, and chromatin condensation in mouse spermatozoa during spermiogenesis.

Reviewer #3 (Public Review):

This work is carried out by the research group led by Shuiqiao Yuan, who has a long interest in and significant contribution to the field of male germ cell development. The authors study a protein for which limited information existed prior to this work, a component of the E3 ubiquitin ligase complex, FBXO24. The authors generated the first FBXO24 KO mouse model reported in the literature using CRISPR, which they complement with HA-tagged FBXO24 transgenic model in the KO background. The authors begin their study with a very careful examination of the expression pattern of the FBXO24 gene at the level of mRNA and the HA-tagged transgene, and they provide conclusive evidence that the protein is expressed exclusively in the mouse testis and specifically in post-meiotic spermatids of stages VI to IX, which include early stages of spermatid elongation and nuclear condensation. The authors report a fully sterile phenotype for male mice, while female mice are normal. Interestingly, the testis size and the populations of spermatogenic cells in the KO mutant mice show a small (but significant) reduction compared to the WT testis. Importantly, the mature sperm from KO animals show a series of defects that were very thoroughly documented in this work by scanning and transmission electron microscopy; this data constitutes a very strong point in this paper. FBXO24 KO sperm have severe defects in the mitochondrial sheath with missing mitochondria near the annulus, and missing outer dense fibers. Collectively these defects cause abnormal bending of the flagellum and severely reduced sperm motility. Moreover, defects in nuclear condensation are observed with faint nuclear staining of elongating and elongated spermatids, and reduction of protein levels of protamine 2 combined with increased levels of histones and transition protein 1. All of the above are in line with the observed male sterility phenotype.

The authors also performed RNAseq in the KO animal, and found profound changes in the abundance of thousands of mRNAs; and changes in mRNA splicing patterns as well. The data reveal deeply affected gene expression patterns in the FBXO24 KO testis, which further supports the essential role that this factor serves in spermiogenesis. Unfortunately, a molecular explanation of what causes these changes is missing; it is still possible that they are an indirect consequence of the KO and not directly caused by the KO.

A well-reasoned narrative on if and how the absence of FBXO24 as an E3 ubiquitin ligase is responsible for the observed mRNA and protein differential expression is missing. If FBXO24-mediated ubiquitination is required for normal protein degradation during spermiogenesis, protein level increase should be the direct consequence of genuine FBXO24 targets in the KO testis. Importantly, besides the Miwi ubiquitination experiment which is performed in a heterologous and therefore may not be ideal for extracting conclusions, the possible involvement of ubiquitination was not shown for any other proteins that the authors found that interact with FBXO24. For example splicing factors SRSF2, SRSF3, SRSF9, or any of the other proteins whose levels were found to be changed (reduced, thus the change in the KO is less likely due to the absence of ubiquitination) such as ODF2, AKAP3, TSSK4, PHF7, TSSK6, and RNF8. Interestingly, the authors do observe increased amounts of histones and transition proteins, but reduced amounts of protamines, which directly shows that histone to protamine transition is indeed affected in the FBXO24 KO testis, and is in agreement with the observed less condensed nuclei of spermatozoa. Could histones and transition proteins be targets of the proposed ubiquitin ligase activity of FBXO24, and in its absence, histone replacement is abrogated? Providing experimental evidence to address this possibility would greatly expand our understanding of why FBXO24 is essential during spermiogenesis.

Regarding the results on Miwi protein and piRNAs, the following remarks can be made:

The finding that the Miwi protein is upregulated is an important point in this work, and it is in agreement with the observed increased size of the chromatoid body, where most of the Miwi protein is accumulated in round spermatids. This finding needs to be further supported and verified with experiments done in WT and KO mice. Miwi should be immunoprecipitated and Miwi ubiquitination should be detected (with WB or mass spec) in WT testis. It should be expected that Miwi ubiquitination is reduced in KO testis. The experiments that the authors performed in HEK293T cells are informative but experiments with tissue/cells normally expressing Miwi and FBXO24 are missing. With regard to piRNA expression, it is an exaggeration to call the observed increase in piRNA expression remarkable, especially since one replicate small RNA library per condition was sequenced. Although the library is constructed from total small RNA (which includes Mili-bound piRNAs as well), it does seem that the upregulated piRNAs are Miwi-bound piRNAs because the size of the upregulated piRNAs is mostly 29-32 bases. However, the direct comparison of the number of upregulated piRNAs with upregulated miRNAs is not in support of the claim that the increase in piRNA expression is higher compared to miRNAs: there are approximately a few hundred miRNAs expressed in mice, but hundreds of thousands of different piRNA sequences, so upregulation of ~10 times more piRNA species than miRNAs is a smaller proportional increase. Moreover, the observed increase in the overall piRNA levels could be just an epiphenomenon of the increased abundance of the Miwi protein; it has been documented that Piwi proteins stabilize their piRNA cargo, so most likely the increase in iRNA levels in 29-32 nt sizes is probably not a result of altered biogenesis, but increased half-life of the piRNAs as a result of Miwi upregulation. Therefore, the claim that FBXO24 is essential for piRNA biogenesis/production (lines 308, 314) is not appropriately supported.