PIWI (P element-induced wimpy testis in Drosophila) family proteins are evolutionarily conserved and have an essential role in male fertility in most of the animal species studied so far [1]. All three murine PIWI orthologs (MIWI, MILI, and MIWI2) are required for spermatogenesis and male fertility, but the relevance of the four human PIWI proteins (HIWI, HILI, HIWI2, and PIWIL3) in human male fertility has not been demonstrated until now. A group led by Mofang Liu recently published a paper in Cell [2], reporting that point mutations in the D-box domain of the N-terminus of HIWI cause male infertility by impeding the histone-to-protamine exchange during late spermiogenesis. Since these point mutations only alter one or two amino acids in the D-box region while other domains known to be critical for HIWI’s piRNA functions, including PAZ, MID, and PIWI, remain intact, it is not surprising that the piRNA-related functions of the mutant HIWI protein are normal, suggesting that the D-box domain has a novel role independent of the known piRNA functions. Indeed, knockin mice carrying one mutant allele with the same point mutations in the D-box as those in humans, or overexpressing the same mutant MIWI proteins, can recapitulate the human phenotype, i.e. impairment in late spermiogenesis and male infertility, demonstrating the cause–effect relationship between these point mutations and the male infertility phenotype. The male mice carrying one null Miwi allele (Miwi+/) are fertile, whereas those with one mutant Miwi allele containing point mutations and producing mutant MIWI protein in the conserved D-box domain are sterile. This result suggests that reduced levels of wild-type MIWI production (e.g. in Miwi or HIWI heterozygous individuals) do not disrupt spermiogenesis, but the production of the mutant MIWI/HIWI with D-box mutations does, indicating a dominant-negative effect of the mutant MIWI/HIWI proteins in late spermiogenesis. How do these D-box point mutations cause male infertility in both mice and humans? Given that the D-box domain is shared among the substrates for APC/C ubiquitin E3 ligase and MIWI is degraded by ACP/C in elongating spermatids [3], mutations in D-box would impeded ubiquitination-mediated MIWI protein turnover, leading to an accumulation of mutant MIWI in spermatids. Indeed, testicular levels of MIWI are higher in MIWI D-box mutant mice. What are the consequences of excessive levels and persistent presence of MIWI/HIWI in spermatids? Through biochemical analyses, the authors further discovered that MIWI interacts with RNF8 and the binding between MIWI and RNF8 inhibits RNF8 ubiquitin E3 ligase activity by sequestering RNF8 in the cytoplasm, thus preventing it from entering the nucleus and starting the histone degradation program [4]. This notion is further supported by the experiments showing that blocking MIWI-RNF8 interactions using a RNF8 N-terminus polypeptide can rescue the defects in the D-box mutant mice. Together, the authors have convincingly demonstrated that D-box point mutations of MIWI/HIWI lead to prolonged existence of MIWI/HIWI protein in elongating spermatids, which, in turn, inhibit the RNF8-mediated histone degradation, thus causing impaired histone-to-protamine exchange and consequently male infertility. The finding is novel because it reveals, for the first time, that point mutations in the D-box domain of HIWI proteins lead to impaired sperm production and male infertility in humans by impeding the histone-to-protamine exchange, rather than by affecting piRNA production and function.

MIWI is required for normal production and function of thousands of pachytene piRNAs, which have been shown to control the massive turnover of mRNAs in late spermiogenesis [5, 6]. A complete lack of MIWI results in a spermiogenic arrest in early round spermatids (steps 3–4), whereas the impairments occur in elongating spermatids (steps 9–12) in MIWI D-box mutant male mice. The spatiotemporal relationship between the classical function of MIWI (i.e. piRNA production and function) and the novel role revealed in this study (i.e. inhibiting histone-to-protamine exchange through sequestering RNF8 in the cytoplasm), in fact, points us to an elegant mechanism through which the two important cellular events during spermiogenesis, i.e. establishment of the haploid-specific transcriptome and the onset of nuclear condensation program, are temporally coordinated. Specifically, in early round spermatids (steps 1–5 in mice), MIWI-dependent pachytene piRNAs participate in the large-scale degradation of a subgroup of mRNAs [5, 6]. Given that the spermatids-specific transcriptome is characterized by an enrichment of shorter 3΄-UTR transcripts for efficient translation and turnover [7], the piRNA target mRNAs are most likely those with longer 3΄-UTRs. Once the spermatid-specific transcriptome is completely established in late round spermatids (steps 6–8 in mice), MIWI/HIWI degradation through the ACP/C ubiquitination pathway becomes increasingly active [3]; consequently, the reduced MIWI/HIWI levels allow RNF8, which is largely anchored by MIWI in the cytoplasm of round spermatids, to enter the nucleus and to initiate the histone degradation program upon the onset of spermatid elongation (step 9 in mice). In elongating spermatids, the significantly reduced levels of MIWI/HIWI lead to more nuclear RNF8, thereby enhancing histone ubiquitination and its subsequent replacement by transition proteins and protamines. These dose-dependent events allow for well-coordinated, precise spatiotemporal control of the two critical cellular processes during spermiogenesis. In this sense, the D-box domain of MIWI/HIWI appears to function as a self-contained timer, which switches on the histone-to-protamine exchange program when its own levels decline with the progression of spermiogenesis from round to elongating spermatids (Figure 1). Given that the study was designed to only screen for mutations in the D-box region of HIWI, one cannot conclude that the D-box point mutations are more frequent than mutations in other functional domains of HIWI. More interestingly, the fact that ICSI using the D-box mutant sperm leads to normal fertilization and birth rates suggests that excessively retained histones are compatible with embryonic and fetal development. However, given the potential epigenetic roles of sperm retained histones [8, 9], the developmental potential of offspring derived from D-box mutant sperm should be further evaluated for potential long-term adverse epigenetic effects on health.

Figure 1.

Schematic illustration of MIWI control of the timing of two major cellular events during spermiogenesis: establishment of the spermatid-specific transcriptome and histone-to-protamine exchange. In early round spermatids (steps 1–5), MIWI-pachytene piRNAs participate in large-scale degradation of a subgroup of mRNAs (most likely long 3΄-UTR transcripts). MIWI levels decline due to ACP/C-mediated ubiquitination from early (steps 1–5) to late (steps 6–8) round spermatids; the reduced MIWI levels allow for RNF8, which is anchored by the D-box domain of MIWI in the cytoplasm of early round spermatids, to translocate to the nuclei of elongating spermatids (steps 9–12) to degrade histones through ubiquitination followed by replacement by transition proteins and protamines. Note that the steps at which each of the events commences are estimated and may be variable because they appear to occur in a dose-dependent manner.

Figure 1.

Schematic illustration of MIWI control of the timing of two major cellular events during spermiogenesis: establishment of the spermatid-specific transcriptome and histone-to-protamine exchange. In early round spermatids (steps 1–5), MIWI-pachytene piRNAs participate in large-scale degradation of a subgroup of mRNAs (most likely long 3΄-UTR transcripts). MIWI levels decline due to ACP/C-mediated ubiquitination from early (steps 1–5) to late (steps 6–8) round spermatids; the reduced MIWI levels allow for RNF8, which is anchored by the D-box domain of MIWI in the cytoplasm of early round spermatids, to translocate to the nuclei of elongating spermatids (steps 9–12) to degrade histones through ubiquitination followed by replacement by transition proteins and protamines. Note that the steps at which each of the events commences are estimated and may be variable because they appear to occur in a dose-dependent manner.

Acknowledgments

The author would like to thank Drs. P. Jeremy Wang and Qi Chen for critical reading of the text.

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