The process of gametogenesis faces various challenges. The best known steps are germline stem cell maintenance, the halving of the number of chromosomes during meiosis, and the spermiogenesis leading to spermatozoa. Less well known steps occur during gametogenesis, including (i) protection of mRNAs (to ensure ongoing transcription and translation processes after genome inactivation during and after the histone-protamine transition that completes spermiogenesis), (ii) transposon silencing (to ensure genome integrity), and (iii) viral defence (to avoid genome integration). All these processes appears to be wholly or partly linked to the P-element-induced wimpy (PIWI)/PIWI-interacting RNAs (piRNAs) [1, 2] for protection of genome integrity and for protein regulation in germ cells [3]. PIWI proteins and piRNAs are commonly perceived to be germline-specific, even though a somatic function has been documented [4]. Furthermore, piRNAs and PIWI proteins are highly conserved and found in the majority of animals [5], including vertebrates, arthropods, and nematodes (see Ensembl: https://www.ensembl.org/index.html).

PIWI proteins are essential for gametogenesis in animals [6, 7]. In Drosophila, the three Piwi proteins Argonaute, Aubergine, and Piwi are required for germ cell formation and germline stem cell maintenance in both males and females [5, 6]. piRNAs were first identified in the fly testis as a novel class of “small-interfering RNAs” [8] that guided PIWI proteins to cleave target RNA, promote heterochromatin assembly, and methylate DNA.

The mouse genome encodes three Piwi paralogs (Miwi, Mili, and Miwi2), whereas the human genome encodes four PIWI paralogs: PIWIL1 (HIWI), PIWIL2 (HILI), PIWIL3 (HIWI3), and PIWIL4 (HIWI2) [9]; all are highly expressed in the testis. Piwi proteins comprise four distinct domains: the PAZ and MID domains for piRNA loading, the PIWI C-terminal domain with RNase H activity [10,11,12], and the N-terminal domain (N-domain) that binds to Tudor-domain-containing proteins (TDRDs). TDRDs act as scaffolds to form the core of the chromatoid body, a cytoplasmic, non–membrane-bound organelle found in haploid, round spermatids [13, 14]. The N-domain also contains a conserved destruction box (D-box), whose signature is shared by substrates of APC/C ubiquitin E3 ligase.

The most abundant piRNA population in mammals corresponds to pachytene piRNAs, with more than 5 million molecules per spermatocyte [15]. Although pachytene piRNAs cleave hundreds of RNAs, a change in steady-state level is only detectable for a small fraction of transcripts. The cleavage of the few targets whose abundance is reduced significantly by piRNAs might be essential for male fertility – even though the targets’ exact functions are still unknown; it has been suggested that piRNAs are involved in gene regulation, but this is subject to debate [16]. The emergence of these PIWI/piRNA complexes reflects an evolutionary conflict between transposons (which must integrate into germline DNA to ensure their propagation) and the host genome (which, in order to maintain germline integrity, must protect itself from transposon-encoded proteins). During evolution, transposons have participated in genome expansion and have contributed to biological diversity. Transposon insertion can lead to chromosome rearrangement by homologous recombination and/or modifying gene expression in time or space. To reset the epigenome and erase genomic imprinting, gametogenesis requires germline reprogramming [17]. Erasing DNA methylation causes a burst of transposon transcription, which the PIWI/piRNA complex must oppose. It is noteworthy that PIWI/piRNAs reportedly regulate protein-coding genes in mouse spermatids.

In various species, PIWI genes have essential roles in the germline, and knock-out leads to distinct fertility phenotypes. In the golden hamster, both females and males lacking Piwil1 are sterile [18]; the zygotes are arrested at the two-cell stage in females, and males display spermatocyte arrest. Piwil2 and Piwil4 knock-out only result in male infertility, through spermatocyte arrest and spermatid arrest, respectively. In contrast, female Piwil3-knock-out hamsters displayed reduced fertility with significantly fewer offspring and the delayed development of embryos [19].

In mice, Miwi (Piwil1), Mili (Piwil2), and Miwi2 (Piwil4) are all essential for male fertility, but female knock-out mice are unaffected; hence, PIWI function is sex-specific in this species. Miwi-null mutant males display spermatogenesis arrest at the early round spermatid stage after meiosis [20], while Mili and Miwi2 are arrested at the pachytene stage during meiosis [21, 22].

In other species, PIWI genes also appear to have a major or prominent role; for example, null male chickens [23] and zebrafish [24] are often sterile (for a review see [25]).

In humans, PIWIL1 heterozygous missense variants have been identified in the Genome Aggregation Database (gnomAD, https://gnomad.broadinstitute.org). In a mouse model mimicking a complex human variant identified in a small Chinese cohort of infertile men, males were reportedly sterile and had few condensed spermatids [26]; this situation is quite similar to that observed with Miwi knock-out mice. However, this work is subject to debate and has not been replicated. Furthermore, no de novo mutations were observed in a much larger international cohort [27]. Furthermore, the PIWIL1 variant c.1580G > A, p.Arg527Lys (rs1106042) is reportedly associated with non-obstructive azoospermia (NOA; odds ratio [95% confidence interval] = 4.737 [1.314–17.072]) [28]. In men, low PIWIL2 mRNA expression is associated with a low sperm count, and elevated PIWIL1 mRNA expression is associated with low progressive motility of ejaculated spermatozoa [29].

Recently, a homozygous stop-gain mutation in PIWIL1 (c.688C > T, pArg230*) was described in a man presenting a round spermatid arrest, and two homozygous missense variants in PIWIL2 were described for patients with Sertoli-cell-only syndrome (c.839A > C; p.Tyr280Ser) and azoospermia lacking a documented histological phenotype (c.1697G > A, p.Arg566His) [30].

Here, we report on the observation and in silico validation of a new homozygous frameshift mutation in PIWIL1 (c.1176_1179delAACT; p.Thr393fs). Furthermore, given the importance of the mechanism driven by the PIWI gene family in spermatogenesis and the gene’s high level of conservation among the mammals, we compare the mRNA and protein sequences in the human vs. the great apes.