Search

Spermiogenesis is a critical, post-meiotic phase of male gametogenesis, in which the proper gene expression is essential for sperm maturation. However, the underFlying molecular mechanism that controls mRNA expression in the round spermatids remains elusive. Here, we identify that FBXO24, an orphan F-box protein, is highly expressed in the testis of humans and mice and interacts with the splicing factors (SRSF2, SRSF3, and SRSF9) to modulate the gene alternative splicing in the round spermatids. Genetic mutation of FBXO24 in mice causes many abnormal splicing events in round spermatids, thus affecting a large number of critical genes related to sperm formation that were dysregulated. Further molecular and phenotypical analyses revealed that FBXO24 deficiency results in aberrant histone retention, incomplete axonemes, oversized chromatoid body, and abnormal mitochondrial coiling along sperm flagella, ultimately leading to male sterility. In addition, we discovered that FBXO24 interacts with MIWI and SCF subunits and mediates the degradation of MIWI via K48-linked polyubiquitination. Furthermore, we show that FBXO24 depletion could lead to aberrant piRNA production in testes, which suggests FBXO24 is required for normal piRNA counts. Collectively, these data demonstrate that FBXO24 is essential for sperm formation by regulating mRNA alternative splicing and MIWI degradation during spermiogenesis., Competing Interests: ZL, XL, YZ, YL, LZ, SY No competing interests declared, (© 2023, Li et al.)

Background: Sperm is formed through spermiogenesis, a highly complex process involving chromatin condensation that results in cessation of transcription. mRNAs required for spermiogenesis are transcribed at earlier stages and translated in a delayed fashion during spermatid formation. However, it remains unknown that how these repressed mRNAs are stabilized., Results: Here we report a Miwi-interacting testis-specific and spermiogenic arrest protein, Ck137956, which we rename Tssa. Deletion of Tssa led to male sterility and absence of sperm formation. The spermiogenesis arrested at the round spermatid stage and numerous spermiogenic mRNAs were down-regulated in Tssa -/- mice. Deletion of Tssa disrupted the localization of Miwi to chromatoid body, a specialized assembly of cytoplasmic messenger ribonucleoproteins (mRNPs) foci present in germ cells. We found that Tssa interacted with Miwi in repressed mRNPs and stabilized Miwi-interacting spermiogenesis-essential mRNAs., Conclusions: Our findings indicate that Tssa is indispensable in male fertility and has critical roles in post-transcriptional regulations by interacting with Miwi during spermiogenesis., (© 2023. The Author(s).)

PIWI proteins and PIWI-interacting RNAs (piRNAs) are specifically expressed in animal germlines and play essential roles during gametogenesis in animals. The primary function of PIWI/piRNAs is known to silence transposable elements for protecting genome integrity in animal germlines, while their roles beyond silencing transposons are also documented by us and others. In particular, we show that mouse PIWIL1 (MIWI)/piRNAs play a dual role in regulating protein-coding genes in mouse spermatids through interacting with different protein factors in a developmental stage-dependent manner, including translationally activating a subset of AU-rich element-containing mRNAs in round spermatids and inducing massive mRNA degradation in late spermatids. We further show that MIWI is eliminated through the ubiquitin-26S proteasome pathway during late spermiogenesis. By exploring the biological function of MIWI ubiquitination by APC/C, we identified ubiquitination-deficient mutations in human PIWIL1 of infertile men and further established their causative role in male infertility in mouse model, supporting PIWIL1 as a human male infertility-relevant gene. Additionally, we reported that PIWIL1, aberrantly induced in human tumors, functions as an oncoprotein in a piRNA-independent manner in cancer cells. In the current review, we summarize our latest findings regarding the roles and mechanisms of PIWIL1 and piRNAs in mouse spermatids and human diseases, and discuss the related works in the field., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

While an E3 ubiquitin ligase, RNF8, was initially reported to be required for histone-to-protamine exchange in spermiogenesis, we subsequently demonstrated that RNF8 is not involved in this process. Nevertheless, reflecting a lingering misunderstanding in the field, a growing number of studies have continued to postulate a requirement for RNF8 in the histone-to-protamine exchange. For example, a recent study claimed that a mouse PIWI protein, MIWI, controls RNF8-mediated histone-to-protamine exchange. Here, confirming our earlier conclusions, we show that RNF8 is required neither for the establishment of histone H4K16 acetylation, which is an initial step in histone removal during spermiogenesis, nor for the incorporation of two protamine proteins, PRM1 and PRM2. Thus, whereas RNF8 mediates ubiquitination of H2A on the sex chromosomes in meiosis, during the prior stage of spermatogenesis, our genetic evidence underscores that RNF8 is not involved in histone-to-protamine exchange., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Researches have shown that silica nanoparticles (SiNPs) could reduce both the quantity and quality of sperm. However, the mechanism of toxicity induced by SiNPs in the male reproductive system is still unclear. In this study, male mice were randomly divided into a control group, and SiNPs treated group (20 mg/kg dose; n = 30 per group). Half of the mice per group were sacrificed on 35 days and the remaining on 50 days of the SiNPs exposure. SiNPs were found to decrease sperm count and mobility, increase the sperm abnormality rate, and damage the testes' structure. Furthermore, SiNPs decreased the protein levels of Protamine 1(PRM1) and elevated the histones' levels and suppressed the chromatin condensation of sperm. There was a significant reduction of the ubiquitinated H2A (ubH2A)/H2B (ubH2B) and RING finger protein 8 (RNF8) levels in the spermatid nucleus, while the RNF8 level in the spermatid cytoplasm increased evidently. The protein expression levels of PIWI-like protein 1(MIWI) in the late spermatids significantly increased on day 35 of SiNPs exposure. After 15 days of the withdrawal, the sperm parameters and protamine levels, and histones in the epididymal sperm were unrecovered; however, the changes in testis induced by SiNPs were recovered. Our results suggested that SiNPs could decrease the RNF8 level in the nucleus of spermatid either by upregulating of the expression of MIWI or by inhibiting its degradation. This resulted in the detention of RNF8 in the cytoplasm that maybe inhibited the RNF8-mediated ubiquitination of ubH2A and ubH2B. These events culminated in creating obstacles during the H2A and H2B removal and chromatin condensation, thereby suppressing the differentiation of round spermatids and chromatin remodeling, which compromised the sperm quality and quantity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Although MIWI (PIWI in humans) regulates spermatogenesis and translation machinery, its role in peripheral nerve injury is poorly understood. In this study, we characterized the expression profiles of MIWI after sciatic nerve injury. The results revealed that MIWI was downregulated after sciatic nerve injury. MIWI was colocalized with S100 (a Schwan cell marker), and TOM20 (a mitochondrial marker) on uncut nerves, while some MIWI was also colocalized with myelin protein zero (a myelin marker) on injured nerves. Immunofluorescence revealed that some MIWI was colocalized with SOX10 in the nuclear compartment following nerve injury. MIWI depletion by MIWI siRNA resulted in the reduction of EGR2. To characterize the expression of PIWI interacting RNA (piRNA) during sciatic nerve injury, microarray-based piRNA was conducted. The results revealed that 3447 piRNAs were upregulated, while 4117 piRNAs were downregulated after nerve transection. Interestingly, piR 009614 downregulated the mRNA level of MBP and enhanced the migration of RT-4 Schwann cells. Together, our results suggest that the MIWI-piRNA complex may play a role in Schwann cell responses to nerve injury., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Repression of retrotransposons is essential for genome integrity during germ cell development and is tightly controlled through epigenetic mechanisms. In primordial germ cells, protein arginine N -methyltransferase (Prmt5) is involved in retrotransposon repression by methylating Piwi proteins, which is part of the piRNA pathway. Here, we show that in mice, genetic inactivation of coprs (which is highly expressed in testis and encodes a histone-binding protein required for the targeting of Prmt5 activity) affects the maturation of spermatogonia to spermatids. Mass spectrometry analysis revealed the presence of Miwi in testis protein lysates immunoprecipitated with an anti-Coprs antibody. The observed deregulation of Miwi and pachytene pre-piRNAs levels and the derepression of LINE1 repetitive sequences observed in coprs -/- mice suggest that Coprs is implicated in genome surveillance mechanisms.

Small RNAs called PIWI-interacting RNAs (piRNAs) act as an immune system to suppress transposable elements in the animal gonads. A poorly understood adaptive pathway links cytoplasmic slicing of target RNA by the PIWI protein MILI to loading of target-derived piRNAs into nuclear MIWI2. Here we demonstrate that MILI slicing generates a 16-nt by-product that is discarded and a pre-piRNA intermediate that is used for phased piRNA production. The ATPase activity of Mouse Vasa Homolog (MVH) is essential for processing the intermediate into piRNAs, ensuring transposon silencing and male fertility. The ATPase activity controls dissociation of an MVH complex containing PIWI proteins, piRNAs, and slicer products, allowing safe handover of the intermediate. In contrast, ATPase activity of TDRD9 is dispensable for piRNA biogenesis but is essential for transposon silencing and male fertility. Our work implicates distinct RNA helicases in specific steps along the nuclear piRNA pathway., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Pachytene piRNAs are MIWI-/MILI-bound small RNAs abundantly expressed in pachytene spermatocytes and round spermatids in adult mouse testes. Miwi knockout (KO) male mice are sterile due to spermiogenic arrest. In Caenorhabditis elegans , sperm-borne piRNAs appear to have an epigenetic role during fertilization and development because progeny of individuals with piRNA-deficient gametes display a progressive loss of fertility after several generations. In mice, it remains unknown whether pachytene piRNA-deficient round spermatids can produce offspring, and whether the progeny of Miwi mutants also exhibits transgenerational, progressive fertility loss. Here, we report that Miwi KO round spermatids could fertilize both wild-type (WT) and Miwi KO oocytes through round spermatid injection, and could produce healthy and fertile offspring despite the global downregulation of both MIWI-/MILI-bound pachytene piRNAs. Progeny of ROSI-derived heterozygotes, both male and female, displayed normal fertility for at least three generations when bred with either WT or Miwi KO females. Our data indicate that aberrant MIWI-/MILI-bound pachytene piRNA profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting that pachytene piRNAs might not be required for paternal transgenerational epigenetic inheritance in mice., Competing Interests: Conflict of interest statement. None declared.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells., (© 2015 Goh et al.; Published by Cold Spring Harbor Laboratory Press.)