Stress granules (SGs) are discrete assemblies of stalled messenger ribonucleoprotein complexes (mRNPs) that form when eukaryotic cells encounter environmental stress. RNA-binding proteins (RBPs) mediate their condensation by recruiting populations of mRNPs. However, the cellular and molecular mechanisms underlying the role of ubiquitin-associated protein 2-like (UBAP2L) in the regulation of SG dynamics remain elusive. Here, we show that UBAP2L is required for both SG assembly and disassembly. UBAP2L overexpression nucleated SGs under stress-null conditions. The UBAP2L Arg-Gly-Gly (RGG) motif was required for SG competence, and mediated the recruitment of SG components, including mRNPs, RBPs, and ribosomal subunits. The domain of unknown function (DUF) of UBAP2L-mediated interaction with ras GTPase-activating protein-binding protein (G3BP)1/2, and its deletion caused the cytoplasmic-nuclear transport of UBAP2L and G3BP1/2, thereby compromising SG formation. The protein arginine methyltransferase PRMT1 asymmetrically dimethylated UBAP2L by targeting the RGG motif. Increased arginine methylation blocked, whereas its decrease enhanced UBAP2L interactions with SG components, ablating and promoting SG assembly, respectively. These results provide new insights into the mechanisms by which UBAP2L regulates SG dynamics and RNA metabolism.
Mammalian spermatozoa are highly polarized cells characterized by compartmentalized cellular structures and energy metabolism. Adenylate kinase (AK), which interconverts two ADP molecules into stoichiometric amounts of ATP and AMP, plays a critical role in buffering adenine nucleotides throughout the tail to support flagellar motility. Yet the role of the major AK isoform, AK1, is still not well characterized. Here, by using a proteomic analysis of testis biopsy samples, we found that AK1 levels were significantly decreased in non-obstructive azoospermia (NOA) patients. This result was further verified by immunohistochemical staining of AK1 on a tissue microarray. AK1 was found to be expressed in post-meiotic round and elongated spermatids in mouse testis and subsequent mature sperm in the epididymis. We then generated Ak1 knockout mice, which showed that AK1 deficiency did not induce any defects in testis development, spermatogenesis or sperm morphology and motility under physiological conditions. We further investigated detergent-modeled epididymal sperm and included individual or mixed adenine nucleotides to mimic energy stress. When only ADP was available, Ak1 disruption largely compromised sperm motility, manifested as a smaller beating amplitude and higher beating frequency, which resulted in less effective forward swimming. The energy restriction/recover experiments with intact sperm further addressed this finding. Besides, decreased AK activity was observed in sperm of a male fertility disorder mouse model induced by cadmium chloride. These results cumulatively demonstrate that AK1 was dispensable for testis development, spermatogenesis or sperm motility under physiological conditions, but was required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.
In mammals, testis development is triggered by expression of the sex-determining Y-chromosome gene SRY to commit Sertoli cell (SC) fate at gonadal sex determination in the fetus. Several genes have been identified to be required to promote the testis pathway following SRY activation (i.e. SOX9) in embryo; however, largely remains unknown about the genes and mechanisms involved in stabilizing the testis pathway after birth and throughout adulthood. Herein, we report that postnatal males with SC-specific deletion of Raptor demonstrated absence of SC unique identity and adversely acquired granulosa cell-like characteristics, along with loss of tubular architecture and scattered distribution of SCs and germ cells. Subsequent genome-wide analysis by RNA sequencing revealed a profound decrease in the transcripts of testis genes (i.e. Sox9, Sox8, and Amh) and conversely an increase in ovary genes (i.e. Lhx9, Foxl2, and Fst); these changes were further confirmed by immunofluorescence and quantitative reverse-transcription PCR. Importantly, co-immunofluorescence demonstrated that Raptor deficiency induced SCs dedifferentiation into a progenitor state; the Raptor-mutant gonads showed some ovarian somatic cell features, accompanied by enhanced female steroidogenesis and elevated estrogen levels, yet the ZP3-positive terminally feminized oocytes were not observed. In vitro experiments with primary SCs suggested that Raptor is likely involved in FGF9-induced formation of cell junctions among SCs. Taken together, our results established that Raptor is required to maintain SC identity, stabilize the male pathway, and promote testis development.
F-box and WD-40 domain protein 7 (Fbxw7) is a component of the Skp1-Cdc53/Cullin-F-box-protein complex (SCF/β-TrCP), which is an E3 ubiquitin ligase that mediates protein degradation. This complex has recently been shown to negatively regulate spermatogonial stem cell self-renewal; however, its roles in Sertoli cell (SC) proliferation, differentiation, and function remain to be established. In this study, we generated conditional mutant mice with SC-specific deletion of Fbxw7 via the Cre-loxP system. Fbxw7 deficiency in SCs impaired testis development, which is characterized by age-dependent tubular atrophy, excessive germ cell loss, and spermatogenic arrest, and the mutant males were infertile at 7 months old. Fbxw7 ablation also compromised cytoskeletal organization and cell polarity of SCs, as well as integrity of the blood-testis barrier. In addition, the transcript levels of cell markers for germ cells, Leydig cells, and SCs were significantly decreased in Fbxw7 mutant mice. Importantly, protein levels of GATA-4, a transcription factor that plays a crucial role in SC maturation and testis development, were progressively decreased in control SCs after postnatal day 14, whereas levels were aberrantly elevated in Fbxw7-deleted SCs. Interestingly, the Gata-4 messenger RNA levels remained stable following Fbxw7 deletion. Fbxw7 silencing in SCs also induced progressive Leydig cell inefficiency and testosterone insufficiency. Collectively, these results demonstrate that Fbxw7 expression is required for SC maturation and function, potentially through degradation of GATA-4, to support pubertal testis development and spermatogenesis.
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