CCDC157 is essential for sperm differentiation and shows oligoasthenoteratozoospermia-related mutations in men.

Oligoasthenoteratospermia (OAT), characterized by abnormally low sperm count, poor sperm motility, and abnormally high number of deformed spermatozoa, is an important cause of male infertility. Its genetic basis in many affected individuals remains unknown. Here, we found that CCDC157 variants are associated with OAT. In two cohorts, a 21-bp (g.30768132_30768152del21) and/or 24-bp (g.30772543_30772566del24) deletion of CCDC157 were identified in five sporadic OAT patients, and 2 cases within one pedigree. In a mouse model, loss of Ccdc157 led to male sterility with OAT-like phenotypes. Electron microscopy revealed misstructured acrosome and abnormal head-tail coupling apparatus in the sperm of Ccdc157-null mice. Comparative transcriptome analysis showed that the Ccdc157 mutation alters the expressions of genes involved in cell migration/motility and Golgi components. Abnormal Golgi apparatus and decreased expressions of genes involved in acrosome formation and lipid metabolism were detected in Ccdc157-deprived mouse germ cells. Interestingly, we attempted to treat infertile patients and Ccdc157 mutant mice with a Chinese medicine, Huangjin Zanyu, which improved the fertility in one patient and most mice that carried the heterozygous mutation in CCDC157. Healthy offspring were produced. Our study reveals CCDC157 is essential for sperm maturation and may serve as a marker for diagnosis of OAT.

Splicing Mutation in DNALI1 Causes Male Infertility with Severe Oligoasthenoteratozoospermia in Humans.

Oligo-astheno-teratozoospermia (OAT), which is a common cause of male infertility, can be caused by genetic factors. This study reports on a case of a male patient suffering from infertility concomitant with OAT. Whole-exome sequencing (WES) confirmed the presence of a homozygous variant (NM_003462: c.464-1G > A) in the DNALI1 gene via Sanger sequencing. Immunofluorescence staining demonstrated that the DNALI1 signal was nearly undetectable in the patient's sperm. Bioinformatics analysis revealed that this mutation could reverse the splicing of the exon 4 acceptor splice site. A minigene experiment was performed to verify the mutation and the results confirmed that the mutation disrupted the splicing. Our findings show that this rare mutation in DNALI1 contributes to male infertility and OAT in humans, thereby expanding our understanding of the causes and pathogenesis of male infertility. This knowledge facilitates genetic counseling, clinical diagnosis, and therapeutic development of male infertility.

X-linked RBBP7 mutation causes maturation arrest and testicular tumors.

Maturation arrest (MA) is a subtype of non-obstructive azoospermia, and male infertility is a known risk factor for testicular tumors. However, the genetic basis for many affected individuals remains unknown. Here, we identified a deleterious hemizygous variant of X-linked retinoblastoma-binding protein 7 (RBBP7) as a potential key cause of MA, which was also found to be associated with the development of Leydig cell tumors. This mutation resulted in premature protein translation termination, affecting the sixth WD40 domain of the RBBP7 and the interaction of the mutated RBBP7 with histone H4. Decreased BRCA1 and increased γH2AX were observed in the proband. In mouse spermatogonial and pachytene spermatocyte-derived cells, deprivation of rbbp7 led to cell cycle arrest and apoptosis. In Drosophila, knockdown of RBBP7/Caf1-55 in germ cells resulted in complete absence of germ cells and reduced testis size, whereas knockdown of RBBP7/Caf1-55 in cyst cells resulted in hyperproliferative testicular cells. Interestingly, male infertility caused by Caf1-55 deficiency was rescued by ectopic expression of wild-type human RBBP7 but not mutant variants, suggesting the importance of RBBP7 in spermatogenesis. Our study provides insights into the mechanisms underlying the co-occurrence of MA and testicular tumors and may pave the way for innovative genetic diagnostics of these 2 diseases.

Pipsqueak family genes dan/danr antagonize nuclear Pros to prevent neural stem cell aging in Drosophila larval brains.

Neural stem cell aging is a fundamental question in neurogenesis. Premature nuclear Pros is considered as an indicator of early neural stem cell aging in Drosophila. The underlying mechanism of how neural stem cells prevent premature nuclear Pros remains largely unknown. Here we identified that two pipsqueak family genes, distal antenna (dan) and distal antenna-related (danr), promote the proliferation of neural stem cells (also called neuroblasts, NBs) in third instar larval brains. In the absence of Dan and Danr (dan/danr), the NBs produce fewer daughter cells with smaller lineage sizes. The larval brain NBs in dan/danr clones show premature accumulation of nuclear Prospero (Pros), which usually appears in the terminating NBs at early pupal stage. The premature nuclear Pros leads to NBs cell cycle defects and NB identities loss. Removal of Pros from dan/danr MARCM clones prevents lineage size shrinkage and rescues the loss of NB markers. We propose that the timing of nuclear Pros is after the downregulation of dan/danr in the wt terminating NBs. dan/danr and nuclear Pros are mutually exclusive in NBs. In addition, dan/danr are also required for the late temporal regulator, Grainyhead (Grh), in third instar larval brains. Our study uncovers the novel function of dan/danr in NBs cell fate maintenance. dan/danr antagonize nuclear Pros to prevent NBs aging in Drosophila larval brains.

METTL14 Regulates Intestine Cellular Senescence through m6A Modification of Lamin B Receptor.

N-6-Methyladenosine (m6A) modification is involved in multiple biological processes including aging. However, the regulation of m6A methyltransferase-like 14 (METTL14) in aging remains unclear. Here, we revealed that the level of m6A modification and the expression of METTL14 were particularly decreased in the intestine of aged mice as compared to young mice. Similar results were confirmed in Drosophila melanogaster. Knockdown of Mettl14 in Drosophila resulted in a short lifespan, associated disrupted intestinal integrity, and reduced climbing ability. In human CCD-18Co cells, knockdown of METTL14 accelerated cellular senescence, and the overexpression of METTL14 rescued senescent phenotypes. We also identified the lamin B receptor (LBR) as a target gene for METTL14-mediated m6A modification. Knockdown of METTL14 decreased m6A level of LBR, resulted in LBR mRNA instability, and thus induced cellular senescence. Our findings suggest that METTL14 plays an essential role in the m6A modification-dependent aging process via the regulation of LBR and provides a potential target for cellular senescence.

Phosphatase of Regenerating Liver-1 Regulates Wing Vein Formation through TGF-ß Pathway in Drosophila melanogaster.

BACKGROUND: Drosophila Phosphatase of Regenerating Liver-1 (PRL-1) is the only homolog of the mammalian PRLs with which it shares high sequence and structural similarities. Whilst PRLs are most notable for their high expression in malignant cancers and related promotion of cancer progression, the specific biological functions of the PRLs remain largely elusive. METHODS: Here, using a gain-of-function approach, we found that PRL-1 functions during wing vein development in Drosophila melanogaster (Drosophila). Overexpression of Drosophila PRL-1 caused dose-dependent wing vein proliferation. RESULTS: Genetic screening of the main TGF-ß signaling factors, Mad and Smox, showed that the RNAi-mediated knockdown of Mad could alleviate the extra vein phenotype caused by overexpressed PRL-1 and lead to loss of the posterior section of longitudinal veins. However, knockdown of Smox resulted in an identical phenotype with or without the overexpression of Drosophila PRL-1. Clonal analyses revealed that overexpression of PRL-1 led to decreased expressions of activated phospho-Mad protein, as measured by immunostaining. Real-time PCR showed that the transcriptional levels of Smox were significantly increased upon overexpression of the Drosophila PRL-1 in wing discs, with a dose dependent effect. CONCLUSIONS: We propose that the main function of Drosophila PRL-1 in wing development is to affect the phospho-Mad levels and Smox transcriptional levels, therefore influencing the competitive balance for Medea between Mad and Smox. Our study demonstrates the novel role for Drosophila PRL-1 in regulating TGF-ß signaling to influence wing vein formation which may also provide insight into the understanding of the relationship between PRLs and TGF-ß signaling in mammals.

Lipidomic Alterations and PPARα Activation Induced by Resveratrol Lead to Reduction in Lesion Size in Endometriosis Models.

Endometriosis is an estrogen-dependent chronic inflammatory disease that affects approximately 10% of women of reproductive age and up to 50% of women with infertility. The heterogeneity of the disease makes accurate diagnosis and treatment a clinical challenge. In this study, we generated two models of endometriosis: the first in rats and the second using human ectopic endometrial stromal cells (HEcESCs) derived from the lesion tissues of endometriosis patients. We then applied resveratrol to assess its therapeutic potential. Resveratrol intervention had significant efficacy to attenuate lesion size and to rectify aberrant lipid profiles of model rats. Lipidomic analysis revealed significant lipidomic alterations, including notable increases of sphingolipids and decreases of both glycerolipids and most phospholipids. Upon resveratrol application, both proliferation capacity and invasiveness parameters decreased, and the early apoptosis proportion increased for HEcESCs. The activation of PPARα was also noted as a factor potentially contributing to recovery from endometriosis in both models. Our study provides valuable insight into the mechanisms of resveratrol in endometriosis and therefore strengthens the potential for optimizing resveratrol treatment for this disease.

A Novel Neuroprotective Role of Phosphatase of Regenerating Liver-1 against CO2 Stimulation in Drosophila.

Neuroprotection is essential for the maintenance of normal physiological functions in the nervous system. This is especially true under stress conditions. Here, we demonstrate a novel protective function of PRL-1 against CO2 stimulation in Drosophila. In the absence of PRL-1, flies exhibit a permanent held-up wing phenotype upon CO2 exposure. Knockdown of the CO2 olfactory receptor, Gr21a, suppresses the phenotype. Our genetic data indicate that the wing phenotype is due to a neural dysfunction. PRL-1 physically interacts with Uex and controls Uex expression levels. Knockdown of Uex alone leads to a similar wing held-up phenotype to that of PRL-1 mutants. Uex acts downstream of PRL-1. Elevated Uex levels in PRL-1 mutants prevent the CO2-induced phenotype. PRL-1 and Uex are required for a wide range of neurons to maintain neuroprotective functions. Expression of human homologs of PRL-1 could rescue the phenotype in Drosophila, suggesting a similar function in humans.

The autophagy-related gene Atg101 in Drosophila regulates both neuron and midgut homeostasis.

Atg101 is an autophagy-related gene identified in worms, flies, mice, and mammals, which encodes a protein that functions in autophagosome formation by associating with the ULK1-Atg13-Fip200 complex. In the last few years, the critical role of Atg101 in autophagy has been well-established through biochemical studies and the determination of its protein structure. However, Atg101's physiological role, both during development and in adulthood, remains less understood. Here, we describe the generation and characterization of an Atg101 loss-of-function mutant in Drosophila and report on the roles of Atg101 in maintaining tissue homeostasis in both adult brains and midguts. We observed that homozygous or hemizygous Atg101 mutants were semi-lethal, with only some of them surviving into adulthood. Both developmental and starvation-induced autophagy processes were defective in the Atg101 mutant animals, and Atg101 mutant adult flies had a significantly shorter lifespan and displayed a mobility defect. Moreover, we observed the accumulation of ubiquitin-positive aggregates in Atg101 mutant brains, indicating a neuronal defect. Interestingly, Atg101 mutant adult midguts were shorter and thicker and exhibited abnormal morphology with enlarged enterocytes. Detailed analysis also revealed that the differentiation from intestinal stem cells to enterocytes was impaired in these midguts. Cell type-specific rescue experiments disclosed that Atg101 had a function in enterocytes and limited their growth. In summary, the results of our study indicate that Drosophila Atg101 is essential for tissue homeostasis in both adult brains and midguts. We propose that Atg101 may have a role in age-related processes.

Drosophila Pif1A is essential for spermatogenesis and is the homolog of human CCDC157, a gene associated with idiopathic NOA.

The dynamic process of spermatogenesis shows little variation between invertebrate models such as Drosophila, and vertebrate models such as mice and rats. In each case, germ stem cells undergo mitotic division to proliferate and then continue, via meiosis, through various stages of elongation and individualization from spermatogonia to spermatid to finally to form mature sperm. Mature sperm are then stored in the seminal vesicles for fertilization. Errors in any of these stages can lead to male infertility. Here, we identify that Drosophila Pif1A acts as a key regulator for sperm individualization. Loss of Pif1A leads to male sterility associated with irregular individualization complex and empty seminal vesicles without mature sperm. Pif1A is highly expressed in the testes of mated male adult flies and the Pif1A protein is expressed at a higher level in male than in female flies. Pif1A is homologous to mammalian coiled-coil domain-containing protein 157 (CCDC157), which is also enriched in the testes of humans and mice. Human CCDC157, with unknown function, was identified to be downregulated in men with idiopathic non-obstructive azoospermia (NOA). We map the function of Drosophila Pif1A during spermatogenesis, showing that Pif1A is essential for spermatide individualization and involved in the regulation of the lipid metabolism genes. Our findings might be applicable for studying the function of CCDC157 in spermatogenesis and other aspects of human male fertility.

Prominin-like, a homolog of mammalian CD133, suppresses di lp6 and TOR signaling to maintain body size and weight in Drosophila.

CD133 (AC133/prominin-1) has been identified as a stem cell marker and a putative cancer stem cell marker in many solid tumors. Its biologic function and molecular mechanisms remain largely elusive. Here, we show that a fly mutant for prominin-like, a homolog of mammalian CD133, shows a larger body size and excess weight accompanied with higher fat deposits as compared with the wild type. The expression levels of prominin-like are mediated by ecdysone signaling where its protein levels increase dramatically in the fat body during metamorphosis. Prominin-like mutants exhibit higher Drosophila insulin-like peptide 6 (di lp6) levels during nonfeeding stages and increased Akt/ Drosophila target of rapamycin (dTOR) signaling. On an amino acid-restricted diet, prominin-like mutants exhibit a significantly larger body size than the wild type does, similar to that which occurs upon the activation of the dTOR pathway in the fat body. Our data suggest that prominin-like functions by suppressing TOR and dilp6 signaling to control body size and weight. The identification of the physiologic function of prominin-like in Drosophila may provide valuable insight into the understanding of the metabolic function of CD133 in mammals.-Zheng, H., Zhang, Y., Chen, Y., Guo, P., Wang, X., Yuan, X., Ge, W., Yang, R., Yan, Q., Yang, X., Xi, Y. Prominin-like, a homolog of mammalian CD133, suppresses di lp6 and TOR signaling to maintain body size and weight in Drosophila.

RanGAP-mediated nucleocytoplasmic transport of Prospero regulates neural stem cell lifespan in Drosophila larval central brain.

By the end of neurogenesis in Drosophila pupal brain neuroblasts (NBs), nuclear Prospero (Pros) triggers cell cycle exit and terminates NB lifespan. Here, we reveal that in larval brain NBs, an intrinsic mechanism facilitates import and export of Pros across the nuclear envelope via a Ran-mediated nucleocytoplasmic transport system. In rangap mutants, the export of Pros from the nucleus to cytoplasm is impaired and the nucleocytoplasmic transport of Pros becomes one-way traffic, causing an early accumulation of Pros in the nuclei of the larval central brain NBs. This nuclear Pros retention initiates NB cell cycle exit and leads to a premature decrease of total NB numbers. Our data indicate that RanGAP plays a crucial role in this intrinsic mechanism that controls NB lifespan during neurogenesis. Our study may provide insights into understanding the lifespan of neural stem cells during neurogenesis in other organisms.

Drosophila Prominin-like, a homolog of CD133, interacts with ND20 to maintain mitochondrial function.

BACKGROUND: Drosophila Prominin-like is a homolog of mammalian CD133, which is recognized as a biomarker for stem cells. The interacting proteins of CD133 and their biological functions remain elusive. RESULTS: In this study, we using yeast two-hybrid assays, GST pull-down assay and co-immunoprecipitation (Co-IP) methods found that Drosophila Prominin-like interacts with ND20, a subunit of mitochondrial respiratory complex I. Bioinformatics analysis suggests that Prominin-like is a six-transmembrane glycoprotein which localizes on cellular membranes. Immunostaining and mitochondrial fractionation indicate that Drosophila Prominin-like could localize in the mitochondria. The knockdown of prominin-like in S2 cells resulted in transient mitochondrial dysfunctions as evidenced by reduced ATP production, elevated ROS generation and an accompanied reduction in mitochondrial proteins. Mitochondrial dysfunctions were detected in aged prominin-like mutant flies. CONCLUSION: Our data indicates that Prominin-like acts to maintain mitochondrial function through its interaction with ND20 which, itself, is active in the mitochondrial electron transport chain. Our study provides insights into a novel molecular mechanism of Drosophila prominin-like and suggests a similar function of CD133 in mammals.

Premature remodeling of fat body and fat mobilization triggered by platelet-derived growth factor/VEGF receptor in Drosophila.

In Drosophila, fat-body remodeling accompanied with fat mobilization is an ecdysone-induced dynamic process that only occurs during metamorphosis. Here, we show that the activated Drosophila platelet-derived growth factor/VEGF receptor (PVR) is sufficient to induce shape changes in the fat body, from thin layers of tightly conjugated polygonal cells to clusters of disaggregated round-shaped cells. These morphologic changes are reminiscent of those seen during early pupation upon initiation of fat-body remodeling. Activation of PVR also triggers an early onset of lipolysis and mobilization of internal storage, as revealed by the appearance of small lipid droplets and up-regulated lipolysis-related genes. We found that PVR displays a dynamic expression pattern in the fat body and peaks at the larval-prepupal transition under the control of ecdysone signaling. Removal of PVR, although it does not prevent ecdysone-induced fat-body remodeling, causes ecdysone signaling to be up-regulated. Our data reveal that PVR is active in a dual-secured mechanism that involves an ecdysone-induced fat-body remodeling pathway and a reinforced PVR pathway for effective lipid mobilization. Ectopic expression of activated c-kit-the mouse homolog of PVR in the Drosophila fat body-also results in a similar phenotype. This may suggest a novel function of c-kit as it relates to lipid metabolism in mammals.-Zheng, H., Wang, X., Guo, P., Ge, W., Yan, Q., Gao, W., Xi, Y., Yang, X. Premature remodeling of fat body and fat mobilization triggered by platelet-derived growth factor/VEGF receptor in Drosophila.

Fat body remodeling and homeostasis control in Drosophila.

Remarkable advances have been made in recent years in our understanding of the Drosophila fat body and its functions in energy storage, immune response and nutrient sensing. The fat body interplays with other tissues to respond to the physiological needs of the body's growth and coordinates various metabolic processes at different developmental stages and under different environmental conditions. The identification of various conserved genetic functions and signaling pathways relating to the Drosophila fat body may provide clues to lipometabolic disease and other aspects of tissue remodeling in humans. Here, we discuss recent insights into how regulation of fat body remodeling contributes to hemostasis with a special focus on how signaling networks and internal physiological states shape different aspects of the lipid metabolism in Drosophila.

Converting a binding protein into a biosensing conformational switch using protein fragment exchange.

Biosensors can be used in applications ranging from identifying disease biomarkers to detecting spatial and temporal distributions of specific molecules in living cells. A major challenge facing biosensor development is how to functionally couple a biological recognition domain to an output module so that the binding event can be transduced to a visible and quantifiable signal [e.g., Förster resonance energy transfer (FRET)]. Most designs achieve coupling by means of a binding protein that changes conformation upon interacting with its target. This approach is limited by the fact that few proteins possess such natural allosteric mechanisms, and for those that do, the conformational change is frequently not extensive enough to produce a large change in distance between FRET donor and acceptor groups. Here, we introduce protein fragment exchange (FREX) to address both problems. FREX employs two components: a folded binding protein and a fragment duplicated from it, the latter of which can be chosen from many possible fragments. The system is rationally tuned so that addition of ligand induces a conformational change in which the fragment exchanges positions with the corresponding segment of the binding protein. Placing fluorescent donor and acceptor groups on the binding protein and fragment reduces the background level of FRET of the unbound sensor, resulting in a ratiometric FRET response that is expected to be strong and reproducible from protein to protein. FREX is demonstrated using fibronectin III, a monobody binding scaffold that has been tailored to recognize multiple targets. Sensors labeled with Alexa FRET pairs exhibit ratiometric FRET changes of up to 8.6-fold and perform equally well in buffer and serum. A genetically encoded variant of this sensor is shown to be functional in cell lysates and in mammalian cell cultures.

Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis.

In yeast cells, subunit a of the vacuolar proton pump (V-ATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1Δ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1Δ and stv1Δ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.