Upregulation of LHPP by saRNA inhibited hepatocellular cancer cell proliferation and xenograft tumor growth.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide and no pharmacological treatment is available that can achieve complete remission of HCC. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a recently identified HCC tumor suppressor gene which plays an important role in the development of HCC and its inactivation and reactivation has been shown to result in respectively HCC tumorigenesis and suppression. Small activating RNAs (saRNAs) have been used to achieve targeted activation of therapeutic genes for the restoration of their encoded protein through the RNAa mechanism. Here we designed and validated saRNAs that could activate LHPP expression at both the mRNA and protein levels in HCC cells. Activation of LHPP by its saRNAs led to the suppression of HCC proliferation, migration and the inhibition of Akt phosphorylation. When combined with targeted anticancer drugs (e.g., regorafenib), LHPP saRNA exhibited synergistic effect in inhibiting in vitro HCC proliferation and in vivo antitumor growth in a xenograft HCC model. Findings from this study provides further evidence for a tumor suppressor role of LHPP and potential therapeutic value of restoring the expression of LHPP by saRNA for the treatment of HCC.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

STUDY QUESTION: What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? SUMMARY ANSWER: EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. WHAT IS KNOWN ALREADY: EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. STUDY DESIGN, SIZE, DURATION: EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. MAIN RESULTS AND THE ROLE OF CHANCE: EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

STUDY QUESTION: Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER: NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY: Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION: This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE: DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Rapamycin protects Sertoli cells against BPA-induced autophagy disorders.

Bisphenol A (BPA) is a well-known environmental contaminant that can negatively impact reproductive function. Disruption of autophagy is implicated in BPA-induced cell injury, the specific molecular mechanisms through which BPA affects autophagy in Sertoli cells are still unknown. In the present study, TM4 cells were exposed to various doses of BPA (10, 100, and 200 µM), and the results indicated that BPA exposure led to the accumulation of autophagosomes, this change was accompanied by increased expression of p-mTOR and decreased expression of Atg12, a protein involved in regulating autophagy initiation. Additionally, BPA exposure upregulated the expression levels of p62, a protein involved in autophagic degradation. The inhibition of autophagy initiation and autophagic degradation contributes to the accumulation of autophagosomes. Further studies showed that BPA exposure didn't affect the expression of the lysosome protein LAMP1; however, decreased cytoplasmic retention of acridine orange in TM4 cells may explain the disruption of autophagy. The role of rapamycin and chloroquine (CQ), an autophagy inhibitor that impairs lysosomal degradation also confirmed the effect of BPA on autophagy regulation. Specifically, rapamycin can protect Sertoli cells against BPA-induced cell injury by promoting autophagy. These findings contribute to our understanding of the mechanisms underlying reproductive toxicity caused by BPA.

LRRC52 is likely a functional component of human KSper†.

Completion of fertilization is orchestrated by various ion channels in sperm membrane. Hyperpolarization of membrane potential, an indispensable event during the capacitation process, is dominated by sperm potassium channel (KSper). In addition to sperm-specific SLO3, which forms the channel pore, the auxiliary subunit leucine-rich-repeat-containing protein 52 (LRRC52) is required to form mKSper to function under physiological conditions. However, in human sperm, although most evidence supports that hSLO3 is the pore-forming subunit, whether hLRRC52 contributes to hKSper conductance and modulates sperm function remains to be understood. Here, using an extracellular segment that is homologous between mice and humans as an antigen, we developed a polyclonal antibody designed as LID1 that specifically detected mLRRC52 and performed co-immunoprecipitation with mSLO3. Additionally, patch-clamp recordings of mouse sperm showed that, physiological activation of mKSper and sperm functions were dramatically attenuated after treatment with LID1, indicating that LID1 functionally disrupted the regulation of mLRRC52 on mKSper. Next, LID1 was used to investigate the significance of hLRRC52 for hKSper activation. As a result, hLRRC52 was expressed in human sperm and might be assembled with hSLO3. More importantly, LID1 inhibited hKSper currents and depolarized sperm membrane potential, supporting essential modulation of hLRRC52 in hKSper. Ca2+ signaling of human sperm was also compromised in the presence of LID1, which impaired sperm motility and acrosome reaction. Because LID1 specifically inhibited both mKSper and hKSper but not mCatSper or hCatSper, our results suggest that hLRRC52 functions as an important component of hKSper and regulates sperm physiological functions.

Ferroptosis contributes to cyclophosphamide-induced hemorrhagic cystitis.

Cyclophosphamide (CYP) is extensively used in tumor therapy, but its clinical application is limited by its toxic effects on the bladder. Since CYP-induced cystitis is believed to be mediated by acrolein (ACR), a product of lipid peroxidation that triggers ferroptosis, we hypothesized that ferroptosis might be an essential molecular mechanism underlying CYP-induced cystitis. The purpose of this study was to test this hypothesis. Intraperitoneal injection of CYP led to bladder hemorrhage and edema, along with increased oxidation, inflammation, and cell injury. Further analysis revealed these changes were associated with altered ferroptosis markers in the bladder, such as FPN1, ACSL4, SLC7A11, and GPX4, indicating the existence of ferroptosis. Administration of ferroptosis inhibitor dexrazoxane (DXZ) improved ferroptosis and prevented CYP-induced pathological changes in the bladder. Collectively, our study revealed that ferroptosis is an important mechanism underlying CYP-induced cystitis, and therapeutic approaches targeting ferroptosis could be developed to treat CYP-induced cystitis.

Puerarin improves busulfan-induced disruption of spermatogenesis by inhibiting MAPK pathways.

Male infertility is a global concern, with a noticeable increase in the decline of spermatogenesis and sperm quality. However, there are limited clinically effective treatments available. This study aimed to investigate the potential effectiveness of puerarin in treating male infertility, which leads to gonadal changes. The results obtained from various analyses such as CASA, immunofluorescence, DIFF-Quick, hematoxylin and eosin (H&E), and periodic acid-Schiff (PAS) staining demonstrated that puerarin supplementation significantly alleviated the busulfan-induced reduction in spermatogenesis and sperm quality in both young and adult mice. Furthermore, puerarin exhibited a marked improvement in the damage caused by busulfan to the architecture of seminiferous tubules, causal epididymis, blood-testicular barrier (BTB), as well as spermatogonia and Sertoli cells. Similarly, puerarin significantly reduced the levels of total antioxidant capacity (T-AOC), malondialdehyde (MDA), and caspase-3 in the testes of busulfan-induced mice, as determined by microplate reader analysis. Additionally, RNA-seq data, RT-qPCR, and western blotting revealed that puerarin restored the abnormal gene expressions induced by busulfan to nearly healthy levels. Notably, puerarin significantly reversed the impact of busulfan on the expression of marker genes involved in spermatogenesis and oxidative stress. Moreover, puerarin suppressed the phosphorylation of p38, ERK1/2, and JNK in the testes, as observed through testicular analysis. Consequently, this study concludes that puerarin may serve as a potential alternative for treating busulfan-induced damage to male fertility by inactivating the testicular MAPK pathways. These findings may pave the way for the use of puerarin in addressing chemotherapy- or other factors-induced male infertility in humans.

TRAF inhibition drives cancer cell apoptosis and improves retinoic acid sensitivity in multiple cancers models.

TNF receptor-associated factors (TRAFs) are signaling adaptor proteins that play a crucial role in regulating cellular receptors' signaling transduction to downstream pathways and exert multifaceted roles in regulating signaling pathways, cell survival, and carcinogenesis. The 13-cis-retinoic acid (RA), an active metabolite of vitamin A, exhibits anti-cancer properties, but the development of retinoic acid resistance poses a challenge in clinical application. This study aimed to investigate the relationship between TRAFs and retinoic acid sensitivity in various cancers. Here, we revealed that TRAFs' expression varied significantly across The Cancer Genome Atlas (TCGA) cancer cohorts and human cancer cell lines. Additionally, inhibiting TRAF4, TRAF5, or TRAF6 improved retinoic acid sensitivity and reduced colony formation in ovarian cancer and melanoma cells. Mechanistically, knocking down TRAF4, TRAF5, or TRAF6 in retinoic acid-treated cancer cell lines increased the levels of procaspase 9 and induced cell apoptosis. Further in vivo studies using the SK-OV-3 and MeWo xenograft models confirmed the anti-tumor effects of TRAF knockdown combined with retinoic acid treatment. These findings support that combination therapy with retinoic acid and TRAF silencing may offer significant therapeutic advantages in treating melanoma and ovarian cancers.

Novel mutations of TEX11 are associated with non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) affects 10% of infertile men worldwide, and genetic studies revealed that there are plenty of monogenic mutations that responsible for a part of idiopathic NOA cases. Testis-expressed gene 11 (TEX11) is an X-linked meiosis-specific gene, many pathogenic variants in TEX11 have been detected in NOA patients, and the deficiency of this gene can cause abnormal meiotic recombination and chromosomal synapsis. However, many NOA-affected cases caused by TEX11 mutation remain largely unknown. This study reported three novel TEX11 mutations (exon 5, c.313C>T: p.R105*), (exon 7, c.427A>C: p.K143Q) and (exon 29, c.2575G>A: p.G859R). Mutations were screened using whole-exome sequencing (WES) and further verified by amplifying and sequencing the specific exon. Histological analysis of testicular biopsy specimens revealed a thicker basement membrane of the seminiferous tubules and poorly developed spermatocytes, and no post-meiotic round spermatids or mature spermatozoa were observed in the seminiferous tubules of patients with TEX11 mutation. Conclusion: This study presents three novel variants of TEX11 as potential infertility alleles that have not been previously reported. It expanded the variant spectrum of patients with NOA, which also emphasizes the necessity of this gene screening for the clinical auxiliary diagnosis of patients with azoospermia.

Hydrogen sulfide protects Sertoli cells against toxicant Acrolein-induced cell injury.

Acrolein (ACR), a highly toxic α,ß-unsaturated aldehyde, is considered to be a common mediator behind the reproductive injury induced by various factors. However, the understanding of its reproductive toxicity and prevention in reproductive system is limited. Given that Sertoli cells provide the first-line defense against various toxicants and that dysfunction of Sertoli cell causes impaired spermatogenesis, we, therefore, examined ACR cytotoxicity in Sertoli cells and tested whether hydrogen sulfide (H2S), a gaseous mediator with potent antioxidative actions, could have a protective effect. Exposure of Sertoli cells to ACR led to cell injury, as indicated by reactive oxygen species (ROS) generation, protein oxidation, P38 activation and ultimately cell death that was prevented by antioxidant N-acetylcysteine (NAC). Further studies revealed that ACR cytotoxicity on Sertoli cells was significantly exacerbated by the inhibition of H2S-synthesizing enzyme cystathionine γ-lyase (CSE), while significantly suppressed by H2S donor Sodium hydrosulfide (NaHS). It was also attenuated by Tanshinone IIA (Tan IIA), an active ingredient of Danshen that stimulated H2S production in Sertoli cells. Apart from Sertoli cells, H2S also protected the cultured germ cells from ACR-initiated cell death. Collectively, our study characterized H2S as endogenous defensive mechanism against ACR in Sertoli cells and germ cells. This property of H2S could be used to prevent and treat ACR-related reproductive injury.

TRAF4 Silencing Induces Cell Apoptosis and Improves Retinoic Acid Sensitivity in Human Neuroblastoma.

Neuroblastoma (NB) is a pediatric malignancy that arises in the peripheral nervous system, and the prognosis in the high-risk group remains dismal, despite the breakthroughs in multidisciplinary treatments. The oral treatment with 13-cis-retinoic acid (RA) after high-dose chemotherapy and stem cell transplant has been proven to reduce the incidence of tumor relapse in children with high-risk neuroblastoma. However, many patients still have tumors relapsed following retinoid therapy, highlighting the need for the identification of resistant factors and the development of more effective treatments. Herein, we sought to investigate the potential oncogenic roles of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family in neuroblastoma and explore the correlation between TRAFs and retinoic acid sensitivity. We discovered that all TRAFs were efficiently expressed in neuroblastoma, but TRAF4, in particular, was found to be strongly expressed. The high expression of TRAF4 was associated with a poor prognosis in human neuroblastoma. The inhibition of TRAF4, rather than other TRAFs, improved retinoic acid sensitivity in two human neuroblastoma cell lines, SH-SY5Y and SK-N-AS cells. Further in vitro studies indicated that TRAF4 suppression induced retinoic acid-induced cell apoptosis in neuroblastoma cells, probably by upregulating the expression of Caspase 9 and AP1 while downregulating Bcl-2, Survivin, and IRF-1. Notably, the improved anti-tumor effects from the combination of TRAF4 knockdown and retinoic acid were confirmed in vivo using the SK-N-AS human neuroblastoma xenograft model. In conclusion, the highly expressed TRAF4 might be implicated in developing resistance to retinoic acid treatment in neuroblastoma, and the combination therapy with retinoic acid and TRAF4 inhibition may offer significant therapeutic advantages in the treatment of relapsed neuroblastoma.

Puerarin extends the lifespan of Drosophila melanogaster by activating autophagy.

Lifespan longevity has attracted increasing attention with societal development. To counter the effects of aging on longevity, we focused on the natural chemicals of plants. In this study, we investigated the effects of puerarin supplementation on the lifespan of Drosophila melanogaster. Puerarin supplementation significantly extended the lifespan of D. melanogaster at 60 µM and 120 µM by upregulating proteasome subunit beta 5 (prosbeta5) and sirtuin-1 (Sirt1). However, puerarin-induced longevity of male flies (F0 generation) may not be passed on to descendants. Additionally, a puerarin diet for 10 and 25 days did not influence the body weight and food intake of male Canton-S flies. Puerarin significantly improved the climbing ability, starvation resistance, and oxidation resistance of male flies by upregulating the expression of Shaker, catalase (CAT), superoxide dismutase 1 (SOD1), and Methuselah, and downregulating poly [ADP-ribose] polymerase (PARP-1) and major heat shock 70 kDa protein Aa (HSP70). Moreover, 120 µM puerarin supplementation for 25 days significantly increased adenosine 5' triphosphate (ATP) content by increasing adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) levels. Additionally, the puerarin diet for 25 days suppressed male fecundity in male flies by decreasing the levels of Bam and Punt. Mechanistically, puerarin enhanced lysosome-involved autophagy by promoting the expression of lysosome markers [ß-galactosidase and lysosomal associated membrane protein 1 (LAMP1)], and elevating the levels of autophagy-related genes, including autophagy-associated gene 1 (ATG1), ATG5, and ATG8b. However, puerarin decreased the phosphorylation of the target of rapamycin (TOR) protein. In conclusion, puerarin is a promising compound for improving the longevity of D. melanogaster by activating autophagy.

An ultrasensitive FRET-based fluorescent low molecular weight heparin nanoprobe for quantifying heparanase activity.

Heparanase (HPA) is a multifaceted endo-ß-glucuronidase, and its dysregulation facilitates cancer metastasis. Developing techniques for fast and sensitively monitoring HPA enzymatic activity is crucial for searching for molecular therapies targeting HPA. Herein, we developed a novel fluorescence resonance energy transfer (FRET)-based nanoprobe AuNCs-LMWH-AuNRs, with AuNCs@GSH-cys and AuNRs/end-NH2/side-SiO2 attached to the non-reducing terminus and reducing terminus of low molecular weight heparin (LMWH), respectively. AuNCs@GSH-cys exhibited an absolute quantum yield of 1.1%. The absorption spectra of AuNRs/end-NH2/side-SiO2 (825 nm for maximum longitudinal absorption) and the emission spectra of AuNCs@GSH-cys (824 nm for maximum emission) were precisely overlapping, further enhancing the efficiency of FRET. In the presence of HPA, the LMWH nanoprobe exhibited an ultrasensitive response with excitation/emission wavelength (lambda (ex) = 560 nm, lambda (em) = 824 nm). The probe presented a wide linear dynamic detection range (LDR) of 0.125 ng/µL - 0.01 µg/µL in vitro with a limit of detection (LODs) of 82.15 pM (0.43 pg/µL). The excellent selectivity and good fluorescence turn-on efficiency of the probe made it possible for one-step detection of cellular heparanase activity. High throughput screening of HPA inhibitors also can be accomplished using the highly efficient LMWH nanoprobe.

ß-Defensin 19/119 mediates sperm chemotaxis and is associated with idiopathic infertility.

Sperm chemotaxis is required for guiding sperm toward the egg. However, the molecular identity of physiological chemoattractant and its involvement in infertility remain elusive. Here, we identify DEFB19/119 (mouse/human orthologs) as a physiological sperm chemoattractant. The epithelia of the female reproductive tract and the cumulus-oocyte complex secrete DEFB19/119 that elicits calcium mobilization via the CatSper channel and induces sperm chemotaxis in capacitated sperm. Manipulating the level of DEFB19 in mice determines the number of sperm arriving at the fertilization site. Importantly, we identify exon mutations in the DEFB119 gene in idiopathic infertile women with low level of DEFB119 in the follicular fluid. The level of DEFB119 correlates with the chemotactic potency of follicular fluid and predicts the infertile outcome with positive correlation. This study reveals the pivotal role of DEFB19/119 in sperm chemotaxis and demonstrates its potential application in the diagnosis of idiopathic infertility.

Single-Cell Transcriptomics Reveals Longevity Immune Remodeling Features Shared by Centenarians and Their Offspring.

Centenarians, who show mild infections and low incidence of tumors, are the optimal model to investigate healthy aging. However, longevity related immune characteristics has not been fully revealed largely due to lack of appropriate controls. In this study, single-cell transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) derived from seven centenarians (CEN), six centenarians' offspring (CO), and nine offspring spouses or neighbors (Control, age-matched to CO) are performed to investigate the shared immune features between CEN and CO. The results indicate that among all 12 T cell clusters, the cytotoxic-phenotype-clusters (CPC) and the naïve-phenotype-clusters (NPC) significantly change between CEN and ontrol. Compared to Control, both CEN and CO are characterized by depleted NPC and increased CPC, which is dominated by CD8+ T cells. Furthermore, CPC from CEN and CO share enhanced signaling pathways and transcriptional factors associated with immune response, and possesse similar T-cell-receptor features, such as high clonal expansion. Interestingly, rather than a significant increase in GZMK+ CD8 cells during aging, centenarians show accumulation of GZMB+ and CMC1+ CD8 T cells. Collectively, this study unveils an immune remodeling pattern reflected by both quantitative increase and functional reinforcement of cytotoxic T cells which are essential for healthy aging.

Testicular aging, male fertility and beyond.

Normal spermatogenesis and sperm function are crucial for male fertility. The effects of healthy testicular aging and testicular premature aging on spermatogenesis, sperm function, and the spermatogenesis microenvironment cannot be ignored. Compared with younger men, the testis of older men tends to have disturbed spermatogenic processes, sperm abnormalities, sperm dysfunction, and impaired Sertoli and Leydig cells, which ultimately results in male infertility. Various exogenous and endogenous factors also contribute to pathological testicular premature aging, such as adverse environmental stressors and gene mutations. Mechanistically, Y-chromosomal microdeletions, increase in telomere length and oxidative stress, accumulation of DNA damage with decreased repair ability, alterations in epigenetic modifications, miRNA and lncRNA expression abnormalities, have been associated with impaired male fertility due to aging. In recent years, the key molecules and signaling pathways that regulate testicular aging and premature aging have been identified, thereby providing new strategies for diagnosis and treatment. This review provides a comprehensive overview of the underlying mechanisms of aging on spermatogenesis. Furthermore, potential rescue measures for reproductive aging have been discussed. Finally, the inadequacy of testicular aging research and future directions for research have been envisaged to aid in the diagnosis and treatment of testicular aging and premature aging.

lncRNA 1700101O22Rik and NONMMUG030480.1 Are Not Essential for Spermatogenesis in Mice.

Many testis-specific lncRNAs are highly expressed in late spermatogenesis, especially in spermiogenesis. However, their functions and the underlying mechanisms in male fertility are largely unknown. Here, we screened two highly expressed lncRNAs, 1700101O22Rik (O22Rik) and NONMMUG030480.1 (NM480) in testes, to investigate the roles in spermatogenesis using lncRNA knockout (KO) mouse generated by CRISPER/Cas9 technology. Both testis-specific lncRNAs were mainly expressed from secondary spermatocytes to round spermatids, suggesting that they might be involved in spermiogenesis. Phenotypic analysis showed that the deletion of O22Rik or NM480 did not affect the development of testis and epididymis or spermatogenesis. These results were confirmed in both young and middle-aged male mice. In addition, there was no significant difference in sperm morphology and other parameters including concentration and motility between wild type (WT) and KO mice. Fertility tests showed that litter size was significantly lower in O22Rik KO mice compared with WT controls. Although O22Rik did not exert dramatic roles in spermatogenesis, on molecular levels, its surrounding gene expression was disturbed significantly. Gm32773 was decreased; however, Gm32828 was increased in KO mice. In conclusion, lncRNA O22Rik and NM480 are not individually essential for spermatogenesis in mice.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

Involvement of Ca2+ and ROS signals in nickel-impaired human sperm function.

As one of the main environmental pollutants and occupational hazards, nickel has been reported to have mutagenic, carcinogenic, and teratogenic properties, as well as reproductive toxicity. However, how nickel affects human reproduction is still unclear. In this study, the toxicity of nickel on human sperm and the underlying mechanisms were evaluated in vitro. We found that NiCl2 (10, 50, and 250 µM) impaired sperm total motility and progressive motility in a dose- and time-dependent manner. In addition, sperm hyperactivation and the ability of human sperm to penetrate a viscous medium were found to be compromised after nickel exposure. Mechanically, NiCl2 significantly inhibited the basal intracellular Ca2+ signaling. Besides, reactive oxygen species (ROS), superoxide, and malondialdehyde levels were increased in human sperm after exposure to different concentrations of NiCl2. Consistently, eliminating excess ROS by N-acetyl-L-cysteine or tocopherol significantly alleviated nickel-impaired sperm motility. Taken together, these results revealed that nickel could compromise sperm functions by interfering with Ca2+ signaling and inducing excessive oxidative stress. These findings suggest that, in the high and occupational nickel exposure environments, the contribution of nickel toxicity to the males who wish to preserve their fertility is worthy of careful evaluation.