A conserved fertilization complex bridges sperm and egg in vertebrates.

Fertilization, the basis for sexual reproduction, culminates in the binding and fusion of sperm and egg. Although several proteins are known to be crucial for this process in vertebrates, the molecular mechanisms remain poorly understood. Using an AlphaFold-Multimer screen, we identified the protein Tmem81 as part of a conserved trimeric sperm complex with the essential fertilization factors Izumo1 and Spaca6. We demonstrate that Tmem81 is essential for male fertility in zebrafish and mice. In line with trimer formation, we show that Izumo1, Spaca6, and Tmem81 interact in zebrafish sperm and that the human orthologs interact in vitro. Notably, complex formation creates the binding site for the egg fertilization factor Bouncer in zebrafish. Together, our work presents a comprehensive model for fertilization across vertebrates, where a conserved sperm complex binds to divergent egg proteins-Bouncer in fish and JUNO in mammals-to mediate sperm-egg interaction.

Orientation Determination of Cryo-EM Projection Images Using Reliable Common Lines and Spherical Embeddings.

Three-dimensional (3D) reconstruction in single-particle cryo-electron microscopy (cryo-EM) is a critical technique for recovering and studying the fine 3D structure of proteins and other biological macromolecules, where the primary issue is to determine the orientations of projection images with high levels of noise. This paper proposes a method to determine the orientations of cryo-EM projection images using reliable common lines and spherical embeddings. First, the reliability of common lines between projection images is evaluated using a weighted voting algorithm based on an iterative improvement technique and binarized weighting. Then, the reliable common lines are used to calculate the normal vectors and local X-axis vectors of projection images after two spherical embeddings. Finally, the orientations of projection images are determined by aligning the results of the two spherical embeddings using an orthogonal constraint. Experimental results on both synthetic and real cryo-EM projection image datasets demonstrate that the proposed method can achieve higher accuracy in estimating the orientations of projection images and higher resolution in reconstructing preliminary 3D structures than some common line-based methods, indicating that the proposed method is effective in single-particle cryo-EM 3D reconstruction.

An Exact Bayesian Model for Meta-Analysis of the Standardized Mean Difference with Its Simultaneous Credible Intervals.

While Bayesian methodology is increasingly favored in behavioral research for its clear probabilistic inference and model structure, its widespread acceptance as a standard meta-analysis approach remains limited. Although some conventional Bayesian hierarchical models are frequently used for analysis, their performance has not been thoroughly examined. This study evaluates two commonly used Bayesian models for meta-analysis of standardized mean difference and identifies significant issues with these models. In response, we introduce a new Bayesian model equipped with novel features that address existing model concerns and a broader limitation of the current Bayesian meta-analysis. Furthermore, we introduce a simple computational approach to construct simultaneous credible intervals for the summary effect and between-study heterogeneity, based on their joint posterior samples. This fully captures the joint uncertainty in these parameters, a task that is challenging or impractical with frequentist models. Through simulation studies rooted in a joint Bayesian/frequentist paradigm, we compare our model's performance against existing ones under conditions that mirror realistic research scenarios. The results reveal that our new model outperforms others and shows enhanced statistical properties. We also demonstrate the practicality of our models using real-world examples, highlighting how our approach strengthens the robustness of inferences regarding the summary effect.

Specific Knockout of Notch-1 in Macrophages Modulate the Progression of Hepatic Insulin Resistance in HFD Fed Mice via Regulating IRE1α-XBP1 Signals.

OBJECTIVE: To develop an intervention based on Notch-1 signalling pathway blockade by investigating the potential application of the neurogenic locus notch homologue protein 1(Notch-1) signalling pathway as a key regulator of chronic inflammation and adipogenesis in the treatment of hepatic insulin resistance (HIR). STUDY DESIGN: Experimental study. Place and Duration of the Study: Animal Laboratory of the Fourth Hospital of Hebei Medical University, Shijiazhuang, China, from April 2021 to June 2022. METHODOLOGY: HIR models were established in Notch-1WT and Notch-1MAC-KO mice by high fat diet (HFD) for 16 weeks. Haematoxylin and eosin (HE) staining and oil red O (ORO) staining were used to detect inflammatory infiltration and lipid accumulation in each group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of TNF-α and IL-6. Free fatty acid (FFA) and total cholesterol (TC) were measured with relevant kits. Moreover, real-time quantitative polymerase chain reaction (PCR) was performed to detect the relative expressions of F4/80, Mcp1, and CD11b in hepatic tissues. Mass spectrometry was used to analyse the levels of triglyceride (TG), diacylglycerol (DAG) and conformite europeenne (CE) in liver tissue. Western blotting was used to detect the expression of related proteins. RESULTS: Specific knockdown of Notch-1 in macrophages decreases the relative fluorescence intensity of CD68 and attenuates inflammatory infiltration and lipid degeneration. There was no difference in plasma levels of FFA and TG. Specific knockdown of Notch-1 in macrophages decreases the expression of F4/80, Mcp1, and CD11b, as well as the levels of TG, DAG, CE, IL-6, and TNF-α. CONCLUSION: Specific knockout of Notch-1 in macrophages may reduce HIR by inhibiting the IRE1α-XBP1 signalling pathway. KEY WORDS: Hepatic insulin resistance, Macrophages, Notch-1, IRE1α, XBP1.

ZP2 cleavage blocks polyspermy by modulating the architecture of the egg coat.

Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm.

A kinetic model for solving a combination optimization problem in ab-initio Cryo-EM 3D reconstruction.

Cryo-Electron Microscopy (cryo-EM) is a widely used and effective method for determining the three-dimensional (3D) structure of biological molecules. For ab-initio Cryo-EM 3D reconstruction using single particle analysis (SPA), estimating the projection direction of the projection image is a crucial step. However, the existing SPA methods based on common lines are sensitive to noise. The error in common line detection will lead to a poor estimation of the projection directions and thus may greatly affect the final reconstruction results. To improve the reconstruction results, multiple candidate common lines are estimated for each pair of projection images. The key problem then becomes a combination optimization problem of selecting consistent common lines from multiple candidates. To solve the problem efficiently, a physics-inspired method based on a kinetic model is proposed in this work. More specifically, hypothetical attractive forces between each pair of candidate common lines are used to calculate a hypothetical torque exerted on each projection image in the 3D reconstruction space, and the rotation under the hypothetical torque is used to optimize the projection direction estimation of the projection image. This way, the consistent common lines along with the projection directions can be found directly without enumeration of all the combinations of the multiple candidate common lines. Compared with the traditional methods, the proposed method is shown to be able to produce more accurate 3D reconstruction results from high noise projection images. Besides the practical value, the proposed method also serves as a good reference for solving similar combinatorial optimization problems.

Disruption of testis-enriched cytochrome c oxidase subunit COX6B2 but not COX8C leads to subfertility.

Mammalian sperm flagellum contains the midpiece characterized by a mitochondrial sheath that packs tightly around the axoneme and outer dense fibers. Mitochondria are known as the "powerhouse" of the cell, and produce ATP through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). However, the contribution of the TCA cycle and OXPHOS to sperm motility and male fertility is less clear. Cytochrome c oxidase (COX) is an oligomeric complex localized within the mitochondrial inner membrane, and the terminal enzyme of the mitochondrial electron transport chain in eukaryotes. Both COX6B2 and COX8C are testis-enriched COX subunits whose functions in vivo are poorly studied. Here, we generated Cox6b2 and Cox8c knockout (KO) mice using the CRISPR/Cas9 system. We examined their fertility and sperm mitochondrial function to determine the significance of testis-enriched COX subunits in male fertility. The mating test revealed that disrupting COX6B2 induces male subfertility, while disrupting COX8C does not affect male fertility. Cox6b2 KO spermatozoa showed low sperm motility, but mitochondrial function was normal according to oxygen consumption rates. Therefore, low sperm motility seems to cause subfertility in Cox6b2 KO male mice. These results also indicate that testis-enriched COX, COX6B2 and COX8C, are not essential for OXPHOS in mouse spermatozoa.

Assessment of infant gonadal dose irradiated from urine-contaminated diapers during diuretic renal scintigraphy.

OBJECTIVE: To estimate the gonadal doses irradiated from urine- contaminated diapers during diuretic renal scintigraphy. METHODS: Images of 31 patients (18 males and 13 females) with urine-contaminated diapers during 99m Tc-MAG3 renal scintigraphy were analyzed. The count rate of the diapers was converted into a time-activity curve based on the calibrated factor of the gamma camera system. The cumulative activity was determined from the area under the curve. By incorporating dose per unit cumulative activity pre-calculated from Monte Carlo simulation with 0-year phantom, the gonadal dose irradiated from diaper was calculated. To assess the degree of this additionally introduced dose from diapers, the calculated gonadal dose was compared to the internal gonadal dose from injected radiotracer activity. RESULTS: The cumulative activities irradiated from urine-contaminated diapers were 1.12 E04 ±â€…1.29E04 MBq.s in male infants, which was nearly half of the 1.94 E04 ±â€…1.80E04 MBq.s ( P  = 0.15) in female infants. However, the absorbed doses for testes in male infants were 7.37E-01 ±â€…8.50E-01 mGy, which was approximately 10 times the 6.38E-02 ±â€…5.94E-02 mGy for ovaries in female infants ( P  < 0.01). The diaper-introduced dose for testes and ovaries was 91.7% and 3.9% of the gonadal doses from the injected activity in patients with normal renal function, and 99.0% and 4.3% of those in patients with abnormal renal function. CONCLUSION: Urine-contaminated diapers introduced additional radiation doses to infant patients during 99m Tc-MAG3 renal scintigraphy. The gonadal doses were of significance in male infants who had nearly double the absorbed dose for the testes.

Specific knockout of notch-1 attenuates non-alcoholic fatty liver disease by promoting SHP2 phosphorylation.

OBJECTIVE: To investigate the effect of Notch-1 signaling on NAFLD and its molecular mechanism. METHODS: The lipid deposition in liver tissues was detected by oil red O staining. Western blotting was performed to detect the expressions of SREBP1C, SREBP2, LXR, IL-1ß, IL-18, NLRP3, Notch-1, NOX2, NOX4, p-PI3K and p-SHP2 in macrophages, and the expressions of ALIX, CD9, IL-1ß and SREBP1C in exosomes. Macrophages in the Notch-1MAC-KO group and Notch-1WT group were treated with FFA, and those in the Notch-1WT+FFA group and Notch-1MAC-KO+FFA group were treated with SHP2 inhibitors PHPS1 and Relaxin. RESULTS: It was observed by oil red O staining that lipid deposition in mice with NAFLD was reduced in the Notch-1MAC-KO group. The results of Western blotting showed that the expressions of ALIX, CD9, IL-1ß and SREBP1C in macrophage exosomes were significantly lower in the Notch-1MAC-KO group than in the Notch-1WT group. In macrophages, the expressions of SREBP1C, SREBP2, LXR, IL-1ß, IL-18, Notch-1, NOX2, NOX4 and p-PI3K significantly decreased, while the expression of p-SHP2 significantly increased in the Notch-1MAC-KO group compared with the Notch-1WT group. The Notch-1MAC-KO+FFA group had significantly decreased expressions of SREBP1C, NLRP3, IL-1ß, IL-18, SREBP2, NOX2, NOX4 and p-PI3K and a significantly increased expression of p-SHP2 compared with the Notch-1WT+FFA group. However, the differences in the above proteins were all eliminated after PHPS1 and Relaxin were added. CONCLUSION: Specific knockout of Notch-1 attenuates NAFLD, and reduces inflammation and lipid deposition in the liver by promoting SHP2 phosphorylation.

Down-regulation of EZH2 genes targeting RUNX3 affects proliferation, invasion, and metastasis of human colon cancer cells by Wnt/ß-catenin signaling pathway.

In order to detect the effect of EZH2 genes on proliferation, migration, invasion, and apoptosis of colon carcinoma cell strains HCT116 and HT29 by the Wnt/ß-catenin signaling pathway, qRT-PCR was applied to measure relative expressions of EZH2, RUNX3, CEA, CA199, MMP-9, VEGF, ß-catenin, and CyclinD1 in each group; Western-blot was employed with the intention of exploring relative expressions of these proteins in vivo and in vitro; monoclonal proliferation experiments and CCK-8 assay was adopted so as to check cell proliferation; the effect on cell migration was investigated via Transwell assay and cell scratch wound assay; flow cytometry was applied with a view to determining the effect on cell apoptosis. Transfected HCT116 cells are injected subcutaneously into nude mice. In colon cell strains HCT-116 and HT29, contrasted to the si-NC group, the RUNX3 expression was prominently up-regulated in the si-EZH2 group. Besides, expressions of CEA, CA199, MMP-9, and VEGF were significantly reduced; down-regulation of EZH2 genes remarkably inhibited cell proliferation, invasion and migration when facilitating apoptosis; down-regulation of EZH2 genes also significantly reduced expressions of essential proteins ß-catenin and CyclinD1 on the Wnt pathway. The subcutaneous tumor body of nude mice was reduced. EZH2-OE is the opposite trend to si-EZH2; The EZH2 gene may target regulatory RUNX3 regulation via that Wnt/ß-catenin signaling pathway, hence affecting colon carcinoma cell proliferation, invasion, migration, and apoptosis. Therefore, EZH2 may become a promising target for the clinical therapy of colon carcinoma.

CRISPR/Cas9-mediated genome editing reveals seven testis-enriched transmembrane glycoproteins dispensable for male fertility in mice.

BACKGROUND: Mammalian fertilization is mediated by multiple sperm acrosomal proteins, many of which are testis-enriched transmembrane glycoproteins expressed during spermiogenesis (e.g., Izumo sperm-egg fusion 1, Sperm acrosome associated 6, and Transmembrane protein 95). METHODS: We hypothesized that proteins with these features might have a role in sperm-egg interaction and thus carried out an in-silico screen based on multiple public databases. We generated knockout mouse lines lacking seven candidate proteins by the CRISPR/Cas9 system and conducted detailed analyses on the fecundity of the knockout males, as well as their testis appearance and weight, testis and epididymis histology, and sperm motility and morphology. RESULTS: Through the in-silico screen, we identified 4932438H23Rik, A disintegrin and metalloproteinase domain-containing protein 29, SAYSvFN domain-containing protein 1, Sel-1 suppressor of lin-12-like 2 (C. elegans), Testis-expressed protein 2, Transmembrane and immunoglobulin domain-containing 3, and Zinc and ring finger 4. Phenotypic analyses unveiled that the knockout males showed normal testis gross appearance, normal testis and epididymis histology, and normal sperm morphology and motility. Fertility tests further indicated that the knockout male mice could sire pups with normal litter sizes when paired with wild-type females. DISCUSSION AND CONCLUSION: These findings suggest that these seven proteins are individually dispensable for male reproduction and fertilization. Future studies are warranted to devise advanced in-silico screening approaches that permit effective identification of gamete fusion-required sperm proteins.

Macrophage-specific deletion of Notch-1 induced M2 anti-inflammatory effect in atherosclerosis via activation of the PI3K-oxidative stress axis.

OBJECTIVE: Notch-1 signaling is significantly associated with the occurrence and development of atherosclerosis (AS). However, the molecular mechanisms underlying the specific deletion of Notch-1 in AS-associated macrophages are not fully understood. This study aimed to investigate the effects of Notch-1 in AS. METHODS AND RESULTS: Tissue samples were obtained from atherosclerotic segments of human carotid arteries. Immunofluorescence staining showed that Notch-1 was significantly colocalized with macrophages (CD68+), and Notch-1 staining was increased in human vulnerable plaques. Notch-1MAC-KO/ApoE-/- mice were generated in which Notch-1 was selectively inactivated in macrophages, and WT for littermate control mice (ApoE-/-/Notch-1WT). A control group was then established. All mice fed with a high-fat and Oil Red O, Movat, a-SMA, CD68, and Sirius red staining were used to evaluate the morphology. Specific deletion of Notch-1 in macrophages repressed the pathophysiology of AS. Immunofluorescent staining and Western blotting revealed that Notch-1MAC-KO repressed M1 and M2 responses in AS. Here, GSEA revealed that Notch-1 activation and PI3K signaling were statistically significantly correlated with each other, and Notch-1 was involved in the regulation of the PI3K signaling pathway. In the in vitro experiments, the secretion of Arg-1 and exosomes was classified by peritoneal macrophages of Notch-1MAC-KO/ApoE-/- and Notch-1WT/ApoE-/- mice. Immunohistochemistry staining and Western blotting were used to measure the expression levels of Notch1, PI3K, p-PI3K, AKT, p-AKT, Arg-1, IL-6, CD36, SREBP-1, CD206, iNOS, cleaved-caspase-3/-9, Bax, CD9, Alix and TSG101 in the peritoneal macrophages and exosomes, respectively. CONCLUSIONS: The specific deletion of Notch-1 in macrophage represses the formation and development of AS via the PI3K/AKT signaling pathway.

Macrophage-derived SHP-2 inhibits the metastasis of colorectal cancer via Tie2-PI3K signals.

This research aimed to explore the influence of Src homology-2 containing protein tyrosine phosphatase (SHP-2) on the functions of tyrosine kinase receptors with immunoglobulin and EGF homology domains 2 (Tie2)-expressing monocyte/macrophages (TEMs) and the influence of the angiopoietin(Ang)/Tie2-phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) (Ang/Tie2-PI3K/Akt/mTOR) signaling pathway on the tumor microvascular remodeling in an immunosuppressive microenvironment. In vivo, SHP-2-deficient mice were used to construct colorectal cancer (CRC) liver metastasis models. SHP-2-deficient mice had significantly more metastatic cancer and inhibited nodules on the liver surface than wild-type mice, and the high-level expression of p-Tie2 was found in the liver tissue of the macrophages' specific SHP-2-deficient mice (SHP-2MAC-KO) + planted tumor mice. Compared with the SHP-2 wild type mice (SHP-2WT) + planted tumor group, the SHP-2MAC-KO + planted tumor group experienced increased expression of p-Tie2, p-PI3K, p-Akt, p-mTOR, vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), matrix metalloproteinase 2 (MMP2), and MMP9 in the liver tissue. TEMs selected by in vitro experiments were co-cultured with remodeling endothelial cells and tumor cells as carriers. It was found that when Angpt1/2 was used for stimulation, the SHP-2MAC-KO + Angpt1/2 group displayed evident increases in the expression of the Ang/Tie2-PI3K/Akt/mTOR pathway. The number of cells passing through the lower chamber and the basement membrane and the number of blood vessels formed by cells compared with the SHP-2WT + Angpt1/2 group, while these indexes were subjected to no changes under the simultaneous stimulation of Angpt1/2 + Neamine. To sum up, the conditional knockout of SHP-2 can activate the Ang/Tie2-PI3K/Akt/mTOR pathway in TEMs, thereby strengthening tumor micro angiogenesis in the microenvironment and facilitating CRC liver metastasis.

An Unsupervised Classification Algorithm for Heterogeneous Cryo-EM Projection Images Based on Autoencoders.

Heterogeneous three-dimensional (3D) reconstruction in single-particle cryo-electron microscopy (cryo-EM) is an important but very challenging technique for recovering the conformational heterogeneity of flexible biological macromolecules such as proteins in different functional states. Heterogeneous projection image classification is a feasible solution to solve the structural heterogeneity problem in single-particle cryo-EM. The majority of heterogeneous projection image classification methods are developed using supervised learning technology or require a large amount of a priori knowledge, such as the orientations or common lines of the projection images, which leads to certain limitations in their practical applications. In this paper, an unsupervised heterogeneous cryo-EM projection image classification algorithm based on autoencoders is proposed, which only needs to know the number of heterogeneous 3D structures in the dataset and does not require any labeling information of the projection images or other a priori knowledge. A simple autoencoder with multi-layer perceptrons trained in iterative mode and a complex autoencoder with residual networks trained in one-pass learning mode are implemented to convert heterogeneous projection images into latent variables. The extracted high-dimensional features are reduced to two dimensions using the uniform manifold approximation and projection dimensionality reduction algorithm, and then clustered using the spectral clustering algorithm. The proposed algorithm is applied to two heterogeneous cryo-EM datasets for heterogeneous 3D reconstruction. Experimental results show that the proposed algorithm can effectively extract category features of heterogeneous projection images and achieve high classification and reconstruction accuracy, indicating that the proposed algorithm is effective for heterogeneous 3D reconstruction in single-particle cryo-EM.

On the correction of respiratory motion-induced image reconstruction errors in positron-emission tomography-guided radiation therapy.

Background and purpose: Free breathing (FB) positron emission tomography (PET) images are routinely used in radiotherapy for lung cancer patients. Respiration-induced artifacts in these images compromise treatment response assessment and obstruct clinical implementation of dose painting and PET-guided radiotherapy. The purpose of this study is to develop a blurry image decomposition (BID) method to correct motion-induced image-reconstruction errors in FB-PETs. Materials and methods: Assuming a blurry PET is represented as an average of multi-phase PETs. A four-dimensional computed-tomography image is deformably registered from the end-inhalation (EI) phase to other phases. With the registration-derived deformation maps, PETs at other phases can be deformed from a PET at the EI phase. To reconstruct the EI-PET, the difference between the blurry PET and the average of the deformed EI-PETs is minimized using a maximum-likelihood expectation-maximization algorithm. The developed method was evaluated with computational and physical phantoms as well as PET/CT images acquired from three patients. Results: The BID method increased the signal-to-noise ratio from 1.88 ± 1.05 to 10.5 ± 3.3 and universal-quality index from 0.72 ± 0.11 to 1.0 for the computational phantoms, and reduced the motion-induced error from 69.9% to 10.9% in the maximum of activity concentration and from 317.5% to 8.7% in the full width at half maximum of the physical PET-phantom. The BID-based corrections increased the maximum standardized-uptake values by 17.7 ± 15.4% and reduced tumor volumes by 12.5 ± 10.4% on average for the three patients. Conclusions: The proposed image-decomposition method reduces respiration-induced errors in PET images and holds potential to improve the quality of radiotherapy for thoracic and abdominal cancer patients.

1700029I15Rik orchestrates the biosynthesis of acrosomal membrane proteins required for sperm-egg interaction.

Sperm acrosomal membrane proteins, such as Izumo sperm-egg fusion 1 (IZUMO1) and sperm acrosome-associated 6 (SPACA6), play essential roles in mammalian gamete binding or fusion. How their biosynthesis is regulated during spermiogenesis has largely remained elusive. Here, we show that 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik is a type-II transmembrane protein expressed in early round spermatids but not in mature spermatozoa. It interacts with proteins involved in N-linked glycosylation, disulfide isomerization, and endoplasmic reticulum (ER)-Golgi trafficking, suggesting a potential role in nascent protein processing. The ablation of 1700029I15Rik destabilizes non-catalytic subunits of the oligosaccharyltransferase (OST) complex that are pivotal for N-glycosylation. The knockout testes exhibit normal expression of sperm plasma membrane proteins, but decreased abundance of multiple acrosomal membrane proteins involved in fertilization. The knockout sperm show upregulated chaperones related to ER-associated degradation (ERAD) and elevated protein ubiquitination; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway underpinning the biosynthesis of acrosomal membrane proteins during spermiogenesis.

ADAD2 functions in spermiogenesis and piRNA biogenesis in mice.

BACKGROUND: Adenosine deaminase domain containing 2 (ADAD2) is a testis-specific protein composed of a double-stranded RNA binding domain and a non-catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive. OBJECTIVES: This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2. MATERIALS AND METHODS: Adad2 homozygous mutant mouse lines, Adad2-/- and Adad2Δ/Δ , were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co-immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2-interacting proteins in mouse testes. RNA-sequencing analyses were carried out to analyze the transcriptome and PIWI-interacting RNA (piRNA) populations in wildtype and Adad2 mutant testes. RESULTS: Adad2-/- and Adad2Δ/Δ mice exhibit male-specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA-binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co-localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster-derived pachytene piRNAs, and increases expression of ping-pong-derived piRNAs. DISCUSSION AND CONCLUSION: In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.

Eukaryotic fertilization and gamete fusion at a glance.

In sexually reproducing organisms, the genetic information is transmitted from one generation to the next via the merger of male and female gametes. Gamete fusion is a two-step process involving membrane recognition and apposition through ligand-receptor interactions and lipid mixing mediated by fusion proteins. HAP2 (also known as GCS1) is a bona fide gamete fusogen in flowering plants and protists. In vertebrates, a multitude of surface proteins have been demonstrated to be pivotal for sperm-egg fusion, yet none of them exhibit typical fusogenic features. In this Cell Science at a Glance article and the accompanying poster, we summarize recent advances in the mechanistic understanding of gamete fusion in eukaryotes, with a particular focus on mammalian species.