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In recent years, the awareness that pesticides can have other effects apart from generic toxicity is growing. In particular, several pieces of evidence highlight their influence on human fertility. In this study, we investigated, by a virtual screening approach, the binding between pesticides and proteins present in human gametes or associated with reproduction, in order to identify new interactions that could affect human fertility. To this aim, we prepared ligand (pesticides) and receptor (proteins) 3D structure datasets from online structural databases (such as PubChem and RCSB), and performed a virtual screening analysis using Autodock Vina. In the comparison of the predicted interactions, we found that famoxadone was predicted to bind Cellular Retinol Binding Protein-III in the retinol-binding site with a better minimum energy value of -10.4 Kcal/mol and an RMSD of 3.77 with respect to retinol (-7.1 Kcal/mol). In addition to a similar network of interactions, famoxadone binding is more stabilized by additional hydrophobic patches including L20, V29, A33, F57, L117, and L118 amino acid residues and hydrogen bonds with Y19 and K40. These results support a possible competitive effect of famoxadone on retinol binding with impacts on the ability of developing the cardiac tissue, in accordance with the literature data on zebrafish embryos. Moreover, famoxadone binds, with a minimum energy value between -8.3 and -8.0 Kcal/mol, to the IZUMO Sperm-Egg Fusion Protein, interacting with a network of polar and hydrophobic amino acid residues in the cavity between the 4HB and Ig-like domains. This binding is more stabilized by a predicted hydrogen bond with the N185 residue of the protein. A hindrance in this position can probably affect the conformational change for JUNO binding, avoiding the gamete membrane fusion to form the zygote. This work opens new interesting perspectives of study on the effects of pesticides on fertility, extending the knowledge to other typologies of interaction which can affect different steps of the reproductive process.

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Effective wild cat conservation programs with assisted reproductive technologies are being developed in different parts of the world. The flat-headed cat, fishing cat, and Asiatic golden cat are three species among nine wild Felidae in Thailand that are in need of urgent conservation efforts. Here, we assessed routine sperm characteristics and we report the detection of protein biomarkers related to the fertilization process, IZUMO1 and the CRISP family, and apoptotic markers, active or cleaved caspase-3, in semen samples collected from these wild cats. IZUMO1 was located in the equatorial segment of the sperm head, which is the region involved in gamete interaction. The highest levels of IZUMO1 were found in both the sperm pellet and the seminal plasma of the flat-headed cat, as determined by immunoblotting. CRISP2, a sperm-egg fusion assisting protein, and CRISP3 were found in both the sperm pellet and the seminal plasma, and the highest levels were observed in the fishing cat. Positive correlations between certain semen parameters and IZUMO1, CRISP2, and CRISP3 expression were also demonstrated. Cleaved caspase-3 was found in all sperm samples in all three species and was associated with an increase in DNA fragmentation and a decrease in certain semen characteristics such as motility, viability, and intact acrosomes. Our results suggest that the analysis of IZUMO1, the CRISP family, and cleaved caspase-3, along with the routine sperm characteristics, may allow for better success in breeding management in wild Felidae, particularly in the flat-headed cat and the fishing cat.

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Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets.

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IZUMO2 belongs to the testis-expressed IZUMO family of proteins, which are characterized by an N-terminal IZUMO domain. Based on integrated analysis of expression profiles and matched DNA methylation data from a public database, IZUMO2 represents a prognosis-related methylation-driven gene in colorectal cancer. However, it remains unclear whether IZUMO2 protein expression is suppressed or overexpressed in colorectal cancer cells. In this study, we aimed to elucidate the expression of the IZUMO2 protein in colorectal cancer, with a focus on the clinicopathological features. Sixty-four colorectal cancer tissue specimens were immunohistochemically stained using specific antibodies against IZUMO2. IZUMO2 immunoreactivity was detected at the invasion front in 30 of the 64 colorectal cancer samples. Kaplan-Meier analysis demonstrated that patients with IZUMO2 immunoreactivity had a relatively shorter overall and progression-free survival (log-rank test, P = 0.046 and 0.019, respectively). IZUMO2 immunoreactivity served as an independent factor predictive of poor progression-free survival in colorectal cancer (P = 0.025) as determined via the Cox proportional hazard regression model. Moreover, IZUMO2 immunoreactivity represented an independent factor for poor overall survival (P = 0.035) and progression-free survival (P = 0.013) in patients with colon cancer. The present findings suggest that IZUMO2 is expressed in many colorectal cancers, especially at the cancer invasion front, and may represent an indicator of poor prognosis in colorectal cancer.

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The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

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The fusion of mammalian gametes requires the interaction between IZUMO1 on the sperm and JUNO on the oocyte. We have recently shown that ectopic expression of mouse IZUMO1 induces cell-cell fusion and that sperm can fuse to fibroblasts expressing JUNO. Here, we found that the incubation of mouse sperm with hamster fibroblasts or human epithelial cells in culture induces the fusion between these somatic cells and the formation of syncytia, a pattern previously observed with some animal viruses. This sperm-induced cell-cell fusion requires a species-matching JUNO on both fusing cells, can be blocked by an antibody against IZUMO1, and does not rely on the synthesis of new proteins. The fusion is dependent on the sperm's fusogenic capacity, making this a reliable, fast, and simple method for predicting sperm function during the diagnosis of male infertility.

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BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by the loss of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The aggressive clinicopathological features and resistance to currently available therapeutics of the disease warrant an urgent need for the development of novel alternate therapeutic options. We have previously reported adiponectin-expressing regulatory T cells (A-Tregs), which can induce apoptosis in TNBC through the cell-in-cell phenomenon. In this study, we aimed to elucidate the molecule that allows TNBC cells to engulf A-Tregs. METHODS: A monoclonal antibody, which repressed the engulfment of A-Tregs by TNBC cells, was developed. Immunoprecipitation followed by mass spectrometry and small interfering RNAs-mediated gene silencing was performed to characterize the antigen. RESULTS: We successfully generated a monoclonal antibody, designated G1D7, which abrogated the engulfment of A-Tregs by TNBC and subsequent A-Treg-mediated apoptosis. G1D7 detected the immunoglobulin-like type I membrane protein IZUMO2, a molecule related to IZUMO1 that is essential for cell-cell membrane binding and fusion of sperm to oocyte. CONCLUSION: The findings highlight the importance of IZUMO2 on TNBC cells in facilitating the cell-in-cell phenomenon by A-Tregs.

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BACKGROUND: There is still a considerable gap between average life expectancy and healthy life expectancy in Japan. Recent research has revealed that gut frailty may be a worsening factor for various diseases, a cause of chronic inflammation, and a precursor to frailty. SUMMARY: Among self-reported symptoms, constipation is particularly significant as one of the key symptoms of gut frailty. Studies have demonstrated that individuals with constipation have significantly lower survival rates and are also at a higher risk of developing various diseases such as chronic kidney disease, cardiovascular diseases, and neurodegenerative disorders like Parkinson's disease. Various molecular mechanisms could contribute to gut frailty, and the decrease in mucus secretion is an extremely early-stage pathology. Dysbiosis of gut microbiota has a major impact on many conditions associated with gut frailty. Prebiotics, probiotics, post-biotics, and fecal microbiota transplantation are under investigation as a treatment option for gut frailty. KEY MESSAGE: Although the concept of gut frailty has not yet gained widespread recognition, we hope to propose more practical screening methods, diagnostic approaches, and specific interventions in the future.

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IZUMO1 is an acrosome transmembrane protein implicated in the adhesion and fusion of gametes. This study aims to describe the distribution of IZUMO1 in human sperm under different physiological conditions: before capacitation (NCS), at one-hour capacitation (CS1), after a hyaluronic acid (HA) selection test (mature, MS1 and immature, IS1), and induced acrosome reaction from one-hour-capacitated sperm (ARS1). The data obtained in NCS, CS1, and MS1 significantly highlight dotted fluorescence in the acrosomal region (P1) as the major staining pattern (~70%). Moreover, we describe a new distribution pattern (P2) with a dotted acrosomal region and a labelled equatorial region that significantly increases in HA-bound spermatozoa, suggesting the onset of the migration of IZUMO1. In contrast, unbound spermatozoa presented an increase in P3 (equatorial region labelled) and P4 (not labelled). Finally, costaining to observe IZUMO1 distribution and acrosome status was performed in ARS1. Interestingly, we reported a variety of combinations between the IZUMO1 staining patterns and the acrosomal stages. In conclusion, these data show as a novelty the diffusion of the IZUMO1 protein during different physiological conditions that could contribute to the improvement in sperm selection techniques.

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Rodent population control through contraception requires species-specific oral contraceptive vaccines. Therefore, in this study, we produced putative mouse-specific contraceptive peptides, mZP2 (from oocyte) and mIzumo1 (from sperm), in plants using Agrobacterium-mediated transient expression. Peptides were produced separately in Nicotiana benthamiana using constructs encoding antigens containing three copies of each peptide. We also determined the immunogenicity and contraceptive effects of the plant-produced antigens in female BALB/c mice. Mice immunized subcutaneously with a relatively low amount of antigen (5 µg/dose of each peptide in a mixture) showed systemic immune responses against mZP2-3 and mIzumo1-3 antigens. Moreover, the mean litter size of mice treated with the plant-produced antigens was reduced by 39% compared to that of the control mice. Notably, there was a significant negative correlation between the number of pups born and individual antibody levels against both antigens. Immunofluorescence assays demonstrated the binding of induced antibodies to the oocytes of BALB/c and wild-type mice in vivo and in vitro, respectively. Our study demonstrate the feasibility of producing small contraceptive peptides in plants that can be further used to develop oral contraceptive vaccines against mouse populations.

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In spermatozoa, the nuclear F-actin supports the acroplaxome, a subacrosomal structure involved in the correct exposure of several acrosomal membrane proteins; among them, the glycoprotein IZUMO1 is the major protein involved in sperm-oocyte fusion. Nuclear F-actin is also involved in sperm head shaping and chromosome compartmentalization. To date, few notions regarding the bivalent role of F-actin on sperm chromatin organization and IZUMO1 positioning have been reported. In our work, we characterized subcellular organization of F-actin in human high- and low-quality spermatozoa (A- and B-SPZ), respectively, showing that F-actin over-expression in sperm head of B-SPZ affected IZUMO1 localization. A correct IZUMO1 repositioning following in vitro induction of F-actin depolymerization, by cytochalasin D treatment, occurred. Interestingly, F-actin depolymerization was also associated with a correct acrosome repositioning, thus to favor a proper acrosome reaction onset, with changes in sperm nuclear size parameters and histone acetylation rate reaching high-quality conditions. In conclusion, the current work shows a key role of F-actin in the control of IZUMO1 localization as well as chromatin remodeling and acetylation events.

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Combined hormone drugs are the basis for orally administered contraception. However, they are associated with severe side effects that are even more impactful for women in developing countries, where resources are limited. The risk of side effects may be reduced by non-hormonal small molecules which specifically target proteins involved in fertilization. In this study, we present a virtual docking experiment directed to discover molecules that target the crucial fertilization interactions of JUNO (oocyte) and IZUMO1 (sperm). We docked 913,000 molecules to two crystal structures of JUNO and ranked them on the basis of energy-related criteria. Of the 32 tested candidates, two molecules (i.e., Z786028994 and Z1290281203) demonstrated fertilization inhibitory effect in both an in vitro fertilization (IVF) assay in mice and an in vitro penetration of human sperm into hamster oocytes. Despite this clear effect on fertilization, these two molecules did not show JUNO-IZUMO1 interaction blocking activity as assessed by AVidity-based EXtracellular Interaction Screening (AVEXIS). Therefore, further research is required to determine the mechanism of action of these two fertilization inhibitors.

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Gamete membrane fusion is a critical cellular event in sexual reproduction. In addition, the generation of knockout models has provided a powerful tool for testing the functional relevance of proteins thought to be involved in mammalian fertilization, suggesting IZUMO1 and TMEM95 (transmembrane protein 95) as essential proteins. However, the molecular mechanisms underlying the process remain largely unknown. Therefore, the aim of this study was to summarize the current knowledge about IZUMO1 and TMEM95 during mammalian fertilization. Hence, three distinct databases were consulted-PubMed, Scopus and Web of Science-using single keywords. As a result, a total of 429 articles were identified. Based on both inclusion and exclusion criteria, the final number of articles included in this study was 103. The results showed that IZUMO1 is mostly studied in rodents whereas TMEM95 is studied primarily in bovines. Despite the research, the topological localization of IZUMO1 remains controversial. IZUMO1 may be involved in organizing or stabilizing a multiprotein complex essential for the membrane fusion in which TMEM95 could act as a fusogen due to its possible interaction with IZUMO1. Overall, the expression of these two proteins is not sufficient for sperm-oocyte fusion; therefore, other molecules must be involved in the membrane fusion process.

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Sperm IZUMO1 protein was recently found to be a crucial mediator in the interaction and fusion with eggs, indicating an important role in assuring the favourable outcome from long-term preservation of chilled semen. The purpose of this study was to investigate whether supplementation of chilled semen extender with green tea polyphenols together with α-tocopherol would provide synergistic effects to prolong sperm survival and maintain IZUMO1 protein stability in cat spermatozoa. Sperm samples were collected from the cat epididymis before being diluted with semen extender containing various concentrations of α-tocopherol (0, 2.5, 5 and 7.5 µg/ml) and 0.75 mg/ml green tea polyphenols and cooled to 4 °C. One sample without antioxidants served as a control. Sperm characteristics and IZUMO1 protein expression were investigated before and after chilling at 3, 6, 9, 12 and 15 days. Using α-tocopherol at 5 µg/ml together with 0.75 mg/ml green tea polyphenols in the semen extender is the most suitable condition to retain the sperm characteristics up to nine days of preservation. Cat IZUMO1 proteins, 17 kDa, were identified at the equatorial segment of acrosome reacted sperm. Without antioxidant, cold storage can gradually degrade the IZUMO1 protein level. Sperm IZUMO1 protein was markedly conserved by supplementation of 5 µg/ml α-tocopherol together with 0.75 mg/ml green tea polyphenols up to 12 days in cold storage. These findings indicate that green tea polyphenols and α-tocopherol have protective effects on the preservation of sperm characteristics and IZUMO1 protein integrity of cat epididymal sperm during long-term chilling.

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The intergenerational effects from chronic low-dose exposure are matters of concern. It is thus important to elucidate the radiation-induced effects of germ cell maturation, fertilization and embryonic development. It is well known that DNA methylation levels in CpG sites in gametes are reprogrammed in stages during their maturity. Furthermore, the binding of Izumo on the surface of sperm and Juno on the surface of oocytes is essential for fertilization. Thus, there is a possibility that these genes are useful indicators to evaluate fertility in mice after irradiation exposure. Therefore, in this study, we analyzed global DNA methylation patterns in the testes and gene expression of Izumo1 and Izumo1r (Juno) in the gonads of mice after neonatal acute high-dose ionizing radiation (HDR) and chronic low-dose ionizing radiation (LDR). One-week-old male and female mice were irradiated with a total dose of 4 Gy, with acute HDR at 7 days at a dose rate of 30 Gy/h and LDR continuously at a dose rate of 6 mGy/h from 7 to 35 days. Their gonads were subsequently analyzed. The results of global DNA methylation patterns in the testes showed that methylation level increased with age in the control group, the LDR group maintained its DNA methylation level, and the HDR group showed decreased DNA methylation levels with age. In the control group, the gene expression level of Izumo1 in the testis did not show age-related changes, although there was high expression at 100 days of age. However, in the LDR group, the expression level recovered after the end of irradiation, while it remained low regardless of age in the HDR group. Conversely, gene expression of Izumo1r (Izumo1 receptor) in the ovary decreased with age in the control group. Although the gene expression of Izumo1r decreased with age in the LDR group, it remained low in the HDR group. Our results indicate that LDR can induce different DNA methylation patterns, and both high- and low-dose radiation before sexual maturity might affect gametogenesis and fertility.

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BACKGROUND: JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for sperm-oocyte fusion; their interaction is indispensable for fertilization. METHODS: PCR was used to clone the full-length DNA sequence of the Juno gene in sheep. The single nucleotide polymorphism (SNP) loci of Juno were genotyped by Sequenom MassARRAY®. PCR combined with rapid amplification of cDNA Ends were used to clone the full-length cDNA sequence of Juno and Izumo1. Reverse transcriptase-PCR (RT-PCR) and real time-quantitative-PCR (RT-qPCR) were used to analyze the genes' expression in tissues of sheep, and single cell RNA-seq was used to analyze the genes' expression in oocytes, granulosa cells and follicular theca of polytocous and monotocous Small Tail Han ewes. Bioinformatics was used to analyze advanced structure and phylogeny of JUNO and IZUMO1 proteins. RESULTS: The full-length DNA sequence of the Juno gene in sheep was cloned and nine SNPs were screened. We found a significant association between the g.848253 C > A locus of Juno and litter size of Small Tail Han sheep (P < 0.05). The full-length cDNA sequence of Juno and Izumo1 genes from Small Tail Han sheep were obtained. We found a new segment of the Izumo1 CDS consisting of 35 bp, and we confirmed the Izumo1 gene has 9 exons, not 8. RT-qPCR showed that Juno and Izumo1 genes were highly expressed in ovarian and testicular tissues, respectively (P < 0.01). Single cell RNA-seq showed Juno was specifically expressed in oocytes, but not in granulosa cells or follicular theca, while Izumo1 displayed little to no expression in all three cell types. There was no difference in expression of the Juno gene in oocyte and ovarian tissue in sheep with different litter sizes, indicating expression of Juno is not related to litter size traits. Bioinformatic analysis revealed the g.848253 C > A locus of Juno results in a nonconservative missense point mutation leading to a change from Phe to Leu at position 219 in the amino acid sequence. CONCLUSIONS: For the first time, this study systematically analyzed the expression, structure and function of Juno and Izumo1 genes and their encoded proteins in Small Tail Han sheep, providing the basis for future studies of the regulatory mechanisms of Juno and Izumo1 genes.

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Sperm-oocyte binding initiates an outside-in signaling event in the mouse oocyte that triggers recruitment and activation of the cytosolic protein kinase PTK2B in the cortex underlying the bound sperm. While not involved in gamete fusion, PTK2B activity promotes actin remodeling events important during sperm incorporation. However, the mechanism by which sperm-oocyte binding activates PTK2B is unknown, and the present study examined the possibility that sperm interaction with specific oocyte surface proteins plays an important role in PTK2B activation. Imaging studies revealed that as IZUMO1R and CD9 became concentrated at the sperm binding site, activated (phosphorylated) PTK2B accumulated in the cortex underlying the sperm head and in microvilli partially encircling the sperm head. In order to determine whether IZUMO1R and/or CD9 played a significant role in PTK2B recruitment and activation at the sperm binding site, the ability of oocytes null for Izumo1r or Cd9, to initiate an increase in PTK2B content and activation was tested. The results revealed that IZUMO1R played a minor role in PTK2B activation and had no effect on actin remodeling; however, CD9 played a very significant role in PTK2B activation and subsequent actin remodeling at the sperm binding site. These findings suggest the possibility that interaction of sperm surface proteins with CD9 or CD9-associated oocyte proteins triggers PTK2B activation at the sperm binding site.

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The aim of the present study was to investigate the association between the severity of constipation and sarcopenia in elderly adults. We conducted a single-center university hospital-based, retrospective cross-sectional study of consecutive outpatients aged ≥65 years from 2017 to 2020. Patients were included in the study if all of the following information were available from medical records: Patient's profile (age, sex, body mass index), laxative/prokinetics use, evaluation of sarcopenia, nutritional status, and questionnaires concerning the severity of constipation [Constipation Scoring System (CSS)], abdominal symptom-related quality of life (QOL) (Izumo scale) and stool shape [Bristol Stool Form Scale (BSFS)]. Multiple regression analysis of risk factors for high CSS score was performed. The results revealed that of the 310 eligible study subjects, [149 men (48.1%) and 161 women (51.9%); mean age, 75.7±6.1 years; mean body mass index, 23.0±3.6 kg/m2], sarcopenia was noted in 83 cases (26.8%). The CSS score was significantly higher in the sarcopenia group than that noted in the non-sarcopenia group (4.9±4.9 vs. 3.6±3.6, P=0.009). The CSS score was significantly associated with the albumin level (r=-0.148), lymphocyte count (r=-0.118), CONUT score (r=0.130), reflux-related QOL score (r=0.155), upper abdominal pain-related QOL score (r=0.171), fullness-related QOL score (r=0.299), constipation-related QOL score (r=0.615), diarrhea-related QOL score (r=0.235) and BSFS score (r=-0.114). In multiple regression analysis, independent predictors for CSS score were sarcopenia [standardized partial regression coefficient (ß)=0.107, P=0.032], constipation-related QOL score (ß=0.537, P<0.001), laxative/prokinetics use (ß=0.211, P<0.001) and BSFS score (ß=-0.098, P=0.031) (R2=0.436). In conclusion, sarcopenia, constipation-related QOL score, laxative/prokinetics use and BSFS score are associated with the severity of constipation in elderly adults.

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The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization.

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In this study, we performed the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis in the spermatozoa of Landrace boars with different fertility potentials and investigated the ability of sperm acrosome associated 4 (SPACA4) and IZUMO family member 2 (IZUMO2) to predict the reproductive perform of boars. The iTRAQ results revealed that 202 proteins were up-regulated and 43 proteins were down-regulated in the spermatozoa from high fertility boars. SPACA4 and IZUMO2 protein levels were significantly up-regulated in the spermatozoa from high fertility boars. SPACA4 and IZUMO2 expression were specifically detected in the adult boar testis. SPACA4 levels were positively correlated with Sow's farrowing rate and reproductive efficiency, but not litter size. IZUMO2 were positively correlated with litter size, Sow's farrowing rate and reproductive efficiency. Treating the boar semen with SPACA4 or IZUMO2 antibodies for 30 min and 60 min failed to affect the sperm motility; while treating the semen with SPACA4 antibody significantly reduced the fertilization and cleavage rates. Similar results for fertilization and cleavage rates were found in IZUMO2 antibody-treated semen. Collectively, our results indicated that protein levels of SPACA4 and IZUMO2 in the spermatozoa were positively related to the reproductive performance of Landrace boars.

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