Dataset of seminal plasma proteome of Nellore bulls with high and low percentage of abnormalities sperm.

OBJECTIVES: Bovine seminal plasma proteins perform several functions related to sperm function. Changes in the expression pattern or abundance of seminal proteins are related to changes in the fertilizing capacity of bulls. Considering the role of seminal plasma proteins in sperm function and animal reproduction, we investigated changes in the protein abundance profile in response to sperm morphological changes using a proteomic approach. DATADESCRIPTION: In our present investigation, we employed liquid chromatography coupled with mass spectrometry to elucidate the proteomic composition of seminal plasma obtained from Nellore bulls exhibiting varying percentages of sperm abnormalities. Following semen collection, seminal plasma was promptly isolated from sperm, and proteins were subsequently precipitated, enzymatically digested using porcine trypsin, and subjected to analysis utilizing the Acquity nano UHPLC System in conjunction with a mass spectrometer. This dataset encompasses a total of 297 proteins, marking the inaugural instance in which a comparative profile of seminal plasma proteins in young Nellore bulls, categorized by their sperm abnormality percentages, has been delineated using LC-MS/MS. The comprehensive nature of this dataset contributes pivotal proteomic insights, representing a noteworthy advancement in our understanding of the reproductive biology of the Nellore breed.

The correlation between human seminal plasma sialoproteins and ejaculate parameters.

Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.

Sast1-mediated manifold effects inhibit Plutella xylostella fertility.

BACKGROUND: Plutella xylostella (Linnaeus) is a destructive pest of cruciferous crops due to its strong reproductive capacity and extensive resistance to pesticides. Seminal fluid proteins (SFPs) are the main effective factors that determine the reproductive physiology and behaviour of both sexes. Although an increasing number of SFPs have been identified, the effects of astacins in SFPs on agricultural pests have not yet been reported. Here, we elucidated the mechanisms by which Sast1 (seminal astacin 1) regulates the fertility of Plutella xylostella (L.). RESULTS: PxSast1 was specifically expressed in the testis and accesssory gland. CRISPR/Cas9-induced PxSast1 knockout successfully constructed two homozygous mutant strains. Sast1 impaired the fertility of P. xylostella by separately regulating the reproductive capacity of males and females. Loss of PxSast1, on the one hand, significantly decreased the ability of males to mate and fertilize, mainly manifested as shortened mating duration, reduced mating competitiveness and decreased eupyrene sperm production; on the other hand, it significantly inhibited the expression of chorion genes in females, resulting in oogenesis deficits. Simultaneously, for mated females, the differentially expressed genes in signalling pathways related to oogenesis and chorion formation were significantly enriched after PxSast1 knockout. CONCLUSION: These analyses of the functions of PxSast1 as the regulator of spermatogenesis and oogenesis establish its importance in the fertility process of P. xylostella, as well as its potential as a promising target for genetic regulation-based pest control. © 2024 Society of Chemical Industry.

Dynamics of seminal fluid production after mating.

Seminal fluid proteins (SFPs) play vital roles for optimizing reproductive success in diverse animals. Underlining their significance, SFP production and transfer are highly plastic, e.g., depending on the presence of rivals or mating status of partners. However, surprisingly little is known about replenishing SFPs after mating. This is especially relevant in species that mate multiple times, as they continuously produce and use SFPs throughout their reproductive life. Here we examined the expression pattern of SFP genes after mating in the great pond snail, Lymnaea stagnalis. Our results show that two out of the six SFP genes investigated here were upregulated 1 week after mating. Surprisingly, most SFP genes did not change their expression immediately after mating. Even after 1 week, when supposedly seminal fluid is fully replenished, the expression of SFP genes is rather high. In addition, the difference with previous studies hints at the possibility that SFP production after mating is plastic and depends on the mating history of female-acting snails. Our results shed light on unexplored aspects of SFP production, thereby expanding the understanding of reproductive strategies in animals.

Proteomic analysis of boar seminal plasma: Putative markers for fertility based on capacity of semen preservation at 17°C.

Boar seminal plasma (SP) proteins were associated with differences on sperm resistance to cooling at 17°C. However, information about seminal plasma proteins in boars classified by capacity of semen preservation and in vivo fertility remains lacking. Thus, the objective was to evaluate the SP proteome in boars classified by capacity of semen preservation and putative biomarkers for fertility. The ejaculates from high-preservation (HP) showed higher progressive motility during all 5 days than the low-preservation (LP) boars. There was no difference for farrowing rate between ejaculates from LP (89.7%) and HP boars (88.4%). The LP boars presented lower total piglets born (14.0 ± 0.2) than HP (14.8 ± 0.2; p < 0.01). A total of 257 proteins were identified, where 184 were present in both classes of boar, and 41 and 32 were identified only in LP and HP boars, respectively. Nine proteins were differently expressed: five were more abundant in HP (SPMI, ZPBP1, FN1, HPX, and C3) and four in LP boars (B2M, COL1A1, NKX3-2, and MPZL1). The HP boars had an increased abundance of SP proteins related to sperm resistance and fecundation process which explains the better TPB. LP boars had a higher abundance of SP proteins associated with impaired spermatogenesis.

Contrasting effects of molecular crowding on the membrane-perturbing and chaperone-like activities of major bovine seminal plasma protein, PDC-109.

Crowded environments inside cells and biological fluids greatly affect protein stability and activity. PDC-109, a polydisperse oligomeric protein of the bovine seminal plasma selectively binds choline phospholipids on the sperm cell surface and causes membrane destabilization and lipid efflux, leading to acrosome reaction. PDC-109 also exhibits chaperone-like activity (CLA) and protects client proteins against various kinds of stress, such as high temperature and low pH. In the present work, we have investigated the effect of molecular crowding on these two different activities of PDC-109 employing Dextran 70 (D70) - a widely used polymeric dextran - as the crowding agent. The results obtained show that presence of D70 markedly increases membrane destabilization by PDC-109. Isothermal titration calorimetric studies revealed that under crowded condition the binding affinity of PDC-109 for choline phospholipids increases approximately 3-fold, which could in turn facilitate membrane destabilization. In contrast, under identical conditions, its CLA was reduced significantly. The decreased CLA could be correlated to reduced surface hydrophobicity, which was due to stabilization of the protein oligomers. These results establish that molecular crowding exhibits contrasting effects on two different functional activities of PDC-109 and highlight the importance of microenvironment of proteins in modulating their functional activities.

FSIP1 enhances the therapeutic sensitivity to CDK4/6 inhibitors in triple-negative breast cancer patients by activating the Nanog pathway.

CDK4/6 inhibitors are routinely recommended agents for the treatment of advanced HR+HER2- breast cancer. However, their therapeutic effectiveness in triple-negative breast cancer (TNBC) remains controversial. Here, we observed that the expression level of fibrous sheath interacting protein 1 (FSIP1) could predict the treatment response of TNBC to CDK4/6 inhibitors. High FSIP1 expression level was related to a poor prognosis in TNBC, which was associated with the ability of FSIP1 to promote tumor cell proliferation. FSIP1 downregulation led to slowed tumor growth and reduced lung metastasis in TNBC. FSIP1 knockout caused cell cycle arrest at the G0/G1 phase and reduced treatment sensitivity to CDK4/6 inhibitors by inactivating the Nanog/CCND1/CDK4/6 pathway. FSIP1 could form a complex with Nanog, protecting it from ubiquitination and degradation, which may facilitate the rapid cell cycle transition from G0/G1 to S phase and exhibit enhanced sensitivity to CDK4/6 inhibitors. Our findings suggest that TNBC patients with high FSIP1 expression levels may be suitable candidates for CDK4/6 inhibitor treatment.

Porcine spermadhesin AQN-3 binds to negatively charged phospholipids.

The spermadhesin AQN-3 is a major component of porcine seminal plasma. While various studies suggest that this protein binds to boar sperm cells, its attachment to the cells is poorly understood. Therefore, the capacity of AQN-3 to interact with lipids was investigated. For that purpose, AQN-3 was recombinantly expressed in E. coli and purified via the included His-tag. Characterizing the quaternary structure by size exclusion chromatography revealed that recombinant AQN-3 (recAQN-3) is largely present as multimer and/or aggregate. To determine the lipid specificity of recAQN-3, a lipid stripe method and a multilamellar vesicle (MLV)-based binding assay were used. Both assays show that recAQN-3 selectively interacts with negatively charged lipids, like phosphatidic acid, phosphatidylinositol phosphates, and cardiolipin. No interaction was observed with phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or cholesterol. The affinity to negatively charged lipids can be explained by electrostatic interactions because binding is partly reversed under high-salt condition. However, more factors have to be assumed like hydrogen bonds and/or hydrophobic forces because the majority of bound molecules was not released by high salt. To confirm the observed binding behavior for the native protein, porcine seminal plasma was incubated with MLVs comprising phosphatidic acid or phosphatidyl-4,5-bisphosphate. Attached proteins were isolated, digested, and analyzed by mass spectrometry. Native AQN-3 was detected in all samples analyzed and was - besides AWN - the most abundant protein. It remains to be investigated whether AQN-3, together with other sperm associated seminal plasma proteins, acts as decapacitation factor by targeting negative lipids with signaling or other functional roles in fertilization.

Primary structure determination and physicochemical characterization of DSP-3, a phosphatidylcholine binding glycoprotein of donkey seminal plasma.

Major proteins of the seminal plasma in a variety of mammals such as bovine PDC-109, equine HSP-1/2, and donkey DSP-1 contain fibronectin type-II (FnII) domains and are referred to as FnII family proteins. To further our understanding on these proteins, we carried out detailed studies on DSP-3, another FnII protein of donkey seminal plasma. High-resolution mass-spectrometric studies revealed that DSP-3 contains 106 amino acid residues and is heterogeneously glycosylated with multiple acetylations on the glycans. Interestingly, high homology was observed between DSP-1 and HSP-1 (118 identical residues) than between DSP-1 and DSP-3 (72 identical residues). Circular dichroism (CD) spectroscopic and differential scanning calorimetric (DSC) studies showed that DSP-3 unfolds at ~45 °C and binding of phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases the thermal stability. Analysis of DSC data suggested that unlike PDC-109 and DSP-1, which exist as mixtures of polydisperse oligomers, DSP-3 most likely exists as a monomer. Ligand binding studies monitoring changes in protein intrinsic fluorescence indicated that DSP-3 binds lyso-phosphatidylcholine (Ka = 1.08 × 105 M-1) with ~80-fold higher affinity than PrC (Ka = 1.39 × 103 M-1). Binding of DSP-3 to erythrocytes leads to membrane perturbation, suggesting that its binding to sperm plasma membrane could be physiologically significant.

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.

The association of ODF4 with AK1 and AK2 in mice is essential for fertility through its contribution to flagellar shape.

Normal sperm flagellar shape and movement are essential for fertilization. The integral protein outer dense fiber 4 (ODF4) localizes to ODFs, but its function remains unclear. Adenylate kinase (AK) is a phosphotransferase that catalyzes the interconversion and controls the concentration equilibrium of adenine nucleotides. AK shuttles ATP to energy-consuming sites. Here, we report on the relationship of flagellar shape and movement with ODF4, AK1 and AK2 by using Odf4-deletion (Odf4-/-) mice. Soluble ODF4 is coimmunoprecipitated with AK1 and AK2 in Odf4+/+ spermatozoa. ODF4, AK1 and AK2 localize to whole flagella (plasmalemma, mitochondria, ODFs, and residual cytoplasmic droplets (CDs)), principal pieces, and midpieces, respectively. Odf4-/- sperm flagella lose ODF4 and reduce AK1 and AK2 but produce ATP. The flagellum is bent (hairpin flagellum) with a large CD in the midpiece. There is no motility in the midpiece, but the principal piece is motile. Odf4-/- spermatozoa progress backward and fail to ascend in the uterus. Thus, Odf4-/- males are infertile owing to abnormal flagellar shape and movement caused mainly by the loss of ODF4 with AK1 and AK2. This study is supported by the rescue experiment; the abnormalities and male infertility caused by Odf4 deletion were reversed by Odf4 restoration.

Fetal Exposure to Endocrine Disrupting-Bisphenol A (BPA) Alters Testicular Fatty Acid Metabolism in the Adult Offspring: Relevance to Sperm Maturation and Quality.

Daily exposure to bisphenols can affect reproductive functions due to their pseudo-estrogenic and/or anti-androgenic effects. Testicular lipids contain high levels of polyunsaturated fatty acids necessary for sperm maturity, motility, and spermatogenesis. Whether prenatal exposure to bisphenols alters testicular fatty acid metabolism in adult offspring is unknown. Pregnant Wistar rats were gavaged from gestational day 4 to 21 with BPA and BPS (0.0, 0.4, 4.0, 40.0 µg/kg bw/day). Despite increased body and testis weight, the total testicular cholesterol, triglyceride, and plasma fatty acids were unaffected in the offspring. Lipogenesis was upregulated by increased SCD-1, SCD-2, and expression of lipid storage (ADRP) and trafficking protein (FABP4). The arachidonic acid, 20:4 n-6 (ARA) and docosapentaenoic acid, 22:5 n-6 (DPA) levels were decreased in the BPA-exposed testis, while BPS exposure had no effects. The expression of PPARα, PPARγ proteins, and CATSPER2 mRNA were decreased, which are important for energy dissipation and the motility of the sperm in the testis. The endogenous conversion of linoleic acid,18:2 n-6 (LA), to ARA was impaired by a reduced ARA/LA ratio and decreased FADS1 expression in BPA-exposed testis. Collectively, fetal BPA exposure affected endogenous long-chain fatty acid metabolism and steroidogenesis in the adult testis, which might dysregulate sperm maturation and quality.

Characterization of SPINK2, SPACA7 and PDCL2: Effect of immunization on fecundity, sperm function and testicular transcriptome.

Testicular factors play a vital role in spermatogenesis. We characterized the functional role of rat Spink2, Spaca7 and Pdcl2 genes. Their primary, secondary and tertiary structure were deduced in silico. The genes of rat Spink2, Spaca7 and Pdcl2 mRNA were predominantly expressed in the testis. SPINK2, SPACA7 and PDCL2 protein expression was evident in all the cell types of testis and on spermatozoa. Ablation of each of these proteins by active immunization resulted in reduced fecundity and sperm count. Damage to the anatomical architecture of testis and epididymis was evident. In SPINK2 immunized rats, 283 genes were differentially regulated while it was 434 and 872 genes for SPACA7 and PDCL2 respectively. Genes that were differentially regulated in the testis of SPINK2 immunized rats primarily belonged to extracellular exosome formation, extracellular space and response to drugs. SPACA7 ablation affected genes related to extracellular space, oxidation-reduction processes, endoplasmic reticulum membrane and response to drugs. Differential gene expression was observed for nuclear function, protein binding and positive regulation of transcription from RNA polymerase II promoter in testis of PDCL2 immunized rats. Results of our study demonstrate the role of SPINK2, SPACA7 and PDCL2 in spermatogenesis and in important molecular processes that may dictate testicular function and other physiological responses as well.

Contributions of seminal plasma proteins to fertilizing ability of bull sperm: A meta-analytical review.

Seminal plasma is a dynamic, intricate combination of fluids from the testicles, epididymides, seminal vesicles, bulbourethral glands, and prostate, containing molecules that modulate sperm functions, post-fertilization events, and the female reproductive tract physiology. Significant variations in sperm parameters and fertility status of bulls relate to differences in the seminal plasma proteome. In this framework, a meta-analytical study was conducted examining 29 studies (published between 1990 and 2021) to ascertain the effects of seminal fluid proteins on parameters associated with bull fertility and the influence of distinct methodologies on such effects. Our results revealed that seminal proteins ameliorate sperm parameters, such as motility, integrity, capacitation, and fertilizing ability, and favours sperm protection. Seminal binder of sperm proteins and beta-defensin 126 highly favoured sperm protection when cells were collected from the epididymis by retrograde flux and analysed under room temperature conditions. Furthermore, seminal proteins improved the motility and quality of Bos taurus sperm collected by artificial vagina, mainly in the presence of heparin-binding proteins. The key limitations faced by this meta-analysis were the paucity of studies evaluating the effects of whole seminal fluid proteins and the limited number of studies conducted in vivo. In conclusion, the present meta-analytical study confirms that seminal proteins improve fertility-related parameters in the bovine species. However, methodological strategies used by authors are diverse, with distinct endpoints and methods. Thus, the translational aspects of seminal plasma research should be taken into consideration to precisely define how seminal proteins can be harnessed to advance reproductive biotechnology.

Human sperm TMEM95 binds eggs and facilitates membrane fusion.

Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. Tmem95 knockout murine sperm can bind to, but do not fuse with, eggs. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the function of human TMEM95. We show that human TMEM95 binds to hamster egg membranes, providing evidence for a TMEM95 receptor on eggs. Using X-ray crystallography, we reveal an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface. Amino acid substitutions within this region of TMEM95 ablate egg-binding activity. We identify monoclonal antibodies against TMEM95 that reduce the number of human sperm fused with hamster eggs in sperm penetration assays. Strikingly, these antibodies do not block binding of sperm to eggs. Taken together, these results provide strong evidence for a specific, receptor-mediated interaction of sperm TMEM95 with eggs and suggest that this interaction may have a role in facilitating membrane fusion during fertilization.

A comparative proteomic study of high and low semen quality seminal plasma in drakes.

Semen quality is the most important indicator in evaluating drake fecundity. At present, the low semen quality has become a major factor restricting the development of artificial insemination (AI) technology in ducks. Numerous studies have indicated that seminal plasma proteins play a crucial role in semen quality, but the mechanism of seminal plasma proteins regulating semen quality of drakes remains unclear. Thus, the objective of this study was to identify seminal plasma proteins associated with semen quality by comparing the seminal plasma proteomic profile of drakes with high-quality semen (HQS) and low-quality semen (LQS). Using a label-free MS-based method, a total of 745 seminal plasma proteins were identified. Of these, 55 differentially expressed proteins (DEPs) were identified (40 up-regulated and 15 down-regulated). Gene Ontology (GO) analysis showed that the DEPs were mainly enriched in transmembrane transport, extracellular matrix structural constituent, transferase activity, transferring acyl groups other than amino-acyl groups, transmembrane transporter activity, and integral component of membrane (P < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis indicated that the DEPs were significantly enriched in apoptosis, tyrosine metabolism, glycerophospholipid metabolism, and sulfur metabolism pathways (P < 0.05). Moreover, through protein-protein interaction (PPI) network analysis, eight potential candidate proteins were identified, including P19140 (Alpha-enolase), R0KUV7 (Calreticulin), R0K3X3 (Solute carrier family 2, facilitated glucose transporter member 5), R0L6V0 (Proteasome subunit beta), R0JKW0 (Cytochrome c), R0JMC5 (Tubulin alpha chain), R0LCK1 (Cathepsin C), and R0JUP6 (Cathepsin D), which could play crucial roles in semen quality. Notably, further analysis demonstrated that key protein P19140 (Alpha-enolase) might can control the semen quality of drakes by regulating the expression of proteins related to apoptosis pathway. This study is the first systematically comparing the seminal plasma proteome of drakes exhibiting high and low semen quality. These results provide novel insights into the mechanisms regulating semen quality of drakes.

On how to identify a seminal fluid protein: A commentary on Hurtado et al.

Seminal fluid proteins (Sfps) have striking effects on the behaviour and physiology of females in many insects. Some Drosophila melanogaster Sfps are not highly or exclusively expressed in the accessory glands, but derive from, or are additionally expressed in other male reproductive tissues. The full suite of Sfps includes transferred proteins from all male reproductive tissues, regardless of expression level or presence of a signal peptide.

On how to identify a seminal fluid protein: A response to Wigby et al.

The choice of criteria to delimit a group or class is a subjective matter, even though the reasoning, the objectives and the criteria themselves should always be clearly stated. This paper is part of a discussion about the criteria used to identify seminal fluid proteins (SFPs) in Drosophila species. SFPs are proteins that are transferred to females during copulation together with sperm. The only way to ascertain that a protein is an SFP is to prove that it is produced in a male reproductive organ and is found in the female reproductive tract after insemination. Nevertheless, the required methodology is labour-intensive and expensive, and therefore this kind of data is unlikely to be available for many species, precluding comparative and evolutionary studies on the subject. To conduct evolutionary analyses, in a previous study, we capitalized on the accumulated knowledge we have in the model species D. melanogaster to recommend a set of criteria for identifying candidate SFPs in other Drosophila species. Those criteria, based on transcriptomic evidence and in silico predictions from sequences, would allow a good balance between sensitivity (the inclusion of true SFPs) and specificity (the exclusion of false positives). In view of the criticism raised by another group, here we defend our criteria on one hand while accepting there is room for improvement on the other. The results are updated sets of criteria and SFPs that we believe can be useful in future evolutionary studies.

FSIP1 Is Associated with Poor Prognosis and Can Be Used to Construct a Prognostic Model in Gastric Cancer.

Gastric cancer (GC) is one of the most common upper gastrointestinal malignant tumors, and the incidence of the GC shows an increasing trend in the past years. Finding more sensitive markers will help to reveal the mechanism of GC progression and clinic diagnoses. This study first analyzed the mRNA expression level of FSIP1 in TCGA GC samples and the significance in predicting the prognosis. KEGG and GO analyses were used to explore the molecular mechanism of FSIP1 in GC progression. This study further retrospectively analyzed 166 clinical samples of GC from Harbin Medical University Cancer Hospital and evaluated the expression level of FSIP1 by immunohistochemistry. Kaplan-Meier and Cox multivariate analysis was used to investigate the prognostic value of FSIP1 expression in GC patients. We also identified correlations between FSIP1 and clinicopathological characteristics. This study found that the mRNA level of FSIP1 was significantly upregulated in GC compared with nontumor specimens and correlated with poor prognosis. Immunohistochemistry confirmed the results of bioinformatics analysis of the TCGA GC database. FSIP1 was associated with pTNM pathological stage, tumor location, and neural invasion. In addition, multivariate Cox regression analysis showed that FSIP1, T classification, and N classification were independent posterior factors of patients and could be combined with pathological features to construct a nomogram prognostic model. Overall, our results suggest that FSIP1 is expected to be an independent prognostic indicator of GC.

Characterization of reproductive proteins in the Mexican fruit fly points towards the evolution of novel functions.

Seminal fluid proteins (Sfps) modify female phenotypes and have wide-ranging evolutionary implications on fitness in many insects. However, in the Mexican fruit fly, Anastrepha ludens, a highly destructive agricultural pest, the functions of Sfps are still largely unknown. To gain insights into female phenotypes regulated by Sfps, we used nano-liquid chromatography mass spectrometry to conduct a proteomic analysis of the soluble proteins from reproductive organs of A. ludens. The proteins predicted to be transferred from males to females during copulation were 100 proteins from the accessory glands, 69 from the testes and 20 from the ejaculatory bulb, resulting in 141 unique proteins after accounting for redundancies from multiple tissues. These 141 included orthologues to Drosophila melanogaster proteins involved mainly in oogenesis, spermatogenesis, immune response, lifespan and fecundity. In particular, we found one protein associated with female olfactory response to repellent stimuli (Scribble), and two related to memory formation (aPKC and Shibire). Together, these results raise the possibility that A. ludens Sfps could play a role in regulating female olfactory responses and memory formation and could be indicative of novel evolutionary functions in this important agricultural pest.