Search Results (34)

Sea urchin eggs are surrounded by a network of extracellular matrix, consisting of the jelly coat (JC) and vitelline layer (VL). While the voluminous JC evokes acrosomal reaction in the approaching sperm, the tight VL ensheathing the plasma membrane of the subjacent microvilli is known to be the subcellular site where ‘sperm receptors’ reside. In this study, we have examined the roles of JC and VL at fertilization in a combinatorial approach utilizing two different pretreatments of the eggs: (i) incubation with dithiothreitol (DTT) in alkaline seawater to remove JC and VL, (ii) masking the egg extracellular matrix with a carbohydrate-binding protein concanavalin A (Con A). Surprisingly, the results showed that the DTT-denuded eggs still engulfed sperm at fertilization, even more effectively than intact eggs, as multiple sperm entered. On the other hand, Con A appeared to interfere with sperm entry in a dose-dependent manner and to delay the onset of the Ca²⁺ wave in intact eggs after the cortical Ca²⁺ release, representing sperm–egg fusion. This prolonged time lag in triggering the Ca²⁺ wave at fertilization was associated with compromised dynamics of the subplasmalemmal actin filaments in Con A-pretreated eggs. By using Alexa Fluor 633 Con A and BPA-C8-Cy3, respectively, we also report unprecedented fluorescent labeling of the egg JC and the spontaneous ‘acrosomal protrusion’ on the head of Paracentrotus lividus sperm diluted in natural seawater. Combined with electron microscopy observations of intact and denuded eggs, our results suggest that the glycoconjugate on the egg surface contributes to the fertilization signal transduction, affecting the Ca²⁺ wave via actin cytoskeletal changes and sperm entry. Full article

The initial molecular events mediating mammalian sperm binding to the zona pellucida (ZP) of the oocyte are highly complex and still not fully elucidated. Recent advances have identified multiple candidate sperm surface proteins, often functioning as part of high-molecular-weight complexes that mediate this critical fertilization event in a species-specific and coordinated manner. To address a significant gap in the literature, we provide an in-depth overview of the functional assays employed to investigate sperm–ZP interactions, emphasizing their underlying principles, potential applications, and key methodological strengths and limitations. The techniques discussed range from classical in vitro sperm–oocyte and hemizona binding assays, including antibody-blocking and competitive strategies, to cutting-edge in vivo genetic models, each contributing unique insights into the physiological relevance of the proposed ZP receptors. Robust experimental design, including the use of appropriate controls and validation strategies, is essential for accurately interpreting the role of candidate sperm receptors. This review provides a structured overview of current methodologies to support researchers in critically evaluating and applying functional assays in future studies. Full article

Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB) serves as a tethering factor that interacts with various proteins and recruits these proteins to the ER surface, exerting multiple functions, such as organelle membrane tethering, lipid transfer between organelles, regulation of calcium homeostasis, autophagy, and the unfolded protein response (UPR). Its interaction is often mediated by its MSP (major sperm) domain, which binds with FFAT (two phenylalanines in an acidic tract)-motif-containing proteins. However, pathogenic variations, such as P56S, P56H, and T46I, in the VAPB MSP domain lead to the familial form of amyotrophic lateral sclerosis (ALS8). Still, the underlying pathophysiology of ALS8 due to pathogenic variations in the VAPB MSP domain remains elusive. In this study, we conducted molecular dynamics (MD) simulations to understand the pathogenic-variant-derived changes in the structural dynamics of the VAPB MSP domain. We found that pathogenic variants altered the fluctuations and conformational dynamics of the VAPB protein. Analyzing the organizations of the secondary structure revealed that pathogenic variants changed the composition of secondary structure elements, especially increasing the proportion of α-helix while reducing β-sheet formation, which might affect the organelle tethering and other functions of VAPB, as well as VAPB homodimer and heterodimer formation. Taken together, these findings can be further investigated through in vivo and/or in vitro studies to not only clarify the pathophysiology of ALS8 resulting from VAPB MSP domain pathogenic variants but also develop novel therapeutics for the disease that restore the native structural organizations as well as fluctuations and motions. Full article

The creation of transgenic chickens holds significant promise for the agricultural and biotechnological sectors, offering potential improvements in disease resistance and production efficiency. The preferred method for generating gene-edited chickens involves the genetic manipulation of primordial germ cells (PGCs), making the identification and isolation of these cells a growing focus of research. PGCs are the precursors to sperm and oocytes, responsible for transmitting genetic material to the next generation. In humans, PGCs are characterized by their large size, round nuclei, and refractive lipids in the cytoplasm, and can be identified using periodic acid–Schiff (PAS) staining and the surface marker stage-specific embryonic antigen 1 (SSEA1). Similarly, chicken PGCs express SSEA1, but their most specific marker is the chicken vasa homologue (CVH), the avian equivalent of the RNA-binding factor gene vasa. However, SSEA1, along with other known surface markers, does not bind to all PGCs or lacks specificity, while CVH, although highly specific to PGCs, is intracellular and unsuitable for isolating viable cells. This study aims to develop an antibody targeting a PGC surface marker with the same specificity as CVH. Despite the importance of identifying surface markers for PGC characterization, to date, such reagents are limited. To address this, whole chicken PGCs were injected into mice, leading to the generation of a panel of monoclonal antibodies. One antibody was found to bind cultured chicken PGCs and showed reduced expression upon differentiation with retinoic acid, indicating its specificity to PGCs. Immunoprecipitation followed by mass spectrometry identified the antigen as myosin heavy chain-like (MYH9) protein. The antibody, αMYH9, was further characterized and shown to bind circulating PGCs and embryonic gonadal PGCs (Hamburger Hamilton (H-H) stage 30, embryonic day 6.5–7). Whilst our primary aim was to determine the binding to PGCs, further investigation is required to determine potential binding to somatic cells. In conclusion, this study provides the characterization of a surface marker for chicken PGCs, with significant implications for advancements in avian genetic preservation, agriculture, and biotechnology. Full article

Utilizing high-resolution microscopy in conjunction with a new antibody highly specific for rat alpha4 Na,K-ATPase, we describe changes in alpha4 expression during spermatogenesis and in sperm incubated under capacitating and noncapacitating conditions. Immunohistochemical analyses showed alpha4 expression at low levels in spermatogonia and in pachytene spermatocytes. Alpha4 then becomes highly expressed on round spermatids and the midpiece of elongated spermatozoa within the seminiferous tubules. In noncapacitating conditions, alpha4 was confined mainly to the flagellum of mature sperm; however, under capacitating conditions, sperm acquired intense alpha4 staining along the acrosomal region of the sperm head. To visualize the precise localization of alpha4 in the sperm head, we performed an ultrastructural analysis using immuno-scanning electron microscopy. Under capacitating conditions, sperm exhibited alpha4 staining along the dorsal surface of the sperm head associated with the acrosome. In addition, after 4 h of incubation in motility buffer, we observed an increase in alpha4 protein in sperm that could be blocked with chloramphenicol, a mitochondrial-type ribosome inhibitor. These findings demonstrate that both the localization and expression level of alpha4 Na,K-ATPase are dynamic aspects of sperm maturation and suggest that sperm motility and capacitation may be supported by these changes to the location and amount of this protein. Full article

Current sperm sexing methods are costly and largely restricted to cattle, while immunological techniques targeting sex-specific membrane proteins may offer more economical alternatives. To advance these methods, understanding the proteomic differences between the cell membranes of X- and Y-chromosome-bearing spermatozoa is essential. This study aimed to characterize the cell surface proteome of bovine sperm and identify potential targets for sperm sexing through LC-MS/MS analysis. Cell surface protein lysates were extracted from unsexed, X-sperm (BX), and Y-sperm (BY) samples via biotinylation. Promising targets were identified through functional annotation (UniProt, eggNOG-mapper v.2.1.7) and topology prediction (DeepTMHMM v.1.0.13). Additionally, statistical overrepresentation (PANTHER 18.0) and orthology analyses were performed. Excluding contaminants, 130 proteins were detected, of which 64 proteins were detected in the BX samples and not in the BY samples. Of these, five transmembrane proteins stood out as potential X-sperm targets (ADAM2, ATP11C, DG1, MCT1, and PMCA4). They were identified as potential cell surface targets, based on GO terms and topology predictions, detected in at least two replicates of the BX samples, and shown to share orthology with other livestock species. These findings enhance our understanding of bovine sperm proteomics; however, further validation is required to confirm the utility of these five proteins in sperm sexing technologies. Full article

As the most valuable protein in the sperm of testes tissues of Coregonus peled, Coregonus peled protamine (CPP) had a natural antibacterial and antiseptic effect, but its physicochemical properties and functions are easily affected by the ultrasound-assisted extraction process. In this study, ultrasound-assisted extraction of CPP was used to investigate the effects of different ultrasonic intensities (0, 3.03, 6.07, 9.10, 12.13, and 15.16 W/cm²) on the structural and functional properties of CPP. The results showed that at moderate ultrasonic intensity (9.10 W/cm²), the protein was the most successful, as it was subjected to cavitation shear and microjet by ultrasound, which resulted in changes in protein structure, moderate unfolding of peptide chains, and changes in the secondary and tertiary structures of CPP. SEM images confirmed the changes in the microstructure of CPP. Ultrasound oxidized the proteins to varying degrees with the highest sulfhydryl and carbonyl and surface hydrophobicity content at an ultrasonic intensity of 9.10 W/cm². At the same time, the solubility, antimicrobial activity, and heparin binding of CPP were affected. It is worth mentioning that the ultrasonicated CPP exhibited a stronger heparin-binding capacity compared to the non-ultrasonicated CPP. In conclusion, 9.10 W/cm² was determined as the optimal ultrasonic intensity parameter for this study. In conclusion, the incorporation of appropriate ultrasonic intensity in the acidic extraction process could help to improve the functional properties of CPP, and ultrasound-assisted protein extraction has emerged as a reliable technique capable of modifying the structure and function of CPP. Full article

Studying proteins associated with sex chromosomes can provide insights into sex-specific proteins. Membrane proteins accessible through the cell surface may serve as excellent targets for diagnostic, therapeutic, or even technological purposes, such as sperm sexing technologies. In this context, proteins encoded by sex chromosomes have the potential to become targets for X- or Y-chromosome-bearing spermatozoa. Due to the limited availability of proteomic studies on rabbit spermatozoa and poorly annotated databases for rabbits compared to humans, a bioinformatic analysis of the available rabbit X chromosome proteome (RX), as well as the human X (HX) and Y (HY) chromosomes proteome, was conducted to identify potential targets that could be accessible from the cell surface and predict which of the potential targets identified in humans might also exist in rabbits. We identified 100, 211, and 3 proteins associated with the plasma membrane or cell surface for RX, HX, and HY, respectively, of which 61, 132, and 3 proteins exhibit potential as targets as they were predicted to be accessible from the cell surface. Cross-referencing the potential HX targets with the rabbit proteome revealed an additional 60 proteins with the potential to be RX targets, resulting in a total of 121 potential RX targets. In addition, at least 53 possible common HX and RX targets have been previously identified in human spermatozoa, emphasizing their potential as targets of X-chromosome-bearing spermatozoa. Further proteomic studies on rabbit sperm will be essential to identify and validate the usefulness of these proteins for application in rabbit sperm sorting techniques as targets of X-chromosome-bearing spermatozoa. Full article

The spermatozoa have limited antioxidant defences, a high polyunsaturated fatty acids content and the impossibility of synthesizing proteins, thus being susceptible to oxidative stress. High levels of reactive oxygen species (ROS) harm human spermatozoa, promoting oxidative damage to sperm lipids, proteins and DNA, leading to infertility. Coenzyme A (CoA) is a key metabolic integrator in all living cells. Recently, CoA was shown to function as a major cellular antioxidant mediated by a covalent modification of surface-exposed cysteines by CoA (protein CoAlation) under oxidative or metabolic stresses. Here, the profile of protein CoAlation was examined in sperm capacitation and in human spermatozoa treated with different oxidizing agents (hydrogen peroxide, (H₂O₂), diamide and tert-butyl hydroperoxide (t-BHP). Sperm viability and motility were also investigated. We found that H₂O₂ and diamide produced the highest levels of protein CoAlation and the greatest reduction of sperm motility without impairing viability. Protein CoAlation levels are regulated by 2-Cys peroxiredoxins (PRDXs). Capacitated spermatozoa showed lower levels of protein CoAlation than non-capacitation cells. This study is the first to demonstrate that PRDXs regulate protein CoAlation, which is part of the antioxidant response of human spermatozoa and participates in the redox regulation associated with sperm capacitation. Full article

FSHr antibodies have been shown to inhibit the differentiation of spermatogonia to primary spermatocytes, resulting in infertility without a pathological effect on reproductive organs. The aim of this study was to develop single-chain variable fragments (scFvs) against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and to evaluate the effects of intratesticular administration of the anti-FSHr scFv on testicular function and testosterone production. A phage clone against the extracellular domain of FSHr selected from a scFv phagemid library was analyzed for binding kinetics by surface plasmon resonance. Using ultrasound guidance, three adult macaques (M. fascicularis) were administered with 1 mL of 0.4 mg/mL anti-FSHr scFv (treatment) and 1 mL sterile phosphate buffer solution (control) into the left and right rete testis, respectively. Testicular appearance and volume, ejaculate quality, and serum testosterone levels were recorded on day 0 (before injection) and on days 7, 28, and 56 (after injection). Testicular tissue biopsies were performed on day 7 and day 56 to quantify the mRNA expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB), and vascular endothelial growth factor A (VEGFA). The results demonstrated that the anti-FSHr scFv molecule was calculated as 27 kDa with a dissociation constant (K_D) of 1.03 µM. The volume of the anti-FSHr scFv-injected testicle was reduced on days 28 and 56 compared with day 0 (p < 0.05). Total sperm number was reduced from day 0 (36.4 × 10⁶ cells) to day 56 (1.6 × 10⁶ cells) (p < 0.05). The percentage of sperm motility decreased from day 0 (81.7 ± 1.0%) to day 7 (23.3 ± 1.9%), day 28 (41.7 ± 53.4%), and day 56 (8.3 ± 1.9%) (p < 0.05). Sperm viability on day 0 was 86.8 ± 0.5%, which reduced to 64.2 ± 1.5%, 67.1 ± 2.2%, and 9.3 ± 1.1% on days 7, 28, and 56, respectively (p < 0.05). The expression of ABP and VEGFA on days 7 (14.2- and 3.2-fold) and 56 (5.6- and 5.5-fold) was less in the scFv-treated testicle compared with the controls (p < 0.05). On day 56, the expression of IHBB was less (p < 0.05) in the treated testis (1.3-fold) compared with the controls. Serum testosterone levels were unchanged throughout the study period (p > 0.05). This study characterized the anti-FSHr scFv and demonstrated that treatment with anti-FSHr ameliorates testicular function without altering testosterone levels, offering a potential alternative contraceptive for the long-tailed macaques. Full article

Sperm capacitation is a complex process endowing biological and biochemical changes to a spermatozoon for a successful encounter with an oocyte. The present study focused on the role of the ubiquitin–proteasome system (UPS) in the remodeling of the sperm surface subproteome. The sperm surface subproteome from non-capacitated and in vitro capacitated (IVC) porcine spermatozoa, with and without proteasomal inhibition, was selectively isolated. The purified sperm surface subproteome was analyzed using high-resolution, quantitative liquid chromatography–mass spectrometry (LC-MS) in four replicates. We identified 1680 HUGO annotated proteins, out of which we found 91 to be at least 1.5× less abundant (p < 0.05) and 141 to be at least 1.5× more abundant (p < 0.05) on the surface of IVC spermatozoa. These proteins were associated with sperm capacitation, hyperactivation, metabolism, acrosomal exocytosis, and fertilization. Abundances of 14 proteins were found to be significantly different (p < 0.05), exceeding a 1.5-fold abundance between the proteasomally inhibited (100 µM MG132) and vehicle control (0.2% ethanol) groups. The proteins NIF3L1, CSE1L, NDUFB7, PGLS, PPP4C, STK39, and TPRG1L were found to be more abundant; while BPHL, GSN, GSPT1, PFDN4, STYXL1, TIMM10, and UBXN4 were found to be less abundant in proteasomally inhibited IVC spermatozoa. Despite the UPS having a narrow range of targets, it modulated sperm metabolism and binding by regulating susceptible surface proteins. Changes in CSE1L, PFDN4, and STK39 during in vitro capacitation were confirmed using immunocytochemistry, image-based flow cytometry, and Western blotting. The results confirmed the active participation of the UPS in the extensive sperm surface proteome remodeling that occurs during boar sperm capacitation. This work will help us to identify new pharmacological mechanisms to positively or negatively modulate sperm fertilizing ability in food animals and humans. Full article

In the study, a new gene homologous to the known antimicrobial peptide Scygonadin was identified in mud crab Scylla paramamosain and named SCY3. The full-length sequences of cDNA and genomic DNA were determined. Similar to Scygonadin, SCY3 was dominantly expressed in the ejaculatory ducts of male crab and the spermatheca of post-mating females at mating. The mRNA expression was significantly up-regulated after stimulation by Vibrio alginolyticus, but not by Staphylococcus aureus. The recombinant protein rSCY3 had a killing effect on Micrococcus luteus and could improve the survival rate of mud crabs infected with V. alginolyticus. Further analysis showed that rSCY3 interacted with rSCY1 or rSCY2 using Surface Plasmon Resonance (SPR, a technology for detecting interactions between biomolecules using biosensor chips) and Mammalian Two-Hybrid (M2H, a way of detecting interactions between proteins in vivo). Moreover, the rSCY3 could significantly improve the sperm acrosome reaction (AR) of S. paramamosain and the results demonstrated that the binding of rSCY3, rSCY4, and rSCY5 to progesterone was a potential factor affecting the sperm AR by SCYs on. This study lays the foundation for further investigation on the molecular mechanism of SCYs involved in both immunity and physiological effects of S. paramamosain. Full article

In the sexual reproduction of flowering plants, two independent fertilization events occur almost simultaneously: two identical sperm cells fuse with either the egg cell or the central cell, resulting in embryo and endosperm development to produce a seed. GCS1/HAP2 is a sperm cell membrane protein essential for plasma membrane fusion with both female gametes. Other sperm membrane proteins, DMP8 and DMP9, are more important for egg cell fertilization than that of the central cell, suggesting its regulatory mechanism in GCS1/HAP2-driving gamete membrane fusion. To assess the GCS1/HAP2 regulatory cascade in the double fertilization system of flowering plants, we produced Arabidopsis transgenic lines expressing different GCS1/HAP2 variants and evaluated the fertilization in vivo. The fertilization pattern observed in GCS1_RNAi transgenic plants implied that sperm cells over the amount of GCS1/HAP2 required for fusion on their surface could facilitate membrane fusion with both female gametes. The cytological analysis of the dmp8dmp9 sperm cell arrested alone in an embryo sac supported GCS1/HAP2 distribution on the sperm surface. Furthermore, the fertilization failures with both female gametes were caused by GCS1/HAP2 secretion from the egg cell. These results provided a possible scenario of GCS1/HAP2 regulation, showing a potential scheme for capturing additional GCS1/HAP2-interacting proteins. Full article

Sperm-neutrophil binding is an important facet of breeding and significantly impacts fertility. While a specific seminal plasma protein has been found to reduce this binding and improve fertility (CRISP-3), additional molecule(s) appear to promote binding between defective sperm and neutrophils. Recent work has suggested one of these proteins is lactoferrin (LF), an 80 kDa iron-binding protein found throughout the body, but the purity of the protein was not confirmed. It is unknown if LF binds to sperm selectively based on viability, and if receptors for LF are located on equine sperm. To evaluate this, we attempted to purify equine seminal LF from five stallions (n = 5), biotinylate LF, and evaluate potential binding site(s) on spermatozoa. LF was consistently associated with superoxide dismutase (SOD-3), and all attempts to separate the two proteins were unsuccessful. Flow cytometric and microscopic analyses were used to compare LF/SOD-3 binding to viable and nonviable spermatozoa. Additionally, various methods of biotinylation were assessed to optimize this methodology. Biotinylation of seminal plasma protein was an effective and efficient method to study seminal plasma protein properties, and the binding site for LF/SOD-3 was found to be broadly localized to the entire sperm cell surface as well as selective towards nonviable/defective sperm. Although we were not able to determine if the binding to equine spermatozoa was through LF or SOD-3, we can conclude that equine seminal LF is tightly bound to SOD-3 and this protein complex binds selectively to nonviable spermatozoa, possibly to mark them for elimination by neutrophil phagocytosis. Full article

In mammals, β-defensins have been reported to play pivotal roles in sperm protection and fertilization. However, the function and mechanism of porcine β-defensin 129 (pBD129) in the sperm remain unclear. Here, we demonstrate that pBD129 is a glycosylated protein and broadly exists in accessory sex glands and coats the sperm surface. We inhibited the pBD129 protein on the sperm surface with an anti-pBD129 antibody and found that sperm motility was not significantly affected; however, sperm acrosome integrity and tyrosine phosphorylation levels increased significantly with time (p < 0.05) during capacitation. These changes were accompanied by an increase in sperm Ca²⁺ influx, resulting in a significantly reduced in vitro fertilization cleavage rate (p < 0.05). Further investigation revealed that treatment with recombinant pBD129 markedly restored the sperm motility in semen contaminated with Escherichia coli. The results suggest that pBD129 is not only associated with poor sperm motility after genital tract infection but can also protect the spermatozoa from premature capacitation, which may be beneficial for semen preservation. Full article