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.

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.

The present study investigated the decline in human fertility by analyzing the multielemental profile of seminal plasma and its relationship with seminal parameters and sperm biomarkers. Twenty-nine donor seminal plasma samples were examined using inductively coupled plasma–tandem mass spectrometry (ICP-MS/MS). Method optimization demonstrated that robust plasma conditions, including internal standardization and helium (He) collision gas, were essential to achieve reliable quantification. These conditions mitigated matrix effects and spectroscopic interferences, despite lower sensitivity. Elements such as copper (Cu), iron (Fe), manganese (Mn), strontium (Sr), titanium (Ti), vanadium (V), and chromium (Cr) were quantified, and several significant correlations were identified. Specifically, Cu was negatively correlated with seminal volume and positively correlated with sperm concentration and spontaneous acrosome reacted sperm, but negatively correlated with medium mitochondrial membrane potential (MMP); Mn showed negative associations with sperm vitality and medium MMP; Fe showed a negative correlation with motile sperm concentration (4 h); V was positively correlated with acrosome reacted sperm after acrosome reaction induction and with very low/medium MMP, whereas it was negatively associated with tyrosine phosphorylation; and Cr also showed a negative correlation with tyrosine phosphorylation. As, Mo, and Pb were detected in a few samples, limiting correlation analysis. From a functional perspective, elements such as As and Pb, as well as excess Cu or Fe, may contribute to oxidative stress by enhancing reactive oxygen species (ROS) generation and impairing antioxidant defenses. Conversely, essential metals, including Mn and Cu, at physiological concentrations act as cofactors of antioxidant enzymes and play a protective role against oxidative damage.

Background: Infertility is a growing concern, which has led to an increase in the use of assisted reproductive techniques, involving in vitro sperm capacitation. Nonetheless, the optimal capacitation time remains a matter of debate, and it is unknown whether it has an impact on the localization of key membrane proteins. Objective: This study aimed to evaluate the distribution and dynamics of membrane protein domains zonadhesin (ZAN), izumo sperm–egg fusion 1 (IZUMO1), and transmembrane protein 95 (TMEM95) in sperm following 1 and 4 h of in vitro capacitation and subsequent induction of the acrosome reaction. Materials and Methods: Sperm from 16 humans under five physiological conditions were used: noncapacitation, 1‐ and 4 h in vitro capacitation, and acrosome reaction induction. Immunolocalization of ZAN, IZUMO1, and TMEM95 was assessed using confocal microscopy. Results: The univariate and multivariate statistical analysis revealed a clear relationship between physiological conditions and protein dynamics. In particular, noncapacitated spermatozoa were characterized by an absence of ZAN and TMEM95. Whereas, regardless of the capacitation time (1 h vs. 4 h), there was an increase in the key protein presence with ZAN and IZUMO1 in the acrosomal region and TMEM95 in the equatorial region. After induction of acrosome reaction, IZUMO1 was relocated to the equatorial region. Conclusion: Here we illustrate the potential to quantify physiological differences among human spermatozoa by a multibiomarker approach. However, no evidence supports that capacitation time influences ZAN, IZUMO1, and TMEM95 distribution.

Zonadhesin (ZAN) is a sperm protein currently known for its role in binding to the oocyte zona pellucida. Although its localization has been described in ejaculated and capacitated spermatozoa from mice, pigs and rabbits, its distribution following the acrosome reaction remains poorly understood. Here, we characterized ZAN localization in rabbit, boar, and bull spermatozoa under in vitro conditions mimicking early fertilization events. Ejaculated spermatozoa, swim-up-selected spermatozoa in capacitation medium, and acrosome reaction-induced spermatozoa were analyzed. Immunofluorescence was performed using an antibody against the MAM domain of ZAN to determine its localization and PNA-FITC to assess acrosomal status, while protein abundance was assessed by quantitative LC-MS/MS. Consistent with previous reports, ZAN was detected in the anterior region of intact spermatozoa. However, the MAM-specific antibody also revealed post-acrosomal localization. Species-specific patterns were observed in intact spermatozoa: rabbit and bull showed acrosomal staining with or without post-acrosomal labeling, whereas boar displayed predominant pre-equatorial and post-acrosomal localization. Although ZAN labeling decreased after acrosomal exocytosis, the protein was consistently retained in the post-acrosomal region. These findings suggest ZAN contributes not only to sperm–ZP binding but also to later fertilization events, including sperm–oolemma interaction and membrane fusion.

Zonadhesin (ZAN) is a sperm protein currently known to mediate binding to the oocyte zona pellucida. Although its localization has been described in ejaculated and capacitated spermatozoa from mice, pigs, and rabbits, its distribution following the acrosome reaction remains poorly understood. Here, we characterized the localization of ZAN in rabbit, boar, and bull spermatozoa under in vitro conditions designed to mimic early fertilization events. Ejaculated spermatozoa, spermatozoa selected by swim-up in capacitation medium and spermatozoa subjected to acrosome reaction induction were analyzed. Immunofluorescence was performed using an antibody targeting a domain of ZAN distinct from the previously studied domain, together with PNA-FITC to assess acrosomal status; protein abundance was assessed by quantitative LC-MS/MS. ZAN was detected mostly in the anterior region of intact spermatozoa, consistent with previous reports, but the distinct epitope also revealed post-acrosomal localization. Species-specific patterns were observed in intact spermatozoa, whereas following the acrosome reaction, ZAN was consistently detected in the post-acrosomal region. These findings identify a previously unrecognized post-acrosomal distribution of ZAN and suggest that this may contribute not only to sperm–ZP binding but also to later stages of fertilization, including sperm–oolemma interaction and membrane fusion.