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Spermatozoa capacitation is a time-dependent physiological process essential for acquiring the fertilizing capacity. In this context, reorganization of spermatozoa surface sugars and tyrosine phosphorylation are some of the most important biochemical changes involved in capacitation. However, the relationship between these 2 biomarkers remains poorly defined. By cytofluorescence we simultaneously characterized the head concanavalin A (ConA)-binding sites and the flagellar tyrosine phosphorylation before capacitation, during different capacitation times (1 and 4 h), and after acrosome reaction induction in human spermatozoa. The results showed a strong connection between ConA-label patterns and tyrosine phosphorylation according to the spermatozoa capacitation time and acrosomal status. Specifically, the spermatozoa subpopulation with phosphotyrosine presented proper sugar location (label in acrosomal and postacrosomal region) just after 1 h of capacitation, while cells without phosphotyrosine needed 4 h to do it. Moreover, after induction of spermatozoa acrosome reaction, phosphorylation was significantly correlated (p < 0.05) with the relocation of ConA-binding residues to the equatorial region, regardless of capacitation time. Overall, these observations provide novel insights regarding spermatozoa subpopulations based on essential physiological events like capacitation and acrosome reaction, which could have potential implications in the improvement of spermatozoa selection techniques. This research was funded by the Human Fertility Cathedra of the University of Alicante and the VIGROB-186.

The failures of binding to the oocyte zona pellucida are commonly attributed to defects in the sperm recognition, adhesion, and fusion molecules. SPAM1 (sperm adhesion molecule 1) is a hyaluronidase implicated in the dispersion of the cumulus-oocyte matrix. Therefore, the aim of this study was to characterize the SPAM1 distribution in the different physiological conditions of human sperm. Specifically, we evaluated the location of the SPAM1 protein in human sperm before capacitation, at one and four hours of capacitation and after hyaluronic acid (HA) selection test by fluorescence microscopy. Sperm bound to HA were considered mature and those that crossed it immature. Our results detected three SPAM1 fluorescent patterns: label throughout the head (P1), equatorial segment with acrosomal faith label (P2), and postacrosomal label (P3). The data obtained after recovering the mature sperm by the HA selection significantly (p < 0.05) highlighted the P1 in both capacitation times, being 79.74 and 81.48% after one hour and four hours, respectively. Thus, the HA test identified that human sperm require the presence of SPAM1 throughout the sperm head (P1) to properly contact the cumulus-oocyte matrix. Overall, our results provide novel insights into the physiological basis of sperm capacitation and could contribute to the improvement of selection techniques. This research was funded by the Cátedra Human Fertility, Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186) and «Proyectos de Generación de Conocimiento» within the framework of the State Program to Promote Scientific-Technical Research and its Transfer, of the State Plan for Scientific, Technical and Innovation Research 2017–2020 (PGC2018-094781-B-I00).

Sperm glycocalyx and plasma membrane undergo outstanding modifications during fertilization. However, it is unclear how in vitro capacitation time and acrosome reaction affect the specific location of boar sperm glycoconjugates. This study aimed to identify lectin binding patterns and to describe the sequential changes during different in vitro capacitation times (1 and 4 h) and acrosome reaction in boar spermatozoa. With Aleuria aurantia agglutinin (AAA), most uncapacitated cells were labelled in the postacrosomal region. Nevertheless, after 1 h of in vitro capacitation and the acrosome reaction, most AAA binding sites were in the acrosomal region. With Concanavalin A (ConA), most sperm were labeled in the postacrosomal region before and after capacitation. After the acrosome reaction induction, this pattern changed to a highly stained acrosomal and postacrosomal regions. Peanut agglutinin (PNA) binding sites were in the acrosomal region in uncapacitated and capacitated sperm. In acrosome reacted sperm after 4h capacitation, the most frequent pattern showed remaining positive labeling in the central area of the head. With Pisum sativum agglutinin (PSA), most uncapacitated cells showed a postacrosomal region staining. Nevertheless, faint stained all over the head and highly acrosomal region labelling was observed in the major part of capacitated and acrosome reacted sperm respectively. With Wheat germ agglutinin (WGA), the most representative pattern in uncapacitated, capacitated and acrosome reacted sperm was labelled in the acrosomal region. Regarding capacitation time, the most significant changes in the most representative pattern were observed in acrosome reacted spermatozoa after 4 h of in vitro capacitation.Highlights Lectins allow detailed descriptions of eight different populations in boar sperm. Changes in lectin binding patterns during capacitation are time dependent. Lectin binding patterns significantly change after 4 h of in vitro capacitation.

Sperm capacitation includes the reorganization of plasma membrane components and the outstanding modification of the glycocalyx. The α-mannose presence and location during in vitro capacitation have been commonly described in human spermatozoa using Concanavalin A (Con A) lectin. However, it is still unclear to date how in vitro capacitation time affects the α-mannose residues and their topographic spatial distribution on sperm membrane. Here, we characterized the α-mannose density and specific membrane domain locations before and after in vitro capacitation (1–4 h) using high-resolution field emission scanning electron microscopy (FE-SEM). Results showed that α-mannose residues were present preferably on the acrosome domains for all physiological conditions. Uncapacitated sperm comparatively exhibits significant highest labeling densities of α-mannose residues. In addition, as in vitro capacitation takes place, significant and progressive decreasing of sugar residues was combined with their relocation mostly affecting acrosomal domain apical areas. Our findings reveal that combined approach using FE-SEM and gold nanoparticle topographical mapping offers new human sperm biomolecular and structural details during capacitation events.

Spermatozoa motility is a key parameter during the fertilization process. In this context, spermatozoa tyrosine protein phosphorylation and an appropriate cytoskeleton α‐tubulin distribution are some of the most important physiological events involved in motility. However, the relationship between these two biomarkers remains poorly defined. Here, we characterized simultaneously by immunocytochemistry the α‐tubulin (TUBA4A) distribution and the tyrosine phosphorylation at flagellum before capacitation, during different capacitation times (1 and 4 hr), and after acrosome reaction induction in human spermatozoa. We found that the absence of spermatozoa phosphorylation in tyrosine residues positively and significantly correlated (p

Spermatozoa interactions with the female reproductive tract and oocyte are regulated by surface molecules such as glycocalyx. The capacitation process comprises molecular and structural modifications which increase zona pellucida binding affinity. Lectins allowed us to describe glycocalyx changes during maturation, capacitation and acrosome reaction. This study had as its aim to identify lectin binding patterns using four lectins with different carbohydrate affinity in bottlenose dolphin (Tursiops truncatus) spermatozoa both before and after in vitro capacitation. Two semen samples from the same dolphin obtained on consecutive days were used, with four different lectin binding patterns becoming visible in both samples before and after capacitation. A highly stained equatorial segment with prolongations at the edges appeared as the most frequent pattern with Wheat germ agglutinin (WGA) in uncapacitated spermatozoa. However, it was homogeneously distributed over the acrosomal region after capacitation. Instead, the use of Peanut agglutinin (PNA) resulted in most spermatozoa showing high labelling in the acrosomal periphery region before capacitation and a homogeneous staining in the acrosomal region within the population of capacitated spermatozoa. Nevertheless, the most representative patterns with Concavalin A (ConA) and Aleuria aurantia agglutinin (AAA) lectins did not change before and after capacitation, labelling the acrosomal region periphery. These findings could contribute to the understanding of the reproductive biology of cetaceans and the improvement of sperm selection techniques.

To determine the localization and distribution of the ArylsulfataseA receptor (ARSA) in human spermatozoa before and after their incubation in capacitation medium for 1 and 4hours.Semen samples were obtained from five normozoospermic donors. Capacitation was by swim-up technique using capacitation medium for 1 and 4hours. Localization of the ARSA receptor was assessed by indirect immunofluorescence using confocal microscopy. A minimum of 200cells were observed in each physiological condition.Before incubation, no representative pattern was observed among the cells positive for this biomarker (8.61%). This percentage increased significantly after incubation in the capacitation medium for 1 and 4hours (61.86% and 63.38% respectively). A majority pattern was observed among the capacitated cells, with intense labelling in the acrosomal region (27.11% and 28.20% after 1 and 4hours respectively). It should be noted that the pattern corresponding to fluorescence at the level of the periacrosomal region was not observed in the spermatozoa prior to incubation. Only after incubation in capacitation medium for 1 and 4hours, 9.13% and 12.78% of cells with such distribution were detected.In vitro capacitation, regardless of time, favours the immunolocalization of ARSA in the cephalic region of the spermatozoa. The most representative subpopulation after this process was the one in which ARSA was intensely and homogeneously distributed in the acrosome region, involved in primary gamete recognition.

Catalase (CAT) stands out as one of the most efficient natural enzymes when catalysing the split of H2O2 into H2O and O2; H2O2 is one of the reactive oxygen species (ROS) involved in oxidative stress, a process closely related to aging and several health disorders or diseases like male infertility. Some studies have correlated H2O2 with male infertility and catalase with fertility restoration. However, the number of studies conducted with human beings remains scarce. Considering the use of CAT as a molecular target for biochemical analysis, this review summarises the current knowledge on how CAT influences human beings’ male fertility. Thus, three different databases were consulted—Scopus, PubMed and WOS—using single keywords and combinations thereof. A total of 40,823 articles were identified. Adopting inclusion and exclusion criteria, a final database of 197 articles served to conduct this work. It follows from this analysis that CAT could play an important role in male fertility and could become a good target for male infertility diagnosis and monitoring. However, that potential role of CAT as a tool in diagnosis must be confirmed by clinical trials. Finally, guidelines are suggested to reinforce the use of CAT in daily clinical tests for male fertility diagnosis and monitoring. This research was funded by the Human Fertility Chair, the Department of Biotechnology (VIGROB-186) and the Applied Biochemistry research group of the University of Alicante (VIGROB-309).

Spermatozoa interactions with the female reproductive tract and oocyte are regulated by surface molecules such as glycocalyx. The capacitation process comprises molecular and structural modifications which increase zona pellucida binding affinity. Lectins allowed us to describe glycocalyx changes during maturation, capacitation and acrosome reaction. This study had as its aim to identify lectin binding patterns using four lectins with different carbohydrate affinity in bottlenose dolphin (Tursiops truncatus) spermatozoa both before and after in vitro capacitation. Two semen samples from the same dolphin obtained on consecutive days were used, with four different lectin binding patterns becoming visible in both samples before and after capacitation. A highly stained equatorial segment with prolongations at the edges appeared as the most frequent pattern with Wheat germ agglutinin (WGA) in uncapacitated spermatozoa. However, it was homogeneously distributed over the acrosomal region after capacitation. Instead, the use of Peanut agglutinin (PNA) resulted in most spermatozoa showing high labelling in the acrosomal periphery region before capacitation and a homogeneous staining in the acrosomal region within the population of capacitated spermatozoa. Nevertheless, the most representative patterns with Concavalin A (ConA) and Aleuria aurantia agglutinin (AAA) lectins did not change before and after capacitation, labelling the acrosomal region periphery. These findings could contribute to the understanding of the reproductive biology of cetaceans and the improvement of sperm selection techniques. Cátedra Human Fertility of University of Alicante and VIGROB-186.

A cascade of dramatic physiological events is linked to the sperm acrosome reaction and binding to the oocyte's zona pellucida during human sperm capacitation. However, structural and functional sperm changes during capacitation currently remain poorly defined. Here, we performed a multibiomarker approach based on the utilization of sperm concentration, motility, viability, morphology, acrosome reaction, tyrosine phosphorylation, DNA fragmentation, and lectin-binding sites to analyze the impact caused by swim-up selection times (uncapacitated, 1 h capacitated, and 4 h capacitated) on sperm function and structure in normozoospermic samples. We found that a 4 h swim-up capacitation increased sperm quality, because a large number of cells with normal morphology and lower DNA fragmentation rates were recovered. Furthermore, the long-term capacitation induced a higher percentage of cells with tyrosine phosphorylation of the principal piece as well as a redistribution of lectin-binding sites. Overall, the multivariate biomarkers analyzed showed a less variable distribution on spermatozoa recovered after 4 h capacitation than that with the shorter capacitation time. These findings stress the importance of capacitation time as a relevant factor in sperm quality with potential biological reproductive implications both for basic research and in assisted reproduction techniques. This research was funded by the Human Fertility Chair, the Department of Biotechnology of the University of Alicante (VIGROB-186), and the project of the Spanish Ministry of Economy and Competitiveness (AGL2015-70159-P).

Capacitation drives sperm biophysical and biochemical changes for sperm-oocyte interactions. It is a well-known fact that the molecular complex arylsulfatase A (ARSA), hyaluronidase sperm adhesion molecule 1 (SPAM1), and heat shock protein 2 (HSPA2) plays a significant role in sperm–zona pellucida (ZP) binding. However, the time-dependent capacitation effects on the sperm surface ARSA presence and specific topographic distributions remain to be elucidated. Here, we quantified the ARSA density and specific membrane domain locations before (US) and after in vitro capacitation (one and four hours; CS1–CS4) in human sperm using high-resolution field emission scanning electron microscopy (FE-SEM) and immunogold labeling. Our results showed a significant and progressive capacitation-mediated increase of labeled spermatozoa from the US (37%) to CS4 (100%) physiological conditions. In addition, surface mapping revealed a close relationship between the ARSA residues and their acrosomal repositioning. Compared with the ARSA surface heterogeneous distribution found in US, the CS1–4 conditions exhibited clustering on the peri-acrosomal region, showing that time-dependent capacitation also induced a ARSA residue dramatic translocation on sperm surfaces. Our findings provide novel insights into the molecular remodeling events preceding sperm-oocyte interactions. This research was funded by the Human Fertility Cathedra of the University of Alicante and R&D&I projects financed by competitive public entities (ViGrob-186, UAIND17-03 and PGC2018-094781-B-100).

Assess changes in sialic acid distribution during capacitation and acrosome reaction processes, and evaluate head sperm morphometrics modifications in these physiological conditions in human sperm.In this prospective study, we included 6 normozoospermics sperm samples. Sialic acid distribution was evaluated by Wheat germ agglutinin lectin in different physiological conditions: before, after capacitation and after acrosome reaction. Head shape and size of each stage were analyzed by means of geometric morphometric methods.After capacitation, 73.07±21.43% of sperm showed sialic acid in acrosomal region, linked with an acrosome expansion and equatorial segment contraction. Otherwise, after acrosome reaction higher allometric effect between stages was recorded since sperm undergo further expansion of equatorial segment. Regarding Wheat germ agglutinin location, we found that sperm percentage significant decline in acrosomal fluorescence and an increase of equatorial band labeling.Our findings demonstrate that modifications in Wheat germ agglutinin expression covariate with dramatic changes in sperm head morphometry, suggesting important implications in capacitation and acrosome reaction processes.