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During fertilization, sperm hyaluronidase activity is essential for spermatozoa to successfully penetrate the hyaluronic acid-enriched extracellular matrix of the cumulus cells. Since molecular chaperones, as the heat shock protein A2, are typically involved in bringing hyaluronic acid receptors to the cell surface, here we evaluated the presence and spatial location of HSPA2 on human spermatozoa based on its hyaluronic acid binding capacity. This study included 16 normozoospermic sperm samples from volunteering donors. The location of HSPA2 was studied in cells before and after 1-h incubation under capacitating conditions, as well as in spermatozoa selected according to their ability of binding to hyaluronic acid. Our results showed no significant differences in HSPA2 immunofluorescent cells before and after 1 h of incubation in capacitating conditions. Nevertheless, after hyaluronic acid selection, the percentage of HSPA2-labelled cells increased significantly, indicating that the interaction with hyaluronic acid may induce the unmasking of HSPA2 epitopes. Furthermore, after swim-up and hyaluronic acid selection, spermatozoa presented a highly immunostained equatorial band with a homogeneous fluorescence throughout the acrosomal region. This distribution has been previously suggested to have important implications in male fertility. Noteworthy, a homogeneous fluorescence among the acrosomal region with a more intense labelling at the apical region was observed only in hyaluronic acid bound sperm cells, which may be associated with primary gamete recognition. Our findings suggest that the hyaluronic acid selection technique and HSPA2 biomarker should be considered candidates to complement the classic seminal analysis before recommending an appropriate assisted reproduction technique. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by the Human Fertility Cathedra of the University of Alicante and R&D&I projects financed by competitive public entities (ViGrob-186, UAIND17-03, PGC2018-094781-B-100).

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.

Globozoospermia is a rare and severe type of teratozoospermia characterized by the presence of round-headed, acrosomeless spermatozoa with cytoskeleton defects. Current data support a negative relationship between globozoospermia and intracytoplasmic sperm injection (ICSI) outcomes, revealing the need to perform exhaustive studies on this type of sperm disorder. The aim of this study was to evaluate different structural, functional and molecular sperm biomarkers in total globozoospermia with proper embryo development after ICSI. The combination of field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) allowed us to identify and correlate eight morphological patterns with both types of microscopy. Additionally, results reported a high percentage of coiled forms, with cytoplasmic retentions around the head and midpiece. By fluorescent microscopy, we detected that most of the sperm showed tubulin in the terminal piece of the flagellum and less than 1% displayed tyrosine phosphorylation in the flagellum. Moreover, we did not detect chaperone Heat shock-related 70 kDa protein 2 (HSPA2) in 85% of the cells. Overall, these findings provide new insights into globozoospermia, which could have potential implications in improving sperm selection methods for assisted reproductive techniques. This research was funded by the Human Fertility Professorship and Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186).

Spermatozoa motility in freshwater and marine fish is mainly controlled by the difference in osmotic pressure. Specifically, zebrafish (Danio rerio) spermatozoa undergo hypoosmotic shock due to the decrease in extracellular potassium, which leads to membrane hyperpolarization and activation of flagellar motility. Previous studies have concluded that motility activation has a negative effect on the spermatozoa structure. However, no evidence exists about ultrastructural changes in zebrafish spermatozoa after motility activation. In this study, spermatozoa samples were obtained from ten adult zebrafish individuals before and 60 seconds after motility activation and analyzed using Scanning and Transmission Electron Microscopy. Results showed dramatic morphological and ultrastructural alterations of the zebrafish spermatozoa after activation. In particular, the spermatozoa head underwent severe morphological distortion, including swelling of the nucleus, the bursting of the plasma membrane, and the alteration of the genetic material. Midpieces were also affected after activation since rupture of the cell membrane and lysis of mitochondria occurred. Furthermore, after the hypoosmotic shock, most spermatozoa showed a coiled flagellum and a disaggregated plasma membrane. Overall, our findings show that the activation of motility leads to substantial zebrafish spermatozoa morphological and ultrastructural changes, which could modify their physiology and decrease the fertilizing potential. This research was funded by the Human Fertility Cathedra of the University of Alicante and the VIGROB-186.

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. This research was funded by the Human Fertility Professorship and Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186).

Heavy metals are endocrine disruptors which interfere with processes mediated by endogenous hormones of the organism, negatively affecting endocrine functions. Some studies have correlated heavy metal exposure with male infertility. However, the number of studies conducted on humans are limited. Therefore, the aim of this study is to summarize the current knowledge on how heavy metals influence human male fertility. Hence, three distinct databases were consulted—PubMed, Scopus and Web of Science—using single keywords and combinations of them. The total number of identified articles was 636. Nevertheless, by using the inclusion and exclusion criteria, 144 articles were finally included in this work. Results display that the development of adequate instruments for heavy metal assessment may play an important function in human male fertility diagnosis and treatment. Furthermore, clinical trials could be useful to confirm the role of heavy metals in human male fertility diagnosis. Overall, further research is required to fully understand the molecular and cellular basis of the influence of environmental and occupational exposure to heavy metals on human male infertility and reproductive outcomes. This study was funded by Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (UGP-19-042 and UGP-20-039).

Study question Can novel sperm biomarkers, such as α-tubulin and tyrosine phosphorylation, help to predict the fertilizing potential osf a globozoospermic sample? Summary answer The characterization of α-tubulin and tyrosine phosphorylation provide additional information that could be useful to determine the fertilizing capacity of the globozoospermic samples. What is known already Globozoospermia is a severe disorder characterized by the acrosome absence and other sperm alterations such as tail structural disorders and chromatin condensation abnormalities. This set of defects makes globozoospermia correlates with primary infertility and low fertilisation rates. Therefore, additional studies are necessary to know how the globozoospermia affects the disposition of α-tubulin and tyrosine phosphorylation and the relationship between embryo development and pregnancy. In this context, previous studies have independently described different patterns of α-tubulin and tyrosine phosphorylation in normozoospermic samples. Specifically, the continuous α-tubulin distribution along the flagellum and tyrosine phosphorylation have been recently linked to proper sperm functionality. Study design, size, duration We conducted a prospective study. The sample was obtained from a globozoospermic patient in October 2019. This sample was divided into two fractions, one proceeded to ICSI and the another was fixed to characterize α-tubulin and tyrosine phosphorylation using confocal microscopy. A total of 200 sperm were analyzed for each biomarker. Participants/materials, setting, methods A 38-year-old man requested assisted reproduction in IVF Spain after a failed treatment with no fertilized oocytes. The clinical procedure was performed at IVF Spain and the characterization studies were made at the University of Alicante. The flagellar cytoskeleton was assessed using anti-α-tubulin antibody (Sigma-Aldrich) at a 1:600 dilution. Besides, tyrosine phosphorylation was detected using anti-phosphotyrosine primary antibody (PY20, Sigma-Aldrich) at a 1:500 dilution. Spermatozoa were evaluated using a confocal microscope (Zeiss LSM 800). Main results and the role of chance Regarding the α-tubulin characterization, only the 33% of spermatozoa showed continuous labelling in the tail and in the 67% the fluorescence appeared in the terminal piece of the flagellum. Otherwise, we only observed 0.5% of positive tyrosine phosphorylation in the studied cells, whereas the 99.5% of sperm analyzed did not show positive fluorescence. The clinical parameters showed a fertilization rate of 20% with only one embryo after the MII oocyte artificial activation by calcium ionophore. The embryo development was further adequate, and it acquired Bt5AA status on day five. Unfortunately, the pregnancy did not ensue after the embryo transfer. Limitations, reasons for caution The main limitation of this study was that due to the very low frequency of globozoospermia ( Wider implications of the findings: The study of potential candidate biomarkers like α-tubulin and tyrosine phosphorylation in globozoospermia and the linkage with clinical parameters would facilitate the diagnosis and improve the selection of more effective treatment techniques. Trial registration number Not applicable

Objetivo: Conocer la localización y la distribución del receptor arilsulfatasa A (ARSA) en espermatozoides humanos antes y después de incubarlos en medio de capacitación durante 1 y 4 h. Material y métodos: Las muestras seminales se obtuvieron de 5 donantes normozoospérmicos. La capacitación se llevó a cabo mediante la técnica de swim-up usando medio de capacitación durante 1 y 4 h. La localización del receptor ARSA se evaluó mediante inmunofluorescencia indirecta usando microscopia confocal. En cada condición fisiológica se observó un mínimo de 200 células. Resultados: Antes de la incubación no se observó ningún patrón representativo entre las células positivas para este biomarcador (8,61%). Dicho porcentaje aumentó significativamente tras la incubación en medio de capacitación 1 y 4 h (61,86 y 63,38%, respectivamente). Entre las células capacitadas se observó un patrón mayoritario, con marcaje intenso en la región acrosomal (27,11 y 28,20% tras 1 y 4 h, respectivamente). Destacar que previamente a la incubación en los espermatozoides no se observó el patrón correspondiente a la fluorescencia a nivel de la región periacrosomal: solo tras incubar en medio de capacitación durante 1 y 4 h se detectaron un 9,13 y un 12,78% de células con dicha distribución. Conclusiones: La capacitación in vitro, independientemente del tiempo, favorece la inmunolocalización de ARSA en la región cefálica de los espermatozoides. La subpoblación más representativa tras dicho proceso fue aquella en la que ARSA se distribuyó de forma intensa y homogénea en la región acrosomal, implicada en el reconocimiento primario entre gametos. Objective: To determine the localization and distribution of the Arylsulfatase A receptor (ARSA) in human spermatozoa before and after their incubation in capacitation medium for 1 and 4 hours. Material and methods: Semen samples were obtained from five normozoospermic donors. Capacitation was by swim-up technique using capacitation medium for 1 and 4 hours. Localization of the ARSA receptor was assessed by indirect immunofluorescence using confocal microscopy. A minimum of 200 cells were observed in each physiological condition. Results: 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 4 hours (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 4 hours 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 4 hours, 9.13% and 12.78% of cells with such distribution were detected. Conclusions: 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. Este trabajo ha sido subvencionado por la Cátedra Human Fertility de la Universidad de Alicante y el proyecto de I+D+IViGrob-186 y UAIND17-03.

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.

Human fertilization success depends on the ability of the spermatozoa to undergo capacitation. Even though this process can be conducted in vitro, the optimal time for a sperm cell to complete capacitation in vitro is still under discussion due to the lack of proper capacitation biomarkers. Here, we evaluated the influence of in vitro capacitation time on HSPA2 distribution over human sperm head testing this chaperone as a potential capacitation biomarker. The chaperone was assessed in human spermatozoa from 16 normozoospermic donors using indirect immunofluorescence in uncapacitated, one and four-hour capacitated spermatozoa. The percentage of HSPA2 immunofluorescent cells before and after one hour of capacitation did not differ significantly. However, after four hours of capacitation, we observed a significantly higher percentage of HSPA2 labelled cells. In fluorescent cells analysed before capacitation, we could not identify a predominant distribution pattern. Meanwhile, after capacitation, most sperm showed a highly labelled equatorial band accompanied by a homogeneous fluorescence throughout the acrosomal region. Our findings suggest that HSPA2 needs more than one hour of in vitro capacitation for being correctly distributed in the anterior region of the sperm head. In conclusion, the present study provides solid evidences for the utility of HSPA2 as a biomarker of human sperm in vitro capacitation. Due to its importance during egg-sperm recognition, the use of HSPA2 as a biomarker before an artificial reproduction technique may be suggested, in addition to a longer capacitation time during sperm preparation. This work was supported 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 AGL2015).

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.

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. The current study was funded by the Cátedra Human Fertility and Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186).

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.

The modification of sperm glycocalyx is an essential process during sperm capacitation. The presence and redistribution of terminal and linked fucose have been described during in vitro capacitation in humans. However, the influence of the capacitation time on the quantification and localization of terminal and linked fucose is still unknown. In this study, the quantitative and qualitative changes in fucosyl residues during different in vitro capacitation times (1 and 4 h), are simultaneously characterized by using Aleuria aurantia (AAA) lectin–gold labelling and high-resolution field emission scanning electron microscopy (FE-SEM) in human sperm. A significant decrease was found in the number of terminal fucose registered in the whole sperm head during the in vitro capacitation. Nevertheless, the quantification of fucose residues after 1 h of in vitro capacitation was very similar to those found after 4 h. Therefore, the changes observed in terminal and linked fucose during capacitation were not time-dependent. Furthermore, the comprehensive analysis of the topographic distribution showed the preferential fucosyl location in the acrosomal region and the presence of distinct clusters distributed over the head in all the studied conditions. Overall, these findings corroborate the validity of FE-SEM combined with gold labelling to register changes in surface molecules during in vitro sperm capacitation. This research was funded by the Cátedra Human Fertility and Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186).

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.

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).

The success of mammalian fertilization depends largely on spermatozoa physiological events. However, the manner in which endometriosis influence morpho-functional spermatozoa biomarkers is poorly defined. Here, we studied in vitro the effect of peritoneal fluid (PF) from women with endometriosis (PFE) and non-endometriosis (PFNE) on spermatozoa parameters. This research was prospective and double-blind. A total of 45 PF samples were collected from women with (n = 25) and without endometriosis (n = 20). Semen samples were obtained from normozoospermic donors (n = 15) and cultured with 20 % (v/v) of PF or commercial culture medium (controls) during 0, 24, and 48 h. The outcome measures were spermatozoa/viability, motility, tyrosine phosphorylation (TP) and spontaneous acrosomal reaction. In addition, plasma membrane sugars were characterized by lectins [Aleuria aurantia agglutinin (AAA), Concanavalin A (ConA), Peanut agglutinin (PNA), and Wheat germ agglutinin (WGA)]. After a 24 -h culture, results reported a significant decrease in motility in cells cultured with PFE compared to the control, together with differences in the AAA and WGA-binding sites. Moreover, spermatozoa in contact with PFNE presented a significantly lower level of acrosome spontaneous reaction. At 48 h, no differences were observed in the biomarkers studied between the PFNE and the control, excluding ConA-binding sites. On the other hand, cells cultured with PFE exhibited significantly less motility, TP, and differences in the relocation of spermatozoa surface sugars. Viability was not affected in any culture condition. Overall, the effects of PFE could negatively affect spermatozoa quality, contributing to explain and diagnose the infertility associated to male partners of women with endometriosis. This study was supported by the Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO) (UGP-14-221), (UGP-15-154), Cátedra Human Fertility of the University of Alicante and VIGROB-186.

The combination of in vitro maturation (IVM) techniques and oocyte vitrification (OV) could increase the number of useful oocytes in different types of patients. IVM and subsequent OV is the most widely used clinical strategy. Would the results improve if we reverse the order of the techniques? Here, we evaluated survival, in vitro maturation, time to extrude the first polar body (PB), and the metaphase plate configuration of human prophase I (GV) oocytes before or after their vitrification. Specific, 195 GV oocytes from 104 patients subjected to controlled ovarian stimulation cycles were included. We stablished three experimental groups: GV oocytes vitrified and IVM (Group GV-Vit), GV oocytes IVM and vitrified at MII stage (Group MII-Vit), and GV oocytes IVM (Group not-Vit). All of them were in vitro matured for a maximum of 48 h and fixed to study the metaphase plate by confocal microscopy. According to our results, the vitrification of immature oocytes and their subsequent maturation presented similar survival, maturation, and metaphase plate conformation rates, but a significantly higher percentage of normal spindle than the standard strategy. Additionally, the extension of IVM time to 48 h did not seem to negatively affect the oocyte metaphase plate configuration. This research was funded by Department of Biotechnology of the University of Alicante (VIGROB-186).

Sperm function and quality are primary determinants of male reproductive performance and hence fitness. The presence of rival males has been shown to affect ejaculate and sperm traits in a wide range of taxa. However, male physiological conditions may not only affect sperm phenotypic traits but also their genetic and epigenetic signatures, affecting the fitness of the resulting offspring. We investigated the effects of male‐male competition on sperm quality using TUNEL assays and geometric morphometrics in the zebrafish, Danio rerio. We found that the sperm produced by males exposed to high male–male competition had smaller heads but larger midpiece and flagellum than sperm produced by males under low competition. Head and flagella also appeared less sensitive to the osmotic stress induced by activation with water. In addition, more sperm showed signals of DNA damage in ejaculates of males under high competition. These findings suggest that the presence of a rival male may have positive effects on sperm phenotypic traits but negative effects on sperm DNA integrity. Overall, males facing the presence of rival males may produce faster swimming and more competitive sperm but this may come at a cost for the next generation.

Objetivo: Evaluar los cambios de distribución del ácido siálico durante el proceso de capacitación y reacción acrosómica in vitro y estudiar las modificaciones morfométricas en estas condiciones fisiológicas en la región cefálica del espermatozoide humano. Material y método: En este estudio prospectivo, evaluamos 6 muestras espermáticas normozoospérmicas. La distribución de ácido siálico se evaluó mediante la lectina Wheat germ agglutinin en diferentes condiciones fisiológicas: antes, después de la capacitación y tras la reacción acrosómica. La forma y el tamaño cefálico de cada estadio se estudiaron mediante métodos de morfometría geométrica. Resultados: Tras la capacitación, un 73,07 ± 21,43% de espermatozoides presentaron ácido siálico en la región acrosomal en relación directa con una expansión del acrosoma y una contracción del segmento ecuatorial. Por otra parte, después de la reacción acrosómica se registra un mayor efecto alométrico entre los estadios debido a que los espermatozoides experimentaron una marcada expansión del segmento ecuatorial. En relación con la localización de Wheat germ agglutinin, encontramos una disminución significativa en el porcentaje de espermatozoides con fluorescencia en la región acrosomal, así como un incremento del marcaje en la banda ecuatorial. Conclusiones: Nuestros resultados demuestran que la distribución de la lectina Wheat germ agglutinin covaría con importantes cambios en la morfometría de la cabeza del espermatozoide y evidencia importantes implicaciones en los procesos de capacitación y reacción acrosómica. Objective: 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. Material and method: 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. Results: 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. Conclusions: 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. Este trabajo ha sido subvencionado por la Cátedra Human Fertility de la Universidad de Alicante y el proyecto del Ministerio de Economía y Competitividad AGL2015-70159-P.

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.