Extracellular vesicles from oviductal isthmus and ampulla stimulate the induced acrosome reaction and signaling events associated with capacitation in bovine spermatozoa.

Cells can communicate with other neighboring or distant cells through the secretion of extracellular vesicles (EV), composed of a lipid bilayer and bearing surface molecules that allow them to recognize target cells. In this way, EV induce signaling via different mechanisms, modulating the physiological state of the recipient cell. EV have been identified in both male and female reproductive fluids, however, the possible role of EV isolated from female reproductive fluids has become an emerging field only recently. It is known that ejaculated mammalian spermatozoa need to undergo physiological preparation in the female reproductive tract to fertilize the egg. EV secreted by different regions of the female tract constitute signals that may have a key role in regulating sperm functions. The aims of the present study were isolating EV from different regions of the bovine oviduct and analyzing their interaction and physiological effects on spermatozoa. Here, we report the characterization of bovine oviductal fluid EV from the isthmus and ampulla region and their effect on the induced acrosome reaction and signaling events associated with sperm capacitation. EV induced an increase in sperm protein tyrosine phosphorylation, while cell survival of cryopreserved bovine spermatozoa was maintained. We also show that EV uptake regulates the sperm calcium levels by inducing an immediate increase in the intracellular calcium concentration and sperm priming, after a pre-incubation period, of the progesterone-induced intracellular calcium rise. Our data contribute to understand the role of EV in the communication between the female reproductive tract and the sperm physiology, information that may be used to improve the efficiency of reproductive assisted technologies.

Sperm chemorepulsion, a supplementary mechanism to regulate fertilization.

STUDY QUESTION: Are human spermatozoa able of chemorepulsive behaviour? SUMMARY ANSWER: Capacitated human spermatozoa are able to be chemorepelled by synthetic Progesterone Receptor Ligands (sPRL, known as contraceptives) and zinc (a cation released by the oocyte upon fertilization). WHAT IS KNOWN ALREADY: Moving cells can be oriented towards or against a molecular gradient, processes called chemoattraction and chemorepulsion, respectively, which have been described in unicellular organisms such as amoebas and bacteria, to organismic cells such macrophages and developmental cells. In the case of spermatozoa, chemoattraction may help the finding of an oocyte and has been widely studied in various invertebrate and mammalian species; however, chemorepulsion has not yet been verified in spermatozoa. STUDY DESIGN, SIZE, DURATION: This is an in vitro study involving human, rabbit and mouse spermatozoa which were used to perform 3-30 experiments per treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm samples were obtained by masturbation from healthy donors who gave written consent. Only those samples exhibiting normal semen parameters according to current WHO criteria were included in the study. Rabbit spermatozoa were obtained by artificial vagina whereas mice spermatozoa were obtained from epididymis. The sperm selection assay (SSA), originally designed to evaluate sperm chemoattraction towards progesterone (P), and a video-microscopy and computer motion analysis system were used to test sperm chemorepulsion. Additional kinetic parameters were also determined by video-microscopy and computer motion analysis. In some experiments, the level of induced acrosome-reacted spermatozoa was determined. Rabbit mating manipulation was achieved to perform the sperm-oocyte co-incubation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Sperm accumulation in the well containing 100 pg/ml of sPRL was lower than the culture medium negative control (P < 0.05). The percentage of sperm persistence against the well containing 100 pg/ml ulipristal acetate (UPA) (P = 0.001), and the percentage of sperm showing a repulsive pattern of movement (a linear trajectory followed by a transitional one after turning against the UPA), were higher than the culture medium negative control (P = 0.049). Sperm accumulation was diminished when spermatozoa where exposed to a homogeneous distribution of 100 pg/ml sPRL combined with a chemotactic gradient of progesterone (P), with respect to the culture medium negative control (P < 0.05). These results were reverted when non-capacitated spermatozoa were used to perform the same experimental settings. The accumulation of spermatozoa against 100 pg/ml sPRL was lower than the culture medium negative control also in rabbits and mice (P < 0.05). The relative number of rabbit spermatozoa arriving to the vicinity of the oocyte was diminished under the presence of 100 pg/ml UPA (P = 0.004). Sperm accumulation in the well containing zinc was decreased compared to the culture medium negative control (P < 0.05). A homogeneous distribution of zinc combined with a gradient of 10 pM P, was lower than the culture medium negative control (P = 0.016). The results were quite reproducible with two different methodologies (accumulation assay and video-microscopy combined with computer motion analysis), in three mammalian species. LIMITATIONS REASONS FOR CAUTION: The experiments were performed in vitro. Even though a quite complete characterization of sperm chemorepulsion was provided, the molecular mechanism that governs sperm repulsion is currently under investigation. WIDER IMPLICATIONS OF THE FINDINGS: Since the chemorepelled spermatozoa are those physiologically ready to fertilize the oocyte, these findings may have both biological and clinical implications, preventing either polyspermy under natural conditions or fertilization under pharmacological treatment with sPRL. STUDY FUNDING/COMPETING INTEREST(S): The study was financed by the Universidad Nacional de Cordoba (Argentina). The authors declare that they do not have competing financial interests. TRIAL REGISTRATION NUMBER: N/A.

Uterosome-like vesicles prompt human sperm fertilizing capability.

STUDY QUESTION: Does the rapid transit through the uterine environment modulate the sperm physiological state? SUMMARY ANSWER: The uterosome-like vesicles (ULVs) secreted by endometrial epithelial cells (EECs) in vitro are able to fuse with human spermatozoa, prompting their fertilizing capacity. WHAT IS KNOWN ALREADY: Early studies suggest that sperm capacitation begins in the uterus and ends in the oviduct, and that a synergistic effect of both female organs may accelerate this process. Although it has been reported that co-incubation of human spermatozoa with endometrial cell-conditioned medium (CM) stimulates sperm capacitation, the mechanism mediating this communication is unknown. STUDY DESIGN, SIZE, DURATION: Human ULVs secreted by EECs were characterized and their effect on human sperm physiology was analysed. Spermatozoa were incubated with EEC-derived CM or ULV, after which sperm capacitation was evaluated at different time points. In addition, the interaction of spermatozoa with ULV was analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS: ULVs were isolated by ultracentrifugation and identified using electron microscopy and Western blotting to assess the presence of specific protein markers. Following seminal plasma removal, human spermatozoa were incubated CM or ULV, after which sperm capacitation was evaluated as the ability of the sperm to undergo the induced acrosome reaction and the level of protein tyrosine phosphorylation (PY) determined by Western blot and immunocytochemistry. The interaction of spermatozoa with labelled ULV was analysed by fluorescence microscopy. In all cases, at least three biological replicates from different sperm donors were performed for each set of experiments. Significant differences between mean values were determined by one-way ANOVA followed by Tukey's post hoc test. Differences between treatments were considered statistically significant at P ≤ 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: The level of capacitated spermatozoa and those recruited by chemotaxis increased 3- to 4-fold when spermatozoa were incubated in the presence of CM for 4 h. Even a 15 min incubation of spermatozoa with CM was also enough to increase the level of capacitated cells 3- to 4-fold (P < 0.05). Furthermore, a short co-incubation of spermatozoa with ULV stimulates sperm capacitation, as determined by the increase in the level of induced acrosome reaction and the induction of PY. In addition, after the co-incubation of spermatozoa with fluorescent labelled ULV, the sperm cells acquired the fluorescent staining which indicates that ULV might be transferred to the sperm surface by a fusion mechanism. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study performed with human biological material, spermatozoa and endometrial derived cells; the latter being a cell line originally isolated from a uterine adenocarcinoma. WIDER IMPLICATIONS OF THE FINDINGS: The capability of spermatozoa to briefly interact with ULVs supports the hypothesis that any step of sperm transport may have physiological consequences, despite the interaction lasting for only a limited period of time. This way of communication of spermatozoa with cell products of uterine origin opens new frontiers of investigation (e.g. the signalling molecules involved), shedding light on the sperm processes that prepare the male gamete for fertilization, which might have implications for human infertility treatment. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTERESTS: The project was financially supported by SECyT-UNC. The authors declare no conflict of interest.

Disrupting the wall accumulation of human sperm cells by artificial corrugation.

Many self-propelled microorganisms are attracted to surfaces. This makes their dynamics in restricted geometries very different from that observed in the bulk. Swimming along walls is beneficial for directing and sorting cells, but may be detrimental if homogeneous populations are desired, such as in counting microchambers. In this work, we characterize the motion of human sperm cells ∼60 µm long, strongly confined to ∼25 µm shallow chambers. We investigate the nature of the cell trajectories between the confining surfaces and their accumulation near the borders. Observed cell trajectories are composed of a succession of quasi-circular and quasi-linear segments. This suggests that the cells follow a path of intermittent trappings near the top and bottom surfaces separated by stretches of quasi-free motion in between the two surfaces, as confirmed by depth resolved confocal microscopy studies. We show that the introduction of artificial petal-shaped corrugation in the lateral boundaries removes the tendency of cells to accumulate near the borders, an effect which we hypothesize may be valuable for microfluidic applications in biomedicine.

Geometrical guidance and trapping transition of human sperm cells.

The guidance of human sperm cells under confinement in quasi-2D microchambers is investigated using a purely physical method to control their distribution. Transport property measurements and simulations are performed with diluted sperm populations, for which effects of geometrical guidance and concentration are studied in detail. In particular, a trapping transition at convex angular wall features is identified and analyzed. We also show that highly efficient microratchets can be fabricated by using curved asymmetric obstacles to take advantage of the spermatozoa specific swimming strategy.

Effects of the synthetic estrogen 17α-ethinylestradiol on aromatase expression, reproductive behavior and sperm quality in the fish Jenynsia multidentata.

The synthetic estrogen 17α-ethynylestradiol (EE2) has been increasingly detected in sewage effluents in the last two decades. The aim of the present study was determined if EE2 exposure adversely affected reproduction in internally fertilizing fish species Jenynsia multidentata. Sexual behavior, brain and gonadal aromatase expression as well as sperm quality were evaluated. The brain aromatase expression, reproductive behavior, spermatozoa viability and gonadosomatic index were sensitive biomarkers of EE2 effects on this species. The condition factor, hepatosomatic index, gonadal aromatase expression, sperm count and sperm velocities were unaltered after EE2 exposure. The present work highlights the importance of using a combination of several biomarkers to study the effects of estrogenic compounds, especially when trying to link these results to potential population-level effects.

Picomolar gradients of progesterone select functional human sperm even in subfertile samples.

More than 1 million infertility treatments are practiced around the world per year, but only 30% of the couples succeed in taking a baby home. Reproductive technology depends in part on sperm quality, which influences not only fertilization but also embryo development and implantation. In order to provide a better quality sperm subpopulation, innovative sperm selection techniques based on physiological sperm features are needed. Spermatozoa at an optimum state may be selected by following an increasing concentration gradient of picomolar progesterone, a steroid secreted by the cumulus cells at the time of ovulation. In this study we developed a method to recruit spermatozoa at the best functional state, based on sperm guidance toward progesterone. The sperm selection assay (SSA) consists of a device with two wells connected by a tube. One well was filled with the sperm suspension and the other with picomolar progesterone, which diffused inside the connecting tube as a gradient. The sperm quality after the SSA was analyzed in normal and subfertile semen samples. Several sperm parameters indicative of sperm physiological state were determined before and after the SSA: capacitation, DNA integrity and oxidative stress. After the SSA, the mean level of capacitated spermatozoa increased three times in normal and in subfertile samples. The level of sperm with intact DNA was significantly increased, while sperm oxidative stress was decreased after sperm selection. Interestingly, the exposure to a progesterone gradient stimulated the completion of capacitation in some spermatozoa that could not do it by themselves. Thus, the SSA supplies a sperm population enriched with spermatozoa at an optimum physiological state that may improve the assisted reproductive technology outcome.

Assessment of sperm function parameters and DNA fragmentation in ejaculated alpaca sperm (Lama pacos) by flow cytometry.

Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR-14/PI staining; acrosomal membrane integrity using FITC-conjugated Pisum Sativum Agglutinin/PI labelling; mitochondrial membrane potential (Δψm) by staining with JC-1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = -0.41) and with plasma membrane integrity (p = 0.01; r = -0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry.

Sperm transport and retention at the fertilization site is orchestrated by a chemical guidance and oviduct movement.

In mammals, only a few spermatozoa arrive at the fertilization site. During the last step in the journey to the egg, apart from their self-propulsion, spermatozoa may be assisted by oviduct movement and/or a guidance mechanism. The proportion of rabbit spermatozoa that arrive at the fertilization site was determined under in vivo conditions, in which either the ovulation products (secreting chemoattractants) and/or the oviduct movement (causing the displacement of the oviductal fluid) was inhibited. When only one of these components was inhibited, sperm transport to the fertilization site was partially reduced. However, when both the ovulation products and the oviduct movement were inhibited, almost no spermatozoa arrived at the fertilization site. The results suggest that spermatozoa are transported to and retained at the fertilization site by the combined action of a chemical guidance and the oviduct movement. A working model is proposed to explain how these two mechanisms may operate to transport spermatozoa to the fertilization site, probably as an evolutionary adaptation to maximize the chance of fertilizing an egg.

Chemotactic response of frozen-thawed bovine spermatozoa towards follicular fluid.

The aim of this study was to verify whether cattle spermatozoa respond by chemotaxis to follicular fluid (FF). The experimental conditions were defined to maintain a frozen-thawed sperm population with great motility and capacitation, and lesser sperm agglutination. Several sperm preparation conditions were studied: sperm separation from the seminal plasma by Sephadex column or migration-sedimentation, incubation under capacitating conditions in the presence or absence of a superficial layer of mineral oil, and different pH of the culture medium. The percentage of motile and agglutinated spermatozoa was determined in plate dishes under inverted phase contrast microscope. The percentage of capacitated spermatozoa was calculated as the difference between the percentages of acrosome reacted spermatozoa with and without lysophosphatidylcholine stimulation. The most ideal experimental conditions to evaluate chemotaxis in frozen-thawed cattle spermatozoa were: to separate the cells from the seminal plasma by migration-sedimentation and to incubate them under oil, in culture medium at pH 7.2, for less than 2h. The chemotaxis assays were conducted with spermatozoa treated as mentioned above which were confronted to several dilutions of FF (1:10(3), 1:10(4), 1:10(5), 1:10(6)) in a chemotaxis chamber by videomicroscopy and computer image analysis. A subpopulation of capacitated spermatozoa ( approximately 10%) that responded chemotactically to a concentration gradient generated by FF (1:10(4) to 1:10(5)) was observed. Since cryopreserved spermatozoa are regularly used to artificially inseminate the cows, the sperm chemotactic response towards FF would be potentially used to diagnose the bull sperm sample or to select the spermatozoa in the most functional state.

Sperm motility parameters to evaluate the seminal quality of Boa constrictor occidentalis, a threatened snake species.

Semen quality analysis constitutes a powerful tool to evaluate the fertility potential of males in threatened species. The Argentine boa constrictor or lampalagua (Boa constrictor occidentalis) is a threatened snake species and has been included in Appendix I of CITES. The objective of this work is to characterize the sperm of B. c. occidentalis on the bases of dynamic parameters to improve this species conservation. Dynamic parameters were measured in sperm samples using videomicroscopy and image analysis software. The sperm population showed a high degree of heterogeneity in velocity parameter values and 95% of the cells showed a linear pattern of movement. Studies in other species indicate that the number of motile spermatozoa and their movement speed is directly correlated with fertilization success. This work will help to establish basic parameter values for the evaluation of the reproductive potential of populations of B. c. occidentalis and to resolve questions referred to its reproductive strategies.

Increased velocity and induction of chemotactic response in mouse spermatozoa by follicular and oviductal fluids.

The dynamic parameters of mouse sperm cells exposed to follicular and oviductal fluids were assessed. Spermatozoa were tracked on a chemotactic Zigmond chamber and recorded using a videomicroscopy system. The results were evaluated with computer-supported image analysis. Follicular fluid at a dilution of 10(-4) markedly increased the proportion of spermatozoa with high velocity, and stimulated chemotactic behaviour. The highest velocities were observed in sperm cells exposed to oviductal fluid, and a greater proportion of these cells had high velocity compared with those exposed to follicular fluid. Chemotaxis was induced in spermatozoa exposed to oviductal fluid at dilutions of 10(-3) and 10(-5). These results suggest the presence of temporal subpopulations of responsive spermatozoa, considering the distance travelled towards both follicular and oviductal fluids and the proportion of sperm cells migrating towards the gradient in the highest distance ranges. This is the first report on the effect of isolated follicular and oviductal fluids on dynamic parameters and chemotaxis of mouse spermatozoa. The findings support previous work showing that the motility and directionality of mouse sperm cells is increased by factors in the microenvironment of the egg. Although the significance of these factors in vivo is unknown, it is possible that there is a relay mechanism involving sequential activity of both oviductal and follicular fluids to direct the male gametes towards the egg.

Mouse spermatozoa modify their motility parameters and chemotactic response to factors from the oocyte microenvironment.

The aim of this work was to evaluate the period of time required for the induction of changes in motility of mouse spermatozoa in response to factors from the microenvironment of oocytes. To determine the effects of the latency time, the period of preincubation before contact with the oocyte product(s), sperm samples were incubated for 15 or 90 min and then exposed to either Dulbecco's Modified Eagle's Medium (DMEM) or to a crude extract of superovulated oocytes (CE). The assays were performed in a Zigmond chamber by filling one compartment with either DMEM or CE, and the other compartment with the sperm suspension. A videomicroscopy system was used for tracking the spermatozoa. Sperm motility analysis was assessed using a semiautomatic objective method, and the following parameters were determined; (1) dynamic parameters: curvilinear velocity, linear velocity and linearity; (2) progressive motility: percentage of spermatozoa showing either circular or linear patterns of movement; and (3) directional motility: percentage of spermatozoa that moved towards either the DMEM or the CE. The results of this work showed that when the spermatozoa contacted soluble factors in CE after only 15 min of previous incubation, there was a significant increase in their dynamic parameters, change in their progressive motility, and induction of directional movement of the spermatozoa towards the CE components, while a longer period of preincubation did not significantly modify these effects. On the other hand, in the presence of culture medium (with or without addition of bovine serum albumin), the spermatozoa needed a more extended period of incubation to significantly increase their dynamic parameters and to modify their progressive motility, while maintaining a random direction of movement.

Correlation between response to progesterone and other functional parameters in human spermatozoa.

OBJECTIVE: To determine the proportion of human sperm that respond to progesterone and to determine their capacitation state. DESIGN: The sperm population was separated according to motility by means of a Percoll density gradient; three subpopulations of low, medium, and high motility were obtained. SETTING: University-based laboratory. PARTICIPANT(S): Sperm samples from healthy donors with normal spermatogram values were used. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The percentage of viable spermatozoa that increased the intracellular [Ca2+]i in response to progesterone was determined with a fluorescence-activated cell sorter (FACS). The percentage of capacitated spermatozoa was determined as the difference between with and without phorbol 12-myristate 13-acetate stimulus according to fluorescence microscopy and the FACS methods. RESULT(S): A significant linear relationship between the proportion of motile cells and the percentage of sperm that increases the [Ca2+]i in response to progesterone was observed with or without previous phorbol 12-myristate 13-acetate treatment. The slope of the correlation equation corresponding to the phorbol 12-myristate 13-acetate treatment was significantly lower. In addition, a significant correlation between capacitation and motility was observed. CONCLUSION(S): It seems likely that the proportion of capacitated and progesterone-responding human sperm correlates with motility.

Type of rectal contents and infectivity of domiciliary populations of Triatoma infestans (Hemiptera: Reduviidae) in Argentina.

Different types of rectal content in domiciliary populations of Triatoma infestans (Hemiptera: Reduviidae: Triatominae) were associated with differences in the degree of infection with Trypanosoma (Schizotrypanum) cruzi. Field studies were carried out in a region of Argentina (Cordoba) where Chagas' disease is endemic. The rectal contents of domiciliary T. infestans were classified by color (clear, yellow, or brown) and the number of infective metacyclic stages of T. cruzi. The seasonal percentage of triatomines with metacyclics did not vary significantly, except in adults, in which it was greatest in spring (November-December). The mean density of T. cruzi per microliter of rectal material varied during the year, according to instar and color of the rectal contents. Clear recta had most metacyclics. December (late spring) may be more important for vectorial transmission of T. cruzi, because there are more infective adults (788) with higher counts of rectal metacyclics.

Seasonal changes in infectivity of domestic populations of Triatoma infestans.

This paper examines the infection rate of Trypanosoma cruzi in Triatoma infestans, the main vector of Chagas disease in Argentina and neighbouring countries. The study was carried out in 1986-1987 on 5 houses (ranchos) in the endemic area of central Argentina. Domestic T. infestans populations were sampled in each season with a constant capture effort (2.5 man-hours/house) using a chemical irritant. The rectal content of the bugs was examined for the presence of T. cruzi. The vector population density showed seasonal changes with highest values during the hot season (November-April). The percentage of infected bugs was higher in mid-spring (November) and autumn (April) than in winter (August) and early spring (October). The mean number of parasites (epimastigotes and trypomastigotes) per microliter of rectal material was very high during mid- and late spring (December). The percentage and number of metacyclic forms differed between seasons, reaching the highest values in late spring. The percentage of infected bugs in houses with children younger than 10 years old was higher than that in houses without children, during all the seasons. Late spring seemed to be the period when domestic populations of T. infestans had the highest vector potential.