Acrosomal Contents Research Articles

SMA20/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization.

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a-Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.

Addition of l-carnitine to the freezing extender improves post-thaw sperm quality of Okinawan native Agu pig

The objective of the present study was to establish whether the addition of l-carnitine (LC), which exhibits antioxidant activity, to the freezing extender improves the quality of cryopreserved Okinawan native Agu pig sperm. Ejaculated sperm frozen in an extender supplemented with 0, 1, 2.5, or 5 mM LC was thawed, and the integrities of mitochondria and the plasmalemma and other sperm characteristics were evaluated. The treatment with different concentrations of LC effectively improved sperm motility, mitochondrial and plasmalemmal integrities, and the proteolytic activity of acrosomal contents after freeze-thawing (P < 0.05). The proportion of post-thaw sperm possessing intact mitochondria and plasmalemma and higher proteolytic activity of acrosomal contents was markedly higher among sperm frozen in the presence of 2.5 mM LC than among sperm frozen in the extender without LC (P < 0.05). Furthermore, although the addition of LC to the freezing extender had no effect on disturbance of DNA damage and caspase activity, sperm treated with 2.5 mM LC during freezing exhibited significantly higher penetrability into matured oocytes in vitro than untreated sperm. Collectively, these results indicate that the addition of LC to the freezing extender effectively improved the post-thaw quality of Agu pig sperm by preventing mitochondrial dysfunction caused by oxidative stress during cryopreservation.

Discrete Dynamic Model of the Mammalian Sperm Acrosome Reaction: The Influence of Acrosomal pH and Physiological Heterogeneity.

The acrosome reaction (AR) is an exocytotic process essential for mammalian fertilization. It involves diverse physiological changes (biochemical, biophysical, and morphological) that culminate in the release of the acrosomal content to the extracellular medium as well as a reorganization of the plasma membrane (PM) that allows sperm to interact and fuse with the egg. In spite of many efforts, there are still important pending questions regarding the molecular mechanism regulating the AR. Particularly, the contribution of acrosomal alkalinization to AR triggering physiological conditions is not well understood. Also, the dependence of the proportion of sperm capable of undergoing AR on the physiological heterogeneity within a sperm population has not been studied. Here, we present a discrete mathematical model for the human sperm AR based on the physiological interactions among some of the main components of this complex exocytotic process. We show that this model can qualitatively reproduce diverse experimental results, and that it can be used to analyze how acrosomal pH (pHa) and cell heterogeneity regulate AR. Our results confirm that a pHa increase can on its own trigger AR in a subpopulation of sperm, and furthermore, it indicates that this is a necessary step to trigger acrosomal exocytosis through progesterone, a known natural inducer of AR. Most importantly, we show that the proportion of sperm undergoing AR is directly related to the detailed structure of the population physiological heterogeneity.

Pores Formation in Porcine Sperm Incubated in Streptolysin O and Its Post Thawing Viability after Trehalose Treatment

Background: Streptolysin O (SLO), a pore-forming protein in plasma membrane (PM), has been used to internalize a variety of molecules (DNA and RNA) in cells. In sperm, however, SLO has only been used to release acrosomal contents. Its possible use as biotechnology in the cryopreservation of pig semen. However, porcine sperm are very sensitive to the freeze-thaw process. The study aimed to evaluate the pore formation in the PM, the addition of trehalose and the post cryopreservation viability of porcine spermatozoa using SLO.Methods: Research period was spring 2017- summer 2018. Thirty ejaculates from five mature boars were used. Semen was incubated in SLO 0.6 IU/ml and trehalose (added at 100, 200 and 400 ìM). Semen diluted in commercial diluent as control group. Presence of pores was checked by scanning electronic microscopy. To evaluate sperm membrane integrity and functional status the Coomassie stain with HOST test and the Chlortetracycline test were used. Result: It was found that SLO could form pores in the sperm cell membrane The addiction of 200 ìM trehalose to the freezing medium have different effects on the quality of boar sperm, showing highest motility and viability during the cooling process.

Different Approaches to Record Human Sperm Exocytosis.

Acrosome reaction is an exocytic process that enables a sperm to penetrate the zona pellucida and fertilize an egg. The process involves the fenestration and vesiculation of the sperm plasma membrane and outer acrosomal membrane, releasing the acrosomal content. Given the importance of the acrosome secretion in fertilization, many different methods have been developed to detect the acrosome reaction of sperm. In this chapter, we describe detailed practical procedures to assess the acrosomal status of human spermatozoa. To do this, we resorted to light optical and epifluorescence microscopy, flow cytometry, and transmission electron microscopy. We also itemize the protocol for real-time measurements of the acrosome reaction by confocal microscopy. Further, we discuss the level of complexity, costs, and the reasons why a researcher should choose each technique.This chapter is designed to provide the user with sufficient background to measure acrosomal exocytosis in human sperm.

Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead's surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.

In vitro Induction and Detection of Acrosomal Exocytosis in Human Spermatozoa.

The acrosome reaction is a highly regulated exocytotic event that primes spermatozoa for successful fertilization. Upon induction, acrosomal exocytosis proceeds via a wave of vesiculation that radiates across the sperm head, destabilizing the acrosomal vesicle and resulting in the release of the acrosomal contents. Having shed their acrosome, spermatozoa are then capable of penetrating the outer vestments of the oocyte and initiating fertilization. Accordingly, the failure of spermatozoa to complete an acrosome reaction represents a relatively common etiology in male infertility patients, and the ability to induce acrosomal exocytosis has found clinical utility in the evaluation of sperm fertilizing capacity. Here, we firstly describe protocols for driving the capacitation of human spermatozoa in vitro using chemically defined media in order to prime the cells for completion of acrosomal exocytosis. We then describe methodology routinely used for the induction of acrosomal exocytosis incorporating either a physiological agonist (i.e., the steroidal hormone, progesterone) or pharmacological reagent (i.e., the divalent cation ionophore, A23187). Finally, we describe the application of histochemical and immunofluorescence techniques that can be applied to study the completion of the acrosome reaction. Such protocols have important diagnostic utility for sperm function testing in both clinical and andrological research laboratories.

Cyclin-CDK Complexes are Key Controllers of Capacitation-Dependent Actin Dynamics in Mammalian Spermatozoa.

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional maturation process to acquire the competence to fertilize the female egg. During this process, called capacitation, the actin cytoskeleton dramatically changes its organization. First, actin fibers polymerize, forming a network over the anterior part of the sperm cells head, and then it rapidly depolymerizes and disappears during the exocytosis of the acrosome content (the acrosome reaction (AR)). Here, we developed a computational model representing the actin dynamics (AD) process on mature spermatozoa. In particular, we represented all the molecular events known to be involved in AD as a network of nodes linked by edges (the interactions). After the network enrichment, using an online resource (STRING), we carried out the statistical analysis on its topology, identifying the controllers of the system and validating them in an experiment of targeted versus random attack to the network. Interestingly, among them, we found that cyclin-dependent kinase (cyclin–CDK) complexes are acting as stronger controllers. This finding is of great interest since it suggests the key role that cyclin–CDK complexes could play in controlling AD during sperm capacitation, leading us to propose a new and interesting non-genomic role for these molecules.

Human sperm acrosomal status, acrosomal responsiveness, and acrosin are predictive of the outcomes of in vitro fertilization: A prospective cohort study

The sperm acrosome reaction (AR) is a physiological secretory course of membrane fusion and hydrolytic enzymes, as well as matrix protein release, enabling spermatozoa to penetrate the egg surroundings. An instable acrosomal status before a specific stimulus, insufficient acrosomal responsiveness, or inadequate enzymatic activity of acrosomal content can be detrimental to male fertility. This prospective cohort study was designed to determine whether three human sperm acrosome evaluation parameters—including spontaneous AR rate, AR after calcium ionophore A23187 challenge (ARIC) rate, and modified Kennedy acrosin activity—can predict fertilization outcomes in vitro and are correlated with male characteristics. A total of 485 eligible couples undergoing in vitro fertilization (IVF) therapy were included in two phases of this study. In a ‘construction phase’, three acrosome evaluation parameters were determined simultaneously in 132 cases, whereas in a ‘validation phase’, the spontaneous AR rate was determined in 353 cases. The results of the ‘construction phase’ revealed that the spontaneous AR rate was the only significant predictor of fertilization outcome (unadjusted odds ratio [OR] = 0.68, 95% confidence interval [CI]: 0.53–0.88, P = 0.003; adjusted OR = 0.64, 95% CI: 0.43–0.95, P = 0.03), and the cut-off value for total fertilization failure (TFF) prediction, determined by ROC curve analysis, was 9.91%; higher acrosin activity was shown to predict a higher fertilization rate only when patients were divided into groups (≥25 μIU/106 spermatozoa, 14–25 μIU/106 spermatozoa, <14 μIU/106 spermatozoa). The spontaneous AR rate was negatively correlated with sperm motility, forward progression motility, and normal morphology; modified Kennedy acrosin activity was positively correlated with normal morphology; and the ARIC rate was not correlated with any of the male characteristics. A similar result was obtained for the spontaneous AR rate in the ‘validation phase’, and the cut-off value in predicting TFF was calibrated for 9.52%. Clinically, patients can voluntarily choose spontaneous AR rate alone or in combination with modified Kennedy acrosin activity to predict TFF, and early rescue intracytoplasmic sperm injection (ICSI), half ICSI, or full ICSI should be considered in advance for men with spontaneous AR rates ≥9.52% or spontaneous AR rates ≥9.52% and AE activities <25 μIU/106 spermatozoa.

Epididymal cysteine-rich secretory proteins are required for epididymal sperm maturation and optimal sperm function.

What is the role of epididymal cysteine-rich secretory proteins (CRISPs) in male fertility? While epididymal CRISPs are not absolutely required for male fertility, they are required for optimal sperm function. CRISPs are members of the CRISP, Antigen 5 and Pathogenesis related protein 1 (CAP) superfamily and are characterized by the presence of an N-terminal CAP domain and a C-terminal CRISP domain. CRISPs are highly enriched in the male reproductive tract of mammals, including in the epididymis. Within humans there is one epididymal CRISP, CRISP1, whereas in mice there are two, CRISP1 and CRISP4. In order to define the role of CRISPs within the epididymis, Crisp1 and Crisp4 knockout mouse lines were produced then interbred to produce Crisp1 and 4 double knockout (DKO) mice, wherein the expression of all epididymal CRISPs was ablated. Individual and DKO models were then assessed, relative to their own strain-specific wild type littermates for fertility, and sperm output and functional competence at young (10-12 weeks of age) and older ages (22-24 weeks). Crisp1 and 4 DKO and control mice were also compared for their ability to bind to the zona pellucida and achieve fertilization. Knockout mouse production was achieved using modified embryonic stem cells and standard methods. The knockout of individual genes was confirmed at a mRNA (quantitative PCR) and protein (immunochemistry) level. Fertility was assessed using breeding experiments and a histological assessment of testes and epididymal tissue. Sperm functional competence was assessed using a computer assisted sperm analyser, induction of the acrosome reaction using progesterone followed by staining for acrosome contents, using immunochemical and western blotting to assess the ability of sperm to manifest tyrosine phosphorylation under capacitating conditions and using sperm-zona pellucida binding assays and IVF methods. A minimum of three biological replicates were used per assay and per genotype. While epididymal CRISPs are not absolutely required for male fertility, their production results in enhanced sperm function and, depending on context, CRISP1 and CRISP4 act redundantly or autonomously. Specifically, CRISP1 is the most important CRISP in the establishment of normally motile sperm, whereas CRISP4 acts to enhance capacitation-associated tyrosine phosphorylation, and CRISP1 and CRISP4 act together to establish normal acrosome function. Both are required to achieve optimal sperm-egg interaction. The presence of immune infiltrates into the epididymis of older, but not younger, DKO animals also suggests epididymal CRISPs function to produce an immune privileged environment for maturing sperm within the epididymis. Caution should be displayed in the translation of mouse-derived data into the human wherein the histology of the epididymis is someone what different. The mice used in the study were housed in a specific pathogen-free environment and were thus not exposed to the full range of environmental challenges experienced by wild mice or humans. As such, the role of CRISPs in the maintenance of an immune privileged environment, for example, may be understated. The combined deletion of Crisp1 and Crisp4 in mice is equivalent to the removal of all CRISP expression in humans. As such, these data suggest that mammalian CRISPs, including that in humans, function to enhance sperm function and thus male fertility. These data also suggest that in the presence of an environmental challenge, CRISPs help to maintain an immune privileged environment and thus, protect against immune-mediated male infertility. Not applicable. This study was funded by the National Health and Medical Research Council, the Victorian Cancer Agency and a scholarship from the Chinese Scholarship Council. The authors have no conflicts of interest to declare.

Sugars Require Rigid Multivalent Displays for Activation of Mouse Sperm Acrosomal Exocytosis.

As a prerequisite to mammalian fertilization, the sperm acrosomal vesicle fuses with the plasma membrane and the acrosome contents are exocytosed. Induction occurs through engagement of the sperm receptors by multiple sugar residues. Multivalent polymers displaying mannose, fucose, or GlcNAc are effective synthetic inducers of mouse sperm acrosomal exocytosis (AE). Each carbohydrate is proposed to have a distinct binding site on the sperm cell surface. To determine the role of the scaffold structure in the efficiency of AE induction, different polymer backbones were employed to display the different activating sugar residues. These glycopolymers were prepared by ruthenium-catalyzed ring-opening metathesis of 5-substituted norbornene or cyclooctene. The conformations of the glycopolymers were characterized by small-angle X-ray scattering. Polynorbornene displaying mannose, fucose, or GlcNAc forms flexible cylinders in aqueous solution. However, polycyclooctenes displaying any of these same sugars are much more flexible and form random coils. The flexible polycyclooctenes displaying fucose or GlcNAc were less effective inducers of AE than their norbornene counterparts. In contrast, polycyclooctene displaying mannose was the most effective AE inducer and had a more collapsed spherelike structure. Our results suggest that the AE efficacy of fucose, GlcNAc, and mannose polymers relies on a relatively rigid polymer that can stabilize receptor signaling complexes.

Dynamic of contribution of UBPy-sorted cargo to acrosome biogenesis: effects of its derailment in a mouse model of globozoospermia, the infertile Vps54 (L967Q) mutant.

The sperm acrosome is a specialized vacuole, a member of the family of cell-specific lysosome-related organelles. Its exocytosis, the acrosome reaction, is a crucial event during fertilization. The released acrosomal contents promote sperm penetration through the investments of the oocyte, whereas the membranous components of the acrosome are involved in sperm-oocyte interaction/fusion and oocyte activation. The way that these functionally distinct acrosomal costituents reach the vacuole during its biogenesis remains poorly understood. The biosynthetic pathway and a consistent supply from the endosomal system have recently been documented. We use immunogold electron microscopy to determine the contribution of endosome cargo-sorting during step-by-step mouse acrosomogenesis. The chosen proteins of this study were UBPy (ESCRT-DUB), together with endosome compartment markers EEA1 and pallidin. The latter is described here for the first time in male germ cells. This new insight expands our knowledge of acrosomogenesis, confirming the plasticity of the endosomal system in supporting cell-type-specific functions. We also study wobbler mice, whose Vps54 mutation causes motor neuron degeneration and male infertility. Use of electron/immunoelectron microscopy and immunofluorescence enabled us to establish that the lack of an acrosome in wobbler spermatozoa is attributable to an early block in acrosome biogenesis and that the mislocalization of acrosome-destined proteins, potentially involved in the signaling events leading to oocyte activation, is possibly responsible for wobbler infertility, even after intracytoplasmic sperm injection.

The Rab3A-22A Chimera Prevents Sperm Exocytosis by Stabilizing Open Fusion Pores

At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction.

Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100

In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (P < 0.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (P < 0.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (P < 0.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (P < 0.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique.

The Acrosome Reaction: A Historical Perspective.

Acrosome reaction is often referred to as acrosomal exocytosis, but it differs significantly from normal exocytosis. While the vesicle membrane initially holding excreting molecules remains on the cell surface during exocytosis, the outer acrosomal membrane and plasma membrane are lost by forming vesicles during acrosome reaction. In this context, the latter process resembles a release of exosome. However, recent experimental data indicate that the most important roles of acrosome reaction lie not in the release of acrosomal contents (or "vesiculated" plasma and outer acrosomal membrane complexes) but rather in changes in sperm membrane. This review describes the mechanism of fertilization vis-a-vis sperm membrane change, with a brief historical overview of the half-century study of acrosome reaction.

Role of Ion Channels in the Sperm Acrosome Reaction.

The acrosome reaction (AR) is a unique exocytotic process where the acrosome, a single membrane-delimited specialized organelle, overlying the nucleus in the sperm head of many species, fuses with the overlying plasma membrane. This reaction, triggered by physiological inducers from the female gamete, its vicinity, or other stimuli, discharges the acrosomal content modifying the plasma membrane, incorporating the inner acrosomal membrane, and exposing it to the extracellular medium. The AR is essential for sperm-egg coat penetration, fusion with the eggs' plasma membrane, and fertilization. As in most exocytotic processes Ca(2+) is crucial for the AR, as well as intracellular pH and membrane potential changes. Thus, among the required processes needed for this reaction, ion permeability changes involving channels are pivotal. In spite of the key role ion channels play in the AR, their identity and regulation is not fully understood. Though molecular and pharmacological evidence indicates that various ionic channels participate during the AR, such as store-operated Ca(2+) channels and voltage-dependent Ca(2+) channels, whole cell patch clamp recordings have failed to detect some of them until now. Since sperm display a very high resistance and a minute cytoplasmic volume, very few channels are needed to achieve large membrane potential and concentration changes. Functional detection of few channels in the morphologically complex and tiny sperm poses technical problems, especially when their conductance is very small, as in the case of SOCs. Single channel recordings and novel fluorescence microscopy strategies will help to define the participation of ionic channels in the intertwined signaling network that orchestrates the AR.

216 EFFECT OF SPERM PRETREATMENT WITH LYSOLECITHIN AND Triton X-100 ON PRONUCLEAR FORMATION AND QUALITY OF BOVINE EMBRYOS PRODUCED BY INTRACYTOPLASMIC SPERM INJECTION

In cattle, intracytoplasmic sperm injection (ICSI) has low efficiency and has application for sexed semen, semen of poor quality of high genetic value, or semen of endangered breeds. The acrosome content has a harmful effect within the oocyte because of the presence of hydrolytic enzymes. With the aim of removing the acrosome and destabilising the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX). Frozen straws of bull semen were thawed and the spermatozoa were selected by Percoll gradient. The selected spermatozoa were incubated at concentrations of 0.05% LL (ICSI-LL), 0.05% TX (ICSI-TX), and a control group (without treatment) for 1 min with vortex, and were then immediately washed by centrifugation at 400 × g in Sp-TALP medium. Following ICSI, oocytes were activated with 5 mM ionomycin and 5 µg mL–1 cycloheximide. Embryos were cultured in KSOM medium at 38.5°C, 5% CO2, 5% O2, and 90% N2, to Day 8 to record blastocyst rate. Pronuclear formation was evaluated at 19 h after activation, and the presumptive zygotes were fixed in methanol-glacial acetic acid (3 : 1), stained with Hoechst, and analysed with a fluorescence microscope. The results were evaluated by a chi-squared test with Bonferroni’s correction. Significance was set at P &lt; 0.05. The quality of embryos was evaluated by counting the total cell numbers (T), inner cell mass cells (ICM), and trophectoderm cells (TE) after staining with Hoechst and propidium iodide. We injected 345 oocytes in the control group, 335 in ICSI-LL, 304 in ICSI-TX, and 351 were parthenogenetically activated (12 replicates for each group). A higher cleavage rate was observed in ICSI-TX (66%; 231/351) and ICSI-LL (62%; 207/335) compared the control group (53%; 183/345). In addition, a significantly higher blastocyst rate was observed in both treatments: 27% (82/304) and 27.5% (92 of 335) for ICSI-TX and ICSI-LL groups, respectively, compared with the control group (22.6%; 78/345). In the parthenogenetic activation group without ICSI, the cleavage rate was 41.9% (147/351) and blastocyst rate was 15.4% (54/351). Differences among treatments were analysed using one-way ANOVA after arcsine transformation, and significance was set at P &lt; 0.05. No differences were observed in pronuclear formation (with male and female pronuclei and 2 polar bodies) for ICSI-LL (77%; 48/53), ICSI-TX (76.4%; 39/51), and the control group (71.6%; 38/53) with 3 replicates for each group. No significant differences were observed in the quality of the embryos, ICM cell number (control: 35.7 ± 2.8, ICSI-LL: 38 ± 4.5, and ICSI-TX: 40.8 ± 8.2), TE (control: 139 ± 31.5, ICSI-LL: 124 ± 10.4, and ICSI-TX: 129 ± 18.5), T (control: 170 ± 25.6, ICSI-LL: 156 ± 14.3, and ICSI-TX: 176 30.3), with 8 replicates for each group. In conclusion, this study proposed treatments able to improve the rate of development without affecting the quality of embryos produced by ICSI.

ESCRT (Endosomal Sorting Complex Required for Transport) Machinery Is Essential for Acrosomal Exocytosis in Human Sperm.

The sperm acrosome reaction is a unique, regulated exocytosis characterized by the secretion of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal and plasma membranes. In previous reports, we have shown that inward invaginations of the acrosomal membrane delineate ring-shaped membrane microdomains that contact the plasma membrane. We have postulated that the opening and expansion of fusion pores along these rings trigger acrosomal exocytosis. The invaginations of the acrosomal membrane topologically resemble the deformations of the endosomal membrane leading to the assembly of luminal vesicles in multivesicular bodies. In fact, intra-acrosomal vesicles are also formed during acrosomal exocytosis. Endosomal sorting complex required for transport (ESCRT) participates in the organization of membrane microdomains that are invaginated and released as intraluminal vesicles in endosomes. We report here that members of ESCRT I (TSG101), ESCRT III (CHMP4), and the AAA ATPase VPS4 are present in the acrosomal region of the human sperm. Perturbing the function of these factors with antibodies or recombinant proteins inhibited acrosomal exocytosis in permeabilized cells. A similar effect was observed with a dominant-negative mutant of VPS4A cross-linked to a cell-penetrating peptide in nonpermeabilized sperm stimulated with a calcium ionophore. When the function of ESCRTs was inhibited, acrosomes showed abnormal deformation of the acrosomal membrane, and SNARE proteins that participate in acrosomal exocytosis failed to be stabilized in neurotoxin-resistant complexes. However, the growing of membrane invaginations was not blocked, and numerous intra-acrosomal vesicles were observed. These observations indicate that ESCRT-mediated processes are essential for acrosomal secretion, implicating these multifunctional complexes in an exocytic event crucial for sperm-egg fusion.

Evaluation of the acrosomal status in Lama glama sperm incubated with acrosome reaction inducers

The objectives of this study were to evaluate the effect of different acrosome reaction (AR) inducers on viability and acrosomal status in llama spermatozoa, by using the FITC-PNA/PI technique and evaluate if there is a positive correlation between the FITC-PNA/PI and the Coomassie blue (CB) staining techniques. After incubating twenty ejaculates in 0.1% collagenase the centrifuged pellets were resuspended in TALP–BSA medium. An aliquot was sonicated to remove the acrosomal content (positive control). The rest of the sample was incubated for 3h at 38°C with 5% CO2 and 100% humidity. TreatmentsThree aliquots were further incubated 1h with one of the following AR inducers: calcium ionophore, ionomycin or progesterone. ControlsOne without inducers and the other, incubated with dimethyl sulfoxide (vehicle of the inducing agents). Acrosomes were evaluated at time 0 and after 4h incubation. Calcium ionophore was the most potent agent for inducing the AR (67.2±14.4% live+dead AR sperm) (P<0.05). These samples showed no motility and viability was very low (0–30%). Both ionomycin and progesterone presented significantly higher (P<0.05) percentages of total AR sperm than the controls, but had similar percentages of dead reacted sperm to the controls. A positive correlation was observed between the intact acrosome FITC-PNA/PI pattern (live+dead sperm) and the acrosome-present CB pattern (r=0.64; P=0.000) in all the evaluated samples. Conclusionsthe FITC-PNA/PI technique simultaneously evaluates viability and acrosomal status in llama spermatozoa and calcium ionophore could be used as a control of AR.

Kinetics of human sperm acrosomal exocytosis.

The acrosome reaction is a unique event in the lifespan of sperm characterized by the exocytosis of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. This unique regulated exocytosis is mediated by essentially the same membrane fusion machinery present in neuroendocrine cells. However, whereas secretion in neuroendocrine cells occurs in less than a second, the acrosome reaction is normally assessed after several minutes of incubation with inducers. In this report, we measured the kinetics of human sperm exocytosis triggered by two stimuli (calcium ionophore and progesterone) by using electron microscopy and three different approaches based on the incorporation of fluorescent Pisum sativum agglutinin into the acrosome upon opening of fusion pores connecting the extracellular medium with the acrosomal lumen. The results with the different methods are consistent with a slow kinetics (t½ = 14 min). We also manipulated the system to measure different steps of the process. We observed that cytosolic calcium increased with a relatively fast kinetics (t½ = 0.1 min). In contrast, the swelling of the acrosomal granule that precedes exocytosis was a slow process (t½ = 13 min). When swelling was completed, the fusion pore opening was fast (t½ = 0.2 min). The results indicate that acrosomal swelling is the slowest step and it determines the kinetics of the acrosome reaction. After the swelling is completed, the efflux of calcium from intracellular stores triggers fusion pores opening and the release of hybrid vesicles in seconds.